Integrated Clinical, Molecular and Immunological Characterization of Pulmonary Sarcomatoid Carcinomas Reveals an Immune Escape Mechanism That May Influence Therapeutic Strategies.

Pulmonary sarcomatoid carcinoma (PSC) has highly aggressive biological behaviour and poor clinical outcomes, raising expectations for new therapeutic strategies. We characterized 179 PSC by immunohistochemistry, next-generation sequencing and in silico analysis using a deep learning algorithm with respect to clinical, immunological and molecular features. PSC was more common in men, older ages and smokers. Surgery was an independent factor (p < 0.01) of overall survival (OS). PD-L1 expression was detected in 82.1% of all patients. PSC patients displaying altered epitopes due to processing mutations showed another PD-L1-independent immune escape mechanism, which also significantly influenced OS (p < 0.02). The effect was also maintained when only advanced tumour stages were considered (p < 0.01). These patients also showed improved survival with a significant correlation for immunotherapy (p < 0.05) when few or no processing mutations were detected, although this should be interpreted with caution due to the small number of patients studied. Genomic alterations for which there are already approved drugs were present in 35.4% of patients. Met exon 14 skipping was found more frequently (13.7%) and EGFR mutations less frequently (1.7%) than in other NSCLC. In summary, in addition to the divergent genomic landscape of PSC, the specific immunological features of this prognostically poor subtype should be considered in therapy stratification.

Landscape of Genomic Alterations and PD-L1 Expression in Early-Stage Non-Small-Cell Lung Cancer (NSCLC)-A Single Center, Retrospective Observational Study.

Precision oncology and immunotherapy have revolutionized the treatment of advanced non-small-cell lung cancer (NSCLC). Emerging studies show that targeted therapies are also beneficial for patients with driver alterations such as epidermal growth factor receptor (EGFR) mutations in early-stage NSCLC (stages I-IIIA). Furthermore, patients with elevated programmed death-ligand 1 (PD-L1) expression appear to respond favorably to adjuvant immunotherapy. To determine the frequency of genomic alterations and PD-L1 status in early-stage NSCLC, we retrospectively analyzed data from 2066 unselected, single-center patients with NSCLC diagnosed using next-generation sequencing and immunohistochemistry. Nine-hundred and sixty-two patients (46.9%) presented with early-stage NSCLC. Of these, 37.0% had genomic alterations for which targeted therapies have already been approved for advanced NSCLC. The frequencies of driver mutations in the early stages were equivalent to those in advanced stages, i.e., the rates of EGFR mutations in adenocarcinomas were 12.7% (72/567) and 12.0% (78/650) in early and advanced NSCLC, respectively (p = 0778). In addition, 46.3% of early-stage NSCLC cases were PD-L1-positive, with a tumor proportion score (TPS) of ≥1%. With comparable frequencies of driver mutations in early and advanced NSCLC and PD-L1 overexpression in nearly half of patients with early-stage NSCLC, a broad spectrum of biomarkers for adjuvant and neoadjuvant therapies is available, and several are currently being investigated in clinical trials.

Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila.

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.

The Transmembrane Proteins M6 and Anakonda Cooperate to Initiate Tricellular Junction Assembly in Epithelia of Drosophila.

Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained, and remodeled is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here, we show that M6 is essential for the assembly of tricellular, but not bicellular, occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.