An improved assay for detection of theranostic gene translocations and MET exon 14 skipping in thoracic oncology.

Theranostic translocations may be difficult to detect by routine techniques, especially when specimens are exiguous. We recently demonstrated in a series of translocated lung adenocarcinomas that LD-RT-PCR (ligation-dependent reverse transcription polymerase chain reaction) assay could identify ALK, ROS1 and RET rearrangements with 64% sensitivity and 100% specificity. Here, we report an upgraded version of this assay used in a routine prospective cohort of lung carcinomas. Newly diagnosed lung carcinomas referred to the Rouen molecular platform between 15/05/2018 and 15/05/2019 for ALK and ROS1 IHC, genotyping (SNaPshot© +/- high-throughput genotyping) and sometimes FISH (standard routine process) were tested prospectively in parallel with the LD-RT-PCR assay designed to detect at one go ALK, ROS1 and RET translocations and MET exon 14 skipping. 413 tumors from 396 patients were included. LD-RT-PCR had a global sensitivity of 91.43% (standard routine process: 80%), with a specificity of 100%. It detected 15/18 ALK and 4/4 ROS1 translocated tumors, but also 6/6 tumors with MET exon 14 skipping retrieved by genotyping. In addition, it retrieved 7 alterations missed by the routine process, then confirmed by other means: 5 MET exon 14 skipping and 2 RET translocated tumors. Finally, it allowed to deny an effect on MET exon 14 skipping for 8 mutations detected by routine genotyping. We successfully implemented LD-RT-PCR in routine analysis. This technique is cheap, fast, sensitive, specific, and easily upgradable (e.g., NTRK translocations), but still requires IHC to be performed in parallel. Owing to its advantages, we recommend considering it, in parallel with IHC and genotyping, as an excellent cost-effective alternative, for the systematic testing of lung adenocarcinoma, to FISH and to more expensive and complex assays such as RNA-seq.

Anti-PD1/PD-L1 Immunotherapy for Non-Small Cell Lung Cancer with Actionable Oncogenic Driver Mutations.

Anti-PD1/PD-L1 immunotherapy has emerged as a standard of care for stage III-IV non-small cell lung cancer (NSCLC) over the past decade. Patient selection is usually based on PD-L1 expression by tumor cells and/or tumor mutational burden. However, mutations in oncogenic drivers such as EGFR, ALK, BRAF, or MET modify the immune tumor microenvironment and may promote anti-PD1/PD-L1 resistance. In this review, we discuss the molecular mechanisms associated with these mutations, which shape the immune tumor microenvironment and may impede anti-PD1/PD-L1 efficacy. We provide an overview of the current clinical data on anti-PD1/PD-L1 efficacy in NSCLC with oncogenic driver mutation.

The Path of Gallium from Chemical Bath into ZnO Nanowires: Mechanisms of Formation and Incorporation.

ZnO nanowires grown by chemical bath deposition (CBD) are of high interest, but their doping with extrinsic elements including gallium in aqueous solution is still challenging despite its primary importance for transparent electrodes and electronics, and for mid-infrared plasmonics. We elucidate the formation mechanisms of ZnO nanowires by CBD using zinc nitrate and hexamethylenetetramine as standard chemical precursors, as well as gallium nitrate and ammonia as chemical additives. A complete growth diagram, revealing the effects of both the relative concentration of gallium nitrate and pH, is gained by combining a thorough experimental approach with thermodynamic computations yielding theoretical solubility plots as well as Zn(II) and Ga(III) speciation diagrams. The role of Ga(OH)4- complexes is specifically shown as capping agents on the m-plane sidewalls of ZnO nanowires, enhancing their development and hence decreasing their aspect ratio. Additionally, the gallium incorporation into ZnO nanowires is investigated in detail by chemical analyses and Raman scattering. They show the predominant formation of gallium substituting for zinc atoms (GaZn) in as-grown ZnO nanowires and their partial conversion into GaZn-VZn complexes after postdeposition annealing under oxygen atmosphere. The conversion is further related to a significant relaxation of the strain level in ZnO nanowires. These findings reporting the physicochemical processes at work during the formation of ZnO nanowires and the related gallium incorporation mechanisms offer a general strategy for their extrinsic doping and open the way for carefully controlling their physical properties as required for nanoscale device engineering.

