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Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed. In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression. SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors. In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy.

// Daniel Kaemmerer 1 , Tina Trager 1, 2 , Maike Hoffmeister 3 , Bence Sipos 3 , Merten Hommann 1 , Jorg Sanger 4 , Stefan Schulz 2 , Amelie Lupp 2 1 Department of General and Visceral Surgery, Zentralklinik Bad Berka, Bad Berka, Germany 2 Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University, Jena, Germany 3 Institute of Pathology, University Hospital Tuebingen, Germany 4 Institute of Pathology and Cytology, Bad Berka, Germany Correspondence to: Daniel Kaemmerer, e-mail: Daniel.Kaemmerer@zentralklinik.de Keywords: somatostatin receptor, neuroendocrine tumor, neuroendocrine carcinoma, chemokine receptor, CXCR4 Received: April 13, 2015 Accepted: July 03, 2015 Published: July 14, 2015 ABSTRACT Introduction: Somatostatin receptors (SSTR) are widely distributed in well-differentiated neuroendocrine neoplasms (NEN) and serve as primary targets for diagnostics and treatment. An overexpression of the chemokine receptor CXCR4, in contrast, is considered to be present mainly in highly proliferative and advanced tumors. Comparative data are still lacking, however, for neuroendocrine carcinomas (NEC). Methods: SSTR subtype (1, 2A, 3, 5) and CXCR4 expression was evaluated in G1 ( n = 31), G2 ( n = 47), and low (G3a; Ki-67: 21–49%; n = 21) and highly proliferative (G3b; Ki-67: >50%, n = 22) G3 (total n = 43) gastroenteropancreatic NEN samples by performing immunohistochemistry with monoclonal rabbit anti-human anti-SSTR and anti-CXCR4 antibodies, respectively, and was correlated with clinical data. Results: Both CXCR4 and SSTR were widely expressed in all tumors investigated. CXCR4 expression differed significantly between the G1 and G3 specimens and within the G3 group (G3a to G3b), and was positively correlated with Ki-67 expression. SSTR2A, in contrast, exhibited an inverse association with Ki-67. SSTR2A was highly expressed in G1 and G2 tumors, but was significantly less abundant in G3 carcinomas. Additionally, SSTR1 expression was higher in G3a than in G3b tumors. Conclusion: We observed an elevation in CXCR4 and a decrease in SSTR2A expression with increasing malignancy. Interestingly, 23% of the G3 specimens had strong SSTR2A expression. Because CXCR4 was strongly expressed in highly proliferative G3 carcinomas, it is an interesting new target and needs to be validated in larger studies.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are heterogeneous tumors that need to be molecularly defined to obtain novel therapeutic options. Forkheadbox protein M1 (FOXM1) is a crucial transcription factor in neoplastic cells and has been associated with differentiation and proliferation. We found that FOXM1 is strongly associated with tumor differentiation and occurrence of metastases in gastrointestinal NENs. In vitro inhibition by the FOXM1 inhibitor siomycin A led to down-regulation of mitotic proteins and resulted in a strong inhibitory effect. Siomycin A decreased mitosis rate, induced apoptosis in GEP-NEN cell lines and exerts synergistic effects with chemotherapy. FOXM1 is associated with clinical outcome and FOXM1 inhibition impairs survival in vitro. We therefore propose FOXM1 as novel therapeutic target in GEP-NENs.

// Elisa Neubauer 1 , Ralph M. Wirtz 2 , Daniel Kaemmerer 3 , Maria Athelogou 4 , Lydia Schmidt 1 , Jorg Sanger 5 , Amelie Lupp 1 1 Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University, Jena, Germany 2 STRATIFYER Molecular Pathology GmbH Koln, Koln, Germany 3 Department of General and Visceral Surgery, Zentralklinik Bad Berka, Bad Berka, Germany 4 DEFINIENS AG, Munchen, Germany 5 Laboratory of Cytology and Pathology, Bad Berka, Germany Correspondence to: Elisa Neubauer, email: elisa.specht@med.uni-jena.de Keywords: Ki-67, topoisomerase 2 alpha, RacGAP1, lung neuroendocrine neoplasms, IHC/RT-qPCR Received: March 13, 2016 Accepted: May 16, 2016 Published: May 31, 2016 ABSTRACT The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In this regard, we compared the expression of three proliferation markers, Ki-67, Topoisomerase II alpha (TOP2A), and RacGAP1, in a series of tumor samples from 104 BP-NEN patients (24 TC, 21 AC, 52 SCLC, 7 LCNEC) using different evaluation methods (immunohistochemistry (IHC): Average evaluation, Hotspot evaluation, digital image analysis; RT-qPCR). The results indicated that all three markers had increased protein and mRNA expression with poorer differentiation and correlated well with each other, as well as with grading, staging, and poor survival. Compared with Ki-67 and TOP2A, RacGAP1 allowed for a clearer prognostic statement. The cut-off limits obtained for Ki-67-Average (IHC) were TC-AC 1.5, AC-SCLC 19, and AC-LCNEC 23.5. The Hotspot evaluation generated equal to higher, the digital image analysis generally lower between-entity cut-off limits. All three markers enabled a clear-cut differentiation between the BP-NEN entities, and all methods evaluated were suitable for marker assessment. However, to define optimal cut-off limits, the Ki-67 evaluation methods should be standardized. RacGAP1 appeared to be a new marker with great potential.

// Sanaz Taromi 1 , Florentine Lewens 2 , Ruza Arsenic 5 , Dagmar Sedding 2 , Jorg Sanger 4 , Almut Kunze 4 , Markus Mobs 5 , Joana Benecke 2 , Helma Freitag 2, 11 , Friederike Christen 2, 6 , Daniel Kaemmerer 7 , Amelie Lupp 8 , Mareike Heilmann 9 , Hedwig Lammert 5 , Claus-Peter Schneider 9 , Karen Richter 9 , Michael Hummel 5 , Britta Siegmund 2 , Meike Burger 1 , Franziska Briest 2, 3, 10, * and Patricia Grabowski 2, 10, 11 * 1 Department of Medicine, Division of Hematology and Oncology, University Medical Center, Freiburg, Germany 2 Department of Gastroenterology, Infectious Diseases, Rheumatology CC13, Charite-Universitatsmedizin, Berlin, Germany 3 Department of Chemistry and Biochemistry, Freie Universitat (FU), Berlin, Germany 4 Institute of Pathology, Bad Berka, Germany 5 Institute of Pathology, Charite-Universitatsmedizin, Berlin, Germany 6 Institute of Biology, Humboldt-Universitat, Berlin, Germany 7 Department of General and Visceral Surgery, Zentralklinik Bad Berka GmbH, Bad Berka, Germany 8 Institute of Pharmacology and Toxicology, Jena University Hospital, Jena, Germany 9 Department for Oncology, Zentralklinik Bad Berka GmbH, Bad Berka, Germany 10 Department of Gastroenterology and Endocrinology, Zentralklinik Bad Berka GmbH, Bad Berka, Germany 11 Department of Medical Immunology, Charite Universitatsmedizin, Berlin, Germany * These authors have contributed equally to this work Correspondence to: Franziska Briest, email: franziska.briest@charite.de Keywords: FOXM1, in vivo , SCLC, mouse model, lung cancer Received: January 26, 2016 Accepted: August 04, 2017 Published: September 23, 2017 ABSTRACT Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage.