Efficiency of B-RAF-/MEK-inhibitors in B-RAF mutated Ameloblastoma: Case report and review of literature.

Background: Ameloblastoma is a benign but locally invasive and aggressive odontogenic tumor harboring activating BRAF V600E mutations in about two thirds of the cases. Case presentation: Neoadjuvant therapy with Dabrafenib and Trametinib was given to a 42-year-old male patient with recurrent ameloblastoma of the right mandible with a BRAF V600E mutation for 18 months. The patient manifested an excellent response to the therapy with remarkable reduction in tumor size from 72.6 mm to 55.9 mm. Histopathologically, the tumor underwent significant degenerative changes with only a few sparse vital residuals revealing 0 % Ki67 proliferative index. Conclusions: Neoadjuvant therapy with BRAF-inhibitors or BRAF-MEK-inhibitors is an effective means to reduce the size of mandibulary ameloblastomas. We propose the consideration of neoadjuvant therapy in future treatment modalities to minimize post-surgical morbidity and facial deformations.

[Cytopathology and molecular diagnostics of non-small cell lung cancer (NSCLC)]. / Zytopathologie und molekulare Diagnostik von nichtkleinzelligen Lungenkarzinomen (NSCLC).

Cytological specimens from endobronchial aspirates and pleural effusions are frequently used materials in the diagnostics of non-small cell lung cancer (NSCLC). In the same way as histological samples from endobronchial and transbronchial biopsy material or computed tomography (CT)-guided needle biopsies, cytological specimens are eminently suitable for molecular and immunohistological biomarker diagnostics of NSCLC, provided optimal techniques and clear diagnostic algorithms are employed. This article presents the typical processing techniques and a scheme for biomarker analytics and discusses an optimal approach for comprehensive diagnostics of NSCLC. When cytological specimens are processed and used in this way, the analytics are equivalent to those from histopathological specimens. For a detailed and advanced description of cytological and molecular techniques on cytological specimens the reader is referred to our own review articles.

Circulating microRNA-122 in HCV cirrhotic patients with high frequency of genotype 3.

MicroRNA-122 (miR-122) is a liver abundant microRNA that is released upon liver injury. In the present study, we investigated the circulating miR-122 profiles in a Pakistani patients´ cohort with HCV chronic liver disease that was mainly based on HCV genotype 3 infections. From 222 patients with chronic HCV liver disease, classified as mild, moderate, or severe, serum samples were collected. Cell-free RNA was isolated and used for miR-122 quantification by qPCR. More than 60% of 222 patients were infected with HCV genotype 3. ALT values and HCV viral load showed no correlation with the HCV genotype. Circulating miR-122 levels were significantly upregulated in patients with cirrhosis. Notably, HCV patients with mild cirrhosis showed the most marked increase in serum miR-122 levels (p = 0.0001). Furthermore, we proved a positive correlation (r = 0.46) of miR-122 with the ALT values in patients with mild cirrhosis. Importantly, our data of increased miR-122 levels in serum samples obtained from a patient cohort with a high prevalence of chronic genotype 3 HCV infection confirmed the previous findings collected from cohorts with a high prevalence of genotype 1. Therefore, we suggest that miR-122 increase after HCV infection does not depend on the HCV genotype. In conclusion, our findings confirm that serum miR-122 levels are significantly upregulated in the HCV cirrhotic patients serving in particular as a biomarker for the non-advanced stages of cirrhosis, independently of the HCV genotype.

Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking.

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an alpha-helix configuration. In this paper we look at the immune response of CD8(+) T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8(+)CD28(-) T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8(+)CD28(-) T lymphocytes. The Sp1-induced CD8(+)CD28(-) T lymphocytes are CD122(low)CTLA-4(+)CD39(+). They synthesize IL-10 and TGF-beta. The Sp1-induced CD8(+)CD28(-) T cells exhibit immunosuppressive properties on CD4(+) T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8(+) T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8(+) T cell activation by enhancing crosslinking of TCR. The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8(+)CD28(-) T lymphocytes in vivo and in vitro. The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model.

