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Background. Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumor associated with a dismal prognosis. Multimodal therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons underlying the rather poor efficacy of platinum compounds remain largely unknown. Kinase activity might influence cellular response to these regimens.Materials and Methods. For this exploratory study, we screened MPM cell lines (NCI-H2452, NCI-H2052, and MSTO-211H) differing in response to cisplatin and benign control fibroblasts (MRC-5) for overall phosphorylation patterns as well as kinase activity with respect to cellular response to cisplatin-based therapeutics. We analysed the cell lines for cellular kinases in a high-throughput manner using the highly innovative technique PamGene. Cell state analysis including apoptosis, necrosis, and cell viability was performed by using enzyme activity and fluorescent-based assays.Results. Cisplatin alters cellular phosphorylation patterns affecting cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis. In cisplatin-responsive cell lines, phosphorylation of AKT1 and GSK3B was decreased but could not be influenced in cisplatin-resistant NCI-H2452 cells. Cisplatin-responsive cell lines showed increased phosphorylation levels of JNK1/2/3 but decreased phosphorylation in cisplatin-resistant NCI-H2452 cells.Conclusion. Kinase phosphorylation and activity might play a crucial role in cellular response to cytostatic agents. Cisplatin influences phosphorylation patterns with distinct features in cisplatin-resistant cells. These alterations exert a significant impact on cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis of the respective tumor cells. Based on our results, the induction of p38 or JNK1/3, or inhibition of AKT1 by, for example, BIA-6, might offer a positive synergistic effect by induction of an apoptotic response to cisplatin-based treatment, thus potentially enhancing the clinical outcome of MPM patients.

Papillomas of the fallopian tube are exceedingly rare benign tumors, and only very few cases have been reported in the literature. Clinically, they may present as a mass lesion or occur without symptoms. Histomorphologically, they are papillary tumors covered by nonatypical epithelium with occasional ciliated or goblet cells growing in the lumen, and they are most frequently located in the infundibular region of the fallopian tube. They require a number of differential diagnostic evaluations and can be mistaken for either other benign tumors or malignant neoplasms. Because of their rare occurrence, molecular data about this entity have been lacking so far. Herein, a case of a papilloma with a BRAF (c.1799T>A) mutation (V600E) in a 45-yr-old woman with tumor-like dilation of the fallopian tube is presented.

Background Bortezomib is an approved proteasome inhibitor for the treatment of certain lymphoma subtypes. Two clinical trials investigated bortezomib in patients with malignant pleural mesothelioma (MPM) and failed to improve outcome. We present a potential explanation for this event. Methods 171 patients with MPM were analyzed for their mRNA expression of proteasomal subunits PSMA1, PSMA5, PSMB1, PSMB2, PSMB4 and PSMB5 via qPCR (n=84) or sequencing (n=87 TCGA/cBioPortal data set "Mesothelioma"). Outcome and subunit expression were correlated. Four mesothelial and one fibroblast cell line were treated with bortezomib and cisplatin. Cellular response was measured after 0, 6, 12, 24, 48 and 72 hrs. Enzyme activity of proteasomal subunits was assessed via functional enzyme activity assays. Results Patients with MPM presented with elevated expression of proteasomal subunits compared to benign controls (p

This study focused to investigate a possible association of extensive umbilical hypercoiling (displaying an umbilical coiling index [UCI] of at least 1.0 coils/cm), clinical outcome, and associated pathoanatomical placental lesions. Of the 771 singleton placentas from the second and third trimesters submitted for pathoanatomical evaluation, 15 cases (2%) displayed extensive hypercoiling. There was an association of excessive hypercoiling with hypotrophy of fetuses and children (11 cases) and fetal demise (12 cases). Thin cord syndrome and umbilical stricture were observed in 9 cases and 4 cases, respectively. Seven of the 15 cases with excessive umbilical hypercoiling showed increased placental fibrin deposition (47% of the cases with hypercoiling), in 4 cases sufficient for rendering the diagnosis of massive perivillous fibrin deposition. Signs of maternal vascular malperfusion (n = 6) and chorangiosis (n = 2) were also detected in cases with hypercoiling. Recurrence of excessive umbilical hypercoiling was observed in 2 families, suggesting a genetic predisposition for the development of this lesion. Extensive hypercoiling could be a hitherto underrecognized pathogenetic factor for the development of massive perivillous fibrin deposition. A high UCI measured in the second trimester by ultrasound may be predictive of fetal hypotrophy, and intensified fetal monitoring is warranted, particularly if there is a history of hypercoiling and adverse fetal outcome.

