PDF file, 52K, Downregulation of GLI1 target HHIP after treatment of MPM cells during 48h with HhAntag 5 μM.

PDF file, 36K, Table S1: Primers used for RT-PCR.

PDF file, 66K, Western blot analysis of NF2 protein in SDM103T, ZL55SPT cells. SDM71 cells were used as positive control (1). 1. Thurneysen C, Opitz I, Kurtz S, Weder W, Stahel RA, Felley-Bosco E. Functional inactivation of NF2/merlin in human mesothelioma. Lung Cancer. 2009;64:140-7.

PDF file, 50K, GLI1 transfection (GLI1) leads to increased expression of GLI1 target HHIP compared to control vector (V) transfected ZL55SPT cells.

PDF file, 96K, 1A. Expression of HH pathway components in non-tumoral pleural tissue and mesothelioma tumors. Quantitative real-time PCR analysis of SMO, GLI2, IHH, DHH gene expression in non-tumoral pleural tissue (NT) and tumor (T). 1B. Overall survival is inversely correlated with high GLI1 expression (delta Ct below the median) (p=0.042, low GLI1: median survival 22.9 months, 95% confidence interval 0.4- 45.4 months; high GLI1: median survival 17.0 months, 95% confidence interval 9.0- 25.1 months ).

PDF file, 159K, HHIP protein is expressed in all tumor cells and its levels are increased by HhAntag. Immunohistochemistry analysis of HHIP expression was performed on xenograft tumors of control and HhAntag treated mice. H score was determined based as frequency* intensity ( frequency score 1=1-10% cells, 2=11-50% cells, 3=51-100% cells and intensity score1=low; 2= high). Bar represents 50 μm.

PDF file, 117K, Quantitative real-time PCR analysis of HH pathway gene expression in primary cultures grown without serum and 3% oxygen (n=15).

PDF file, 60K, MPM primary cultures maintained without serum in 3% oxygen show a three-fold increase in expression of ABCG2, GLI1 and PATCH1 compared to cultures without serum in 20% oxygen.

Abstract Purpose: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM). Experimental Design: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice. Results: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors. Conclusions: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach. Clin Cancer Res; 18(17); 4646–56. ©2012 AACR.

Background HNSCC has different etiology that influences therapy susceptibility. Infection with human papillomaviruses (HPV) or exposure to toxic substances can lead to cellular transformation. HPV-positive tumors have a better prognosis. The knowledge of HPV association, expression of HPV oncogenes and related cellular biomarkers may be important.

ABSTRACT Chromosomal translocations involving the immunoglobulin loci represent frequent oncogenic events in B-cell lymphoma development. Although IRF8 (ICSBP-1) protein expression has been demonstrated in germinal centre B-cells and related lymphomas in a single report, the IRF8 gene was not described as an IGH translocation partner. In a discovery driven approach we searched for new translocation partners of the IGH in DLBCL by long distance inverse (LDI) PCR and Sanger sequencing. A t(14;16)(q32.33;q24.1) IGH/IRF8 was detected in a CD5+ de novo DLBCL, confirmed by translocation specific PCR and FISH analysis. No further IRF8 aberration could be identified neither by LDI-PCR in additional five CD5+ DLBCL nor by FISH on 78 FFPE biopsies. Subsequent screening for IRF8 by immunohistochemistry revealed IRF8 expression in 18/78 (23%) correlating with a GCB type of DLBCL. This hitherto unknown translocation t(14;16)(q32.33;q24.1) is alike to represent the initiator of a multistep lymphomagenesis in a CD5+ de novo DLBCL. DOI: https://doi.org/10.3109/10428194.2013.793324 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-77888 Accepted Version Originally published at: Tinguely, Marianne; Thies, Svenja; Frigerio, Simona; Reineke, Tanja; Korol, Dimitri; Zimmermann, Dieter R (2014). IRF8 is associated with a GCB-type of DLBCL and exceptionally involved in a translocation t(14;16) (q32.33;q24.1). Leukemia Lymphoma, 55(1):136-142. DOI: https://doi.org/10.3109/10428194.2013.793324 © 2013 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily refl ect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientifi c review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the fi nal print and fi nal online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication. Just Accepted by Leukemia & Lymphoma IRF8 is associated with a GCB-type of DLBCL and exceptionally involved in a translocation t(14;16) (q32.33;q24.1) Marianne Tinguely , Svenja Thies , Simona Frigerio , Tanja Reineke , Dimitri Korol , Dieter R. Zimmermann 10.3109/10428194.2013.793324