In vivo probe-based confocal laser endomicroscopy in chronic interstitial lung diseases: Specific descriptors and correlation with chest CT.

BACKGROUND AND OBJECTIVE: Probe-based confocal laser endomicroscopy (pCLE) enables in vivo microimaging of the distal lung, during bronchoscopy. This study aims at identifying pCLE descriptors of chronic interstitial lung diseases (ILD), their correlations with chest HRCT and assessing inter-observer agreement. METHODS: pCLE was performed in 21 healthy volunteers (HV) and 59 non-smoking ILD patients, including 19 patients with idiopathic pulmonary fibrosis (IPF) or asbestosis, 15 with connective tissue disease-associated ILD (CTD-ILD), 17 with sarcoidosis and 8 with hypersensitivity pneumonitis (HP). pCLE descriptors were identified in ILD on the basis of comparison with HV. RESULTS: Nine pCLE descriptors were more frequent in ILD compared to HV, with good inter-observer agreement, including fluorescent bronchiolar cells (sarcoidosis, CTD-ILD and HP), fluorescent alveolar cells (CTD-ILD and HP), small alveolar entrance rings (IPF or asbestosis and CTD-ILD), enlarged axial elastic fibres (IPF or asbestosis), septal fibres (IPF or asbestosis, CTD-ILD and HP), disorganized acinar network and rigid acinar network (IPF or asbestosis and CTD-ILD), dense elastic network (IPF or asbestosis) and alveolar fluorescent nodular structures (in sarcoidosis) (P < 0.01, Fisher's exact test, all comparisons). The distribution of nodules on computed tomography (CT) appeared to correlate with pCLE alveolar nodular structures, rigid acinar network and septal fibres, while reticulations were associated with septal fibres and disorganized or dense acinar network; ground-glass opacities on CT with small alveolar entrances, rigid elastic network and septal fibres; and honeycombing with septal fibres. CONCLUSION: In the four groups of ILD studied, 9 pCLE descriptors are described, which appear specific and reproducible, and correlate with chest HRCT patterns.

Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer.

BACKGROUND: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). METHODS: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 µM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 µM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. RESULTS: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. CONCLUSION: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma.

Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

Cost-effectiveness of lung volume reduction coil treatment in patients with severe emphysema: results from the 2-year follow-up crossover REVOLENS study (REVOLENS-2 study).

BACKGROUND: The REVOLENS study compared lung volume reduction coil treatment to usual care in patients with severe emphysema at 1 year, resulting in improved quality-adjusted life-year (QALY) and higher costs. Durability of the coil treatment benefit and its cost-effectiveness at 2 years are now assessed. METHODS: After one year, the REVOLENS trial's usual care group patients received coil treatment (second-line coil treatment group). Costs and QALYs were assessed in both arms at 2 years and an incremental cost-effectiveness ratio in cost per QALY gained was calculated. The uncertainty of the results was estimated by probabilistic bootstrapping. RESULTS: The average cost of coil treatment in both groups was estimated at €24,356. The average total cost at 2 years was €9655 higher in the first-line coil treatment group (p = 0.07) and the difference in QALY between the two groups was 0.127 (p = 0.12) in favor of first-line coil treatment group. The 2-year incremental cost-effectiveness ratio (ICER) was €75,978 / QALY. The scatter plot of the probabilistic bootstrapping had 92% of the replications in the top right-hand quadrant. CONCLUSION: First-line coil treatment was more expensive but also more effective than second-line coil treatment at 2 years, with a 2-year ICER of €75,978 / QALY. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01822795 .

Time-gated triplet-state optical spectroscopy to decipher organic luminophores embedded in rigid matrices.

Wet-chemically synthesized inorganic materials often exhibit luminescence behavior. We have recently shown that the amorphous yttrium-aluminium-borate (a-YAB) powders obtained by sol-gel and modified Pechini methods exhibit organic impurities, responsible for their intense visible photoluminescence and phosphorescence afterglow. However, the heterogeneity of impurity organic compounds and difficulties in their intact extraction from the solid inorganic host matrix limit the extraction-based chemical analysis for luminophore identification. Here, we propose a photo-physical route based on time-gated triplet-state optical spectroscopy (TGTSS) to construct the electronic structures of the trapped unknown luminophores, which successfully illustrates the luminescence properties of a-YAB powders in more detail and also provides important insights intrinsic to the nature of the luminophores. The experimental results accompanied with TD-DFT calculations of the theoretical electronic structures thus help us to propose the probable luminophore compounds trapped in rigid a-YAB matrices. We anticipate that the present approach will open new opportunities for analyzing similar complex luminescent materials, including carbon dots, graphene oxides, etc., which is vital for their improvement.

Pleural Dye Marking Using Radial Endobronchial Ultrasound and Virtual Bronchoscopy before Sublobar Pulmonary Resection for Small Peripheral Nodules.

BACKGROUND: Minimally invasive surgery of pulmonary nodules allows suboptimal palpation of the lung compared to open thoracotomy. OBJECTIVE: The objective of this study was to assess endoscopic pleural dye marking using radial endobronchial ultrasound (r-EBUS) and virtual bronchoscopy to localize small peripheral lung nodules immediately before minimally invasive resection. METHODS: The endoscopic procedure was performed without fluoroscopy, under general anesthesia in the operating room immediately before minimally invasive surgery. Then, 1 mL of methylene blue (0.5%) was instilled into the guide sheath, wedged in the subpleural space. Wedge resection or segmentectomy were guided by visualization of the dye on the pleural surface. Contribution of dye marking to the surgical procedure was rated by the surgeon. RESULTS: Twenty-five nodules, including 6 ground glass opacities, were resected in 22 patients by video-assisted thoracoscopic wedge resection (n = 11) or robotic-assisted thoracoscopic surgery (10 segmentectomies and 1 wedge resection). The median greatest diameter of nodules was 8 mm. No conversion to open thoracotomy was needed. The endoscopic procedure added an average 10 min to surgical resection. The dye was visible on the pleural surface in 24 cases. Histological diagnosis and free margin resection were obtained in all cases. Median skin-to-skin operating time was 90 min for robotic segmentectomy and 40 min for video-assisted wedge resection. The same operative precision was considered impossible by the surgeon without dye marking in 21 cases. CONCLUSIONS: Dye marking using r-EBUS and virtual bronchoscopy can be easily and safely performed to localize small pulmonary nodules immediately before minimally invasive resection.

Evidence of Organic Luminescent Centers in Sol-Gel-Synthesized Yttrium Aluminum Borate Matrix Leading to Bright Visible Emission.

Yttrium aluminum borate (YAB) powders prepared by sol-gel process have been investigated to understand their photoluminescence (PL) mechanism. The amorphous YAB powders exhibit bright visible PL from blue emission for powders calcined at 450 °C to broad white PL for higher calcination temperature. Thanks to 13 C labelling, NMR and EPR studies show that propionic acid initially used to solubilize the yttrium nitrate is decomposed into aromatic molecules confined within the inorganic matrix. DTA-TG-MS analyses show around 2 wt % of carbogenic species. The PL broadening corresponds to the apparition of a new band at 550 nm, associated with the formation of aromatic species. Furthermore, pulsed ENDOR spectroscopy combined with DFT calculations enables us to ascribe EPR spectra to free radicals derived from small (2 to 3 rings) polycyclic aromatic hydrocarbons (PAH). PAH molecules are thus at the origin of the PL as corroborated by slow afterglow decay and thermoluminescence experiments.