Cystic echinococcosis is a widely endemic helminthic disease worldwide but occurs only rarely in Central Europe. Humans are infected as ‘aberrant’ hosts by Echinococcus granulosus and develop cysts in numerous different organs. 20%–30% of the affected individuals develop hydatid disease in the lungs with associated complications including pleuritis, lung abscess and pneumothorax. Radiologically, the pulmonary lesions of cystic echinococcosis occasionally pose difficulties in the differential diagnosis of primary lung carcinoma or metastatic disease and vice versa. Herein we report on a case of pulmonary hydatid disease in a 25-year-old Iraqi male presenting with a cystic lesion of the lung associated with thoracic pain and involuntary weight loss. Despite of its rare occurrence in Central Europe, clinicians, radiologists and pathologists should be aware of this entity and its pulmonary manifestations. During frozen section examination, imprint cytology specimens may facilitate the detection of the pathogens.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare malignant tumor associated with asbestos exposure, with infaust prognosis and overall survival below 20 months in treated patients. Platinum is still the backbone of the chemotherapy protocols, and the reasons for the rather poor efficacy of platinum compounds in MPM remain largely unknown. Therefore, we aimed to analyze differences in key signaling pathways and biological mechanisms in therapy-naïve samples and samples after chemotherapy in order to evaluate the effect of platinum-based chemotherapy. METHODS: The study cohort comprised 24 MPM tumor specimens, 12 from therapy-naïve and 12 from patients after platinum-based therapy. Tumor samples were screened using the NanoString nCounter platform for digital gene expression analysis with an appurtenant custom-designed panel comprising a total of 366 mRNAs covering the most important tumor signaling pathways. Significant pathway associations were identified by gene set enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) RESULTS: We have found reduced activity of TNF (normalized enrichment score: 2.03), IL-17 (normalized enrichment score: 1.93), MAPK (normalized enrichment score: 1.51), and relaxin signaling pathways (normalized enrichment score: 1.42) in the samples obtained after platinum-based therapy. In contrast, AMPK (normalized enrichment score: –1.58), mTOR (normalized enrichment score: –1.50), Wnt (normalized enrichment score: –1.38), and longevity regulating pathway (normalized enrichment score: –1.31) showed significantly elevated expression in the same samples. CONCLUSIONS: We could identify deregulated signaling pathways due to a directed cellular response to platinum-induced cell stress. Our results are paving the ground for a better understanding of cellular responses and escape mechanisms, carrying a high potential for improved clinical management of patients with MPM.

Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8–12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.

Villitis of unknown etiology (VUE) and chronic deciduitis with plasma cells (CD) are supposed to be non infectious placental lesions caused by a pathologic immune reaction similar to a host versus graft mechanism. In some investigations, infection of human trophoblastic cells with human papilloma virus (HPV) has been described, and a relationship with miscarriage, preeclampsia, and chronic inflammatory placental lesions has been suspected. Infection with enterovirus, especially Coxsackievirus, has been observed in cases with spontaneous abortion and adverse perinatal outcome, respectively. We investigated 20 cases with VUE and 30 cases with chronic deciduitis with plasma cells. The placenta specimens were analyzed for expression of HPV capsid protein by immunohistochemistry, for presence of HPV DNA via polymerase chain reaction (PCR), and for presence of enterovirus mRNA using RT-PCR, respectively. VUE was associated with maternal diseases: atopic lesions in 21%, other autoimmune diseases in 15.5%, and obesity in 31.5%, respectively. Birth weight below the 10th percentile was detected in 63% of the cases with VUE. Chronic deciduitis was associated with preterm labor and preterm premature rupture of membranes (26%). Intrauterine fetal demise occurred in 5 cases with CD (18.5%). HPV DNA, HPV capsid protein, and enterovirus mRNA were not detected in all investigated VUE or CD cases. Our investigations show that a causal role for enterovirus and human papilloma virus in the development of VUE and CD is unlikely. Therefore, HPV vaccination is unlikely to reduce the incidence of VUE and CD in the future.