Tumor budding has prognostic significance in many carcinomas and is defined as the presence of detached isolated single cells or small cell clusters up to 5 cells at the invasion front (peritumoral budding [PTB]) or within the tumor (intratumoral budding [ITB]). For esophageal adenocarcinomas (EACs), there are currently only few data about the impact of this morphological feature. We investigated tumor budding in a large collective of 200 primarily resected EACs. Pancytokeratin staining was demonstrated to be superior to hematoxylin and eosin staining for the detection of buds with substantial to excellent interobserver agreement and used for subsequent analysis. PTB and ITB were scored across 10 high-power fields (HPFs). The median count of tumor buds was 130/10 HPFs for PTB (range, 2-593) and 80/10 HPFs for ITB (range, 1-656). PTB and ITB correlated significantly with each other (r = 0.9; P < .001). High PTB and ITB rates were seen in more advanced tumor categories (P < .001 each); tumors with lymph node metastases (P < .001/P = .002); and lymphatic, vascular, and perineural invasion and higher tumor grading (P < .001 each). Survival analysis showed an association with worse survival for high-grade ITB (P = .029) but not PTB (P = .385). However, in multivariate analysis, lymph node and resection status, but not ITB, were independent prognostic parameters. In conclusion, PTB and ITB can be observed in EAC to various degrees. High-grade budding is associated with aggressive tumor phenotype. Assessment of tumor budding, especially ITB, may provide additional prognostic information about tumor behavior and may be useful in specific cases for risk stratification of EAC patients.

Background The epithelioid and sarcomatoid histologic variants of malignant pleural mesothelioma (MPM) can be considered as E- and M-parts of the epithelial-mesenchymal transition (EMT) axis; the biphasic being an intermediate. EMT is associated with an increase of stem cell (SC) traits. We correlated the neural crest SC marker nestin and the EMT marker periostin with histology, type of neo-adjuvant chemotherapy (CT) and overall survival (OS) of MPM patients. Patients and Methods Tumor tissues of a historic cohort 1 (320 patients) and an intended induction chemotherapy followed by extrapleural pneumonectomy (EPP) cohort 2 (145 patients) were immunohistochemically H-scored (intensity of immunoreactivity multiplied by frequency of stained cells). Paired chemo-naïve biopsies and -treated surgical specimens were available for 105/145 patients. CT included platinum/gemcitabine (Pla/Gem) or platinum/pemetrexed (Pla/Pem). Results Expression of any cytosolic nestin progressively increased from epithelioid to biphasic to sarcomatoid MPM in cohort 1, whereas the diagnostic markers calretinin and podoplanin decreased. In cohort 2, Pla/Pem CT increased the expression level of nestin in comparison to Pla/Gem, whereas the opposite was found for periostin. In Pla/Pem treated patients, nestin was higher in biphasic MPM compared to epithelioid. In addition to non-epithelioid histology, any expression of nestin in chemo-naïve biopsies (median overall survival: 22 vs. 17 months) and chemo-treated surgical specimens (18 vs. 12 months) as well as high periostin in biopsies (23 vs. 15 months) were associated with poor prognosis. In the multivariate survival analysis, any nestin expression in chemo-naïve biopsies proved to be an independent prognosticator against histology. In both pre- and post-CT situations, the combination of nestin or periostin expression with non-epithelioid histology was particularly/ dismal (all p-values <0.05). Conclusions The SC marker nestin and the EMT marker periostin allow for further prognostic stratification among histologic variants of MPM. Their expression level is influenced by neo-adjuvant chemotherapy.