Molecular analysis of peripheral non-squamous non-small cell lung cancer sampled by radial EBUS.

BACKGROUND AND OBJECTIVE: Treatment optimization of non-squamous non-small-cell lung cancers (nonSq-NSCLC) relies on the molecular analysis of the tumour. We aimed to assess the predictive factors of molecular analysis feasibility (MAF) from samples of peripheral nonSq-NSCLC obtained by radial endobronchial ultrasound bronchoscopy (r-EBUS) and 1.5 mm microbiopsy forceps. METHODS: We reviewed data from consecutive peripheral lung nodules sampled with r-EBUS between January 2012 and July 2014 at a single French University Hospital. nonSq-NSCLC were systematically analysed for EGFR, KRAS, ALK, HER2, PI3K and BRAF throughout the study, and c-MET and ROS1 alterations for the last 10 months. RESULTS: Of 111 nonSq-NSCLC diagnosed by r-EBUS (113 procedures, mean nodule diameter 28 ± 15 mm), 88 were analysed for EGFR and ALK, 87 for KRAS, 86 for HER2, PI3K and BRAF and 14 for c-MET. Forty-one mutations were identified (23 KRAS, 10 EGFR, 2 BRAF, 1 HER2 and 5 ALK rearrangements). Four c-MET overexpressions were noted. MAF rose from 67% for the first 57 procedures to 89% for the last 56 procedures (P = 0.02) likely due to a higher number of biopsies performed (2 ± 1 vs 3 ± 2, P = 0.005). Upper or middle lobe location (OR 1.19, 95% CI: 1.02-1.38, P = 0.03), and at least three biopsies (OR 1.20, 95% CI: 1.04-1.40, P = 0.02) were predictive factors of MAF. Percentage of tumour cells, size of lesion and distance to the pleura did not correlate with MAF. CONCLUSION: Multi-gene molecular analysis could be performed in nearly 80% of paraffin-embedded biopsies or smear specimens sampled by r-EBUS assisted bronchoscopy of peripheral tumoral lung nodules.

Erlotinib versus carboplatin and paclitaxel in advanced lepidic adenocarcinoma: IFCT-0504.

The IFCT-0504 phase II trial evaluated the efficacy of erlotinib versus carboplatin-paclitaxel (CP) as first-line treatment in 130 cases of advanced lepidic-predominant adenocarcinoma (ADC).The primary objective of the study was treatment efficacy, evaluated based on an end-point of disease control at 16 weeks.The primary objective was met, with a disease control in 35 (53%) out of 66 patients treated with CP and in 25 (39.1%) out of 64 patients treated with erlotinib. Median progression-free survival (PFS) for the total population was 3.6 months. The disease control rate did not differ between either the therapeutic arms or pathological subtypes, whereas there was a strong interaction between treatment arms and tumour pathological subtypes for PFS (p=0.009). Mucinous tumour patients treated with erlotinib exhibited an increased progression risk (hazard ratio 3.4, 95% CI 1.7-6.5; p≤0.001). The PFS for nonmucinous tumour patients was similar in both arms. Median overall survival was 20.1 months and did not differ between therapeutic arms. These findings were not further elucidated by molecular analyses and the toxicity profiles were as expected.Our study demonstrated the dominant role of CP alongside erlotinib in the management of advanced lepidic ADC. Based on these findings, erlotinib should not be administered in first-line therapy to patients with lepidic ADC in the absence of an epidermal growth factor receptor mutation.

Safety and performance analysis of acriflavine and methylene blue for in vivo imaging of precancerous lesions using fibered confocal fluorescence microscopy (FCFM): an experimental study.