The aim of this study was to analyze the diagnostic role of BAP1 in effusion cytology. Effusions (n = 258), consisting of 53 malignant mesotheliomas and 205 other cancers, the majority carcinomas (62 breast, 60 ovarian, 31 lung, 51 carcinomas of other origin, 1 melanoma), were analyzed for BAP1 expression using immunohistochemistry. BAP1 was lost in 46 (87%) mesotheliomas compared with 4 (2%) of 205 other cancers (P < .001), resulting in sensitivity and specificity of 87% and 98%, respectively. There was no significant difference between peritoneal (n = 14) and pleural (n = 39) mesotheliomas. The 4 carcinomas with loss of BAP1 included 1 ovarian, 1 breast, 1 uterine cervical, and 1 gastric carcinoma. The present study supports the role of BAP1 as a highly sensitive and specific marker for malignant mesothelioma in serous effusions and argues for inclusion of this test in all specimens in which this diagnosis is considered.

Non-classical Human Leukocyte Antigen (HLA)-E preferentially presents leader peptides derived from classical HLA-class I molecules. HLA-E can trigger opposed immune responses by interacting with inhibitory NKG2A or by activating NKG2C receptors on NK and T-cells. We studied the impact of HLA-E on renal allograft survival during acute cellular rejection. HLA-E expression was up-regulated in acute cellular rejection (ACR) biopsies (n=12) compared to biopsies from 13 renal allografts with no rejection-signs. HLA-E up-regulation was correlated with numbers of HLA-class I leader peptide mismatches (p=0.04). CD8+ and CD56+ infiltrating cells correlated with HLA-E expression (p0.0001 and p=0.0009, respectively). Activating NKG2C receptor dominated on effector cells in biopsies and peripheral blood during ACR potentially allowing HLA-E-mediated immune activation. Moreover, HLA-E expression correlated with deterioration in renal allograft function (p0.008) and reduced allograft survival (p=0.002). Our findings provide evidence that during renal allograft rejection HLA-E along with high numbers of mismatched HLA-class I leader peptides might represent additional targets for immune-activating responses.

Introduction Hepatocellular carcinoma (HCC) in cirrhosis is a widely accepted indication for liver transplantation (LT). Many scoring systems have been proposed intending to an extension of the established Milan criteria. Bridging treatments are systematically applied in order to maintain or to downstage such patients to the listing criteria. The objective of our study was to estimate the feasibility of the prediction of microvascular tumor invasion in transplant candidates. Patients and methods Data corresponding to transplanted HCC patients were reviewed for the purposes of this study. All tumor slices were blindly re-evaluated by a single pathologist in order to score for tumor necrosis and microvascular invasion. Recipients of pediatric or split LT were excluded. Results Eighty patients (30 women and 50 men) were included in the study. Tumor necrosis was absent in 29 of 80 liver explants (36.25%). In the majority of instances (63.75%) tumor necrosis was evident in proportions between 5% and 100%. In 58 liver explants showing 0%-60% tumor necrosis and 22 liver explants showing > 60% tumor necrosis, microvascular tumor invasion was detectable in 11 and 0 cases, respectively (P = .0385). Conclusion In about one-fourth of the cases (27.5%) microvascular tumor invasion could not be detected due to extended areas of tumor necrosis. Preoperative detection of microvascular invasion is misleading.

Background Malignant pleural mesothelioma (MPM) is a tumour arising from pleural cavities with poor prognosis. Multimodality treatment with pemetrexed combined with cisplatin shows unsatisfying response-rates of 40%. The reasons for the rather poor efficacy of chemotherapeutic treatment are largely unknown. However, it is conceivable that DNA repair mechanisms lead to an impaired therapy response. We hypothesize a major role of homologous recombination (HR) for genome stability and survival of this tumour. Therefore, we analysed genes compiled under the term “BRCAness”. An inhibition of this pathway with olaparib might abrogate this effect and induce apoptosis. Methods We investigated the response of three MPM cell lines and lung fibroblasts serving as a control to treatment with pemetrexed