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

Expression profiling on subclasses of primary parotid gland carcinomas.

INTRODUCTION: The underlying molecular mechanisms of parotid gland carcinomas (PGC) are still unknown. Knowledge about the tumor-driving signaling pathways is necessary either for diagnostics or developing new therapeutic options in this heterogeneous and rare entity. MATERIAL AND METHODS: 94 matching RNA formalin-fixed and paraffin-embedded tissue samples from PGC and the corresponding non-tumor area, RNA quality and quantity were sufficient for gene expression profiling of 770 genes using the NanoString's nCounter technology. Oncogenic and tumor suppressor genes were examined in the three common PGC tumor entities: adenoid cystic carcinoma (ACC), adenocarcinoma NOS (AC-NOS), and mucoepidermoid carcinoma (MEC). RESULTS: Expression profiling and subsequent hierarchical cluster analysis clearly differentiated between non-tumor gland tissue samples and PGC. In addition expression pattern of all three entities differed. The extensive pathway analysis proved a prominent dysregulation of the Wnt signaling pathway in the three PGC entities. Moreover, transcript upstream analysis demonstrated a pronounced activation of the PI3K pathway in ACC and MEC. DISCUSSION: Our findings revealed divergent molecular expression profiles in MEC, ACC and AC-NOS that are presently studied for their potential application in PGC diagnostics. Importantly, identification of Wnt and PI3K signaling in PGC revealed novel options of PGC therapy.

Oligoclonal CD4+ T cells promote host memory immune responses to Zwitterionic polysaccharide of Streptococcus pneumoniae.

Zwitterionic polysaccharides of the normal flora bacteria represent a novel class of antigens in that they correct systemic CD4(+) T-cell deficiencies and direct lymphoid organogenesis during colonization of the host. Presentation of these polysaccharides to CD4(+) T cells depends on major histocompatibility complex class II- and DM-dependent retrograde transport from lysosomes to the cell surface. Yet the phenotype and clonality of the immune response to the polysaccharide in the mature host immune system have not been studied. Using the zwitterionic capsular polysaccharide Sp1 of Streptococcus pneumoniae, a transient member of the bacterial flora, in an experimental mouse model of cellular immunity, we demonstrated the accumulation of TH1- and TH17-polarized CD4(+) CD44(high) CD62(low) CD25(-) memory T cells. Subcutaneous immunization with Sp1 resulted in an increase of serum immunoglobulin G (IgG), predominantly of the IgG1 subclass, and suggested the presence of a humoral memory response to the polysaccharide. CD4(+) T cells stimulated with polysaccharide in vitro and in vivo showed a nonrestricted pattern for the T-cell receptor (TCR) beta-chain variable region, as demonstrated by semiquantitative reverse transcription-PCR and flow cytometry. Clonotype mapping of in vivo and in vitro polysaccharide-activated CD4(+) T cells revealed clonotypic TCR transcripts. Taken together, the data show the induction of clonal expansion of CD4(+) T cells by polysaccharides of commensal bacteria. Cellular and humoral memory host responses imply the ability of these polysaccharides to mediate the expansion of T cells via recognition within the CDR3 region of the TCR.

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors.

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c(+) CD11b(+) DCs derived from murine CD34-positive precursors. DCs derived from CD34(+) cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34(+) cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.

LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway.

Lysine-specific demethylase 1 (LSD1) is a histone modifier that is highly overexpressed in lung adenocarcinoma, which results in aggressive tumor biology. Tumor cell proliferation and migration analysis after LSD1 inhibition in the lung adenocarcinoma cell line PC9, using the LSD1 inhibitor HCI-2509 and siRNA, demonstrated that LSD1 activity was essential for proliferation and migration capacities of tumor cells. Moreover, reduced proliferation rates after LSD1 inhibition were shown to be associated with a cell-cycle arrest of the tumor cells in the G2-M-phase. Expression profiling followed by functional classification and pathway analysis indicated prominent repression of the polo-like kinase 1 (PLK1) pathway upon LSD1 inhibition. In contrast, transient overexpression of exogenous PLK1 plasmid rescued the LSD1 inhibition-mediated downregulation of PLK1 pathway genes. Mechanistically, LSD1 directly regulates expression of PLK1 by binding to its promoter region that subsequently affects expression of its downstream target genes. Notably, using lung adenocarcinoma TCGA datasets a significant correlation between LSD1 and PLK1 along with its downstream targets was observed. Furthermore, the LSD1/PLK1 linkage was confirmed by IHC analysis in a clinical lung adenocarcinoma cohort (n = 43). Conclusively, this is the first study showing a direct transcriptional link between LSD1 and PLK1. IMPLICATIONS: These findings point to a role of LSD1 in regulating PLK1 and thus efficient G2-M-transition-mediating proliferation of tumor cells and suggest targeting the LSD1/PLK1 axis as a novel therapeutic approach for lung adenocarcinoma treatment.

Preclinical studies reveal that LSD1 inhibition results in tumor growth arrest in lung adenocarcinoma independently of driver mutations.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer. Despite the development of novel targeted and immune therapies, the 5-year survival rate is still only 21%, indicating the need for more efficient treatment regimens. Lysine-specific demethylase 1 (LSD1) is an epigenetic eraser that modifies histone 3 methylation status, and is highly overexpressed in LUAD. Using representative human cell culture systems and two autochthonous transgenic mouse models, we investigated inhibition of LSD1 as a novel therapeutic option for treating LUAD. The reversible LSD1 inhibitor HCI-2509 significantly reduced cell growth with an IC50 of 0.3-5 µmin vitro, which was linked to an enhancement of histone 3 lysine methylation. Most importantly, growth arrest, as well as inhibition of the invasion capacities, was independent of the underlying driver mutations. Subsequent expression profiling revealed that the cell cycle and replication machinery were prominently affected after LSD1 inhibition. In addition, our data provide evidence that LSD1 blockade significantly interferes with EGFR downstream signaling. Finally, our in vitro results were confirmed by preclinical therapeutic approaches, including the use of two autochthonous transgenic LUAD mouse models driven by either EGFR or KRAS mutations. Importantly, LSD1 inhibition resulted in significantly lower tumor formation and a strong reduction in tumor progression, which were independent of the underlying mutational background of the mouse models. Hence, our findings provide substantial evidence indicating that tumor growth of LUAD can be markedly decreased by HCI-2509 treatment, suggesting its use as a single agent maintenance therapy or combined therapeutical application in novel concerted drug approaches.

Interleukin-6 is essential for zwitterionic polysaccharide-mediated abscess formation.

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4+ T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4+ T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4+ T-cells and causes IL-17 expression by CD4+ T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6-specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation.

Anti-inflammatory arene--chromium complexes acting as specific inhibitors of NOD2 signalling.

Inflammation is a hallmark of microbial infection in mammals and is the result of a pathogen-induced release of inflammatory effectors. In humans a variety of germ-line encoded receptors, so-called pattern-recognition receptors, respond to conserved signatures on invading pathogens, which results in the transcriptional activation of pro-inflammatory responses. Inflammation is often detrimental to the host and leads to tissue damage and/or systemic dysfunctions. Thus, specific inhibitors of these pathways are desirable for medical interventions. Herein we report on the synthesis and use of some chromium-containing compounds (arene--Cr(CO)3 complexes) with a core structure related to anti-inflammatory diterpenes produced by the sea whip Pseudopterogorgia elisabethae. By using cell-based reporter assays we identified complexes with a potent inhibitory activity on tumour necrosis factor (TNF), Toll-like receptor (TLR), and nucleotide binding domain, leucine-rich repeat-containing receptor (NLR) pathways. Moreover, we found one complex to be a specific inhibitor of inflammatory responses mediated by the NLR protein NOD2, a pivotal innate immune receptor involved in bacterial recognition. Synthesis and characterisation of a set of derivatives of this substance revealed structural requirements for NOD2 specificity. Taken together, our studies suggest this type of arene--Cr(CO)3 complex as a potential lead for the development of antiphlogistica and pharmacologically relevant NOD2 inhibitors.