BACKGROUND: Fibered confocal fluorescence microscopy (FCFM) allows in vivo investigation of pulmonary microstructures. However, the bronchial epithelium can only be imaged using exogenous fluorophores. The objective of this study is to compare methylene blue (MB) and acriflavine genotoxicity and to assess FCFM performance for in vivo imaging of precancerous lesions. METHODS: Genotoxicity was assessed using the comet assay on both cultured human lymphocytes and NCI-H460 cells, which had been exposed to MB or acriflavine before being illuminated at 660 or 488 nm, respectively. FCFM was performed on precancerous lesions in the hamster cheek pouch model, following topical application of the fluorophores. FCFM data were analyzed according to histology. RESULTS: No genotoxicity was found using 0.01% (w/v) MB after illumination at 660 nm for 2 and 15 min (5 mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15 min of light exposure at 448 nm in both lymphocytes (83.4 to 88%, p = 0.021) and NCI H460 cell populations (79.9 to 84.6%, p = 0.045). In total, 11 invasive carcinoma, 24 reserve cell hyperplasia, and 17 dysplasia lesions were imaged using FCFM in vivo. With both fluorophores, the cellular density increased from hyperplasia to high-grade dysplasia (p < 0.05). With MB, the cellular diameter significantly decreased (48.9 to 13.9 µm) from hyperplasia to carcinoma (p < 0.05). In this model, a cut-off diameter of 30 µm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%. CONCLUSION: Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.

Order quantification of hexagonal periodic arrays fabricated by in situ solvent-assisted nanoimprint lithography of block copolymers.

Directed self-assembly of block copolymer polystyrene-b-polyethylene oxide (PS-b-PEO) thin film was achieved by a one-pot methodology of solvent vapor assisted nanoimprint lithography (SAIL). Simultaneous solvent-anneal and imprinting of a PS-b-PEO thin film on silicon without surface pre-treatments yielded a 250 nm line grating decorated with 20 nm diameter nanodots array over a large surface area of up to 4' wafer scale. The grazing-incidence small-angle x-ray scattering diffraction pattern showed the fidelity of the NIL stamp pattern replication and confirmed the periodicity of the BCP of 40 nm. The order of the hexagonally arranged nanodot lattice was quantified by SEM image analysis using the opposite partner method and compared to conventionally solvent-annealed block copolymer films. The imprint-based SAIL methodology thus demonstrated an improvement in ordering of the nanodot lattice of up to 50%, and allows significant time and cost reduction in the processing of these structures.

Correlation of programmed death-ligand 1 expression in tumour cells between diagnostic small biopsies performed by radial EBUS and surgical specimens of peripheral lung cancer.

BACKGROUND AND OBJECTIVE: Expression of programmed death-ligand 1 (PD-L1) in tumour cells (TCs) is predictive of immunotherapy efficacy in non-small cell lung cancer (NSCLC). Small biopsy samples collected by bronchoscopy are often used to diagnose peripheral lung cancer. It is questionable whether these small samples from radial endobronchial ultrasonography (r-EBUS) procedures are representative of PD-L1 expression in TCs. METHODS: We retrieved data of consecutive patients who had surgery for NSCLC and previous r-EBUS biopsy sampling, from 2017 to 2019 in our centre. PD-L1 expression in tumour cells was categorised as <1%, 1%-49% and ≥50%. PD-L1 expression was compared between r-EBUS samples and surgical specimens. RESULTS: Among 1026 patients who had r-EBUS, 521 had a diagnosis of lung cancer on r-EBUS sample. PD-L1 testing was indicated in 356 cases and results were considered contributive in 325 cases (91%). 82 patients with PD-L1 expression in r-EBUS samples had subsequent surgical resection of the nodule and were included in the study. PD-L1 expression was identical between r-EBUS samples and surgical specimens in 67% of cases, with kappa 0.44 (p<0.001). 82% of patients with PD-L1≥50% in surgical specimens were identified in r-EBUS samples. Nonetheless, 31% of patients with no PD-L1 expression in r-EBUS samples had some expression in surgical specimens. CONCLUSION: Small samples obtained by r-EBUS are adequate for assessment of PD-L1 expression in tumour cells, with moderate concordance compared to surgical specimens. Reassessment of PD-L1 expression in larger samples may be useful to guide therapy in patients with no PD-L1 expression in r-EBUS samples.

Detection of Tumor DNA in Bronchoscopic Fluids in Peripheral NSCLC: A Proof-of-Concept Study.

Introduction: DNA genotyping from plasma is a useful tool for molecular characterization of NSCLC. Nevertheless, the false-negative rate justifies the development of methods with higher sensitivity, especially in difficult-to-reach peripheral lung tumors. Methods: We aimed at comparing molecular analysis from the supernatant of guide sheath flush fluid collected during radial-EndoBronchial UltraSound (r-EBUS) bronchoscopy with plasma sampling and tumor biopsies in patients with peripheral NSCLC. The DNA was genotyped using high-throughput sequencing or the COBAS mutation test. There were 65 patients with peripheral lung tumors subjected to concomitant sampling of guide sheath flush supernatant, plasma tumor DNA, and tumor biopsy and cytology using r-EBUS. There were 33 patients (including 24 newly diagnosed with having NSCLC) with an identifiable tumor mutation in the primary lesion selected for the comparative analysis. Results: Guide sheath flush-based genotyping yielded a mutation detection rate of 61.8% (17 of 24 mutated EGFR, one of two ERBB2, one of one KRAS, one of one MAP2K, one of four MET, and zero of one STK11), compared with 33% in plasma-based genotyping (p = 0.0151). Furthermore, in eight of 34 r-EBUS without tumor cells on microscopic examination, we were able to detect the mutation in four paired guide sheath flush supernatant, compared with only two in paired plasma. Conclusion: The detection of tumor DNA in the supernatant of guide sheath flush fluid collected during r-EBUS bronchoscopy represents a sensitive and complementary method for genotyping NSCLC.

In vivo probe-based confocal laser endomicroscopy in amiodarone-related pneumonia.

Probe-based confocal laser endomicroscopy (pCLE) allows microscopic imaging of the alveoli during bronchoscopy. The objective of the study was to assess the diagnostic accuracy of pCLE for amiodarone-related pneumonia (AMR-IP). Alveolar pCLE was performed in 36 nonsmoking patients, including 33 consecutive patients with acute or subacute interstitial lung disease (ILD), of which 17 were undergoing treatment with amiodarone, and three were amiodarone-treated patients without ILD. Nine out of 17 patients were diagnosed with high-probability AMR-IP (HP-AMR-IP) by four experts, and three separate observers. Bronchoalveolar lavage findings did not differ between HP-AMR-IP and low-probability AMR-IP (LP-AMR-IP) patients. In HP-AMR-IP patients, pCLE showed large (>20 µm) and strongly fluorescent cells in 32 out of 38 alveolar areas. In contrast, these cells were observed in only two out of 39 areas from LP-AMR-IP patients, in one out of 59 areas from ILD patients not receiving amiodarone and in none of the 10 areas from amiodarone-treated patients without ILD (p<0.001; HP-AMR-IP versus other groups). The presence of at least one alveolar area with large and fluorescent cells had a sensitivity, specificity, negative predictive value and positive predictive value for the diagnosis of AMR-IP of 100%, 88%, 100% and 90%, respectively. In conclusion, pCLE appears to be a valuable tool for the in vivo diagnosis of AMR-IP in subacute ILD patients.

Local reorganization of diblock copolymer domains in directed self-assembly monitored by in situ high-temperature AFM.

In situ high-temperature AFM was used to locally follow dynamic processes, leading to directed self-assembly of copolymers in the context of graphoepitaxy. We focused on the effect of heating for temperatures much higher than the Tg of the used PS-b-PMMA polymer. We showed that such conditions favors the block rearrangement, leading to very regular and perfectly aligned structures in limited times. The use of in situ AFM allowed us to locally investigate the self-organization process at high temperature, thus bringing new insights into self-assembly of block copolymers by graphoepitaxy. In particular, we demonstrate that a slight increase of temperature between 180 and 200 °C allowed overpassing an energy barrier and considerably improves the long distance arrangement, even for relatively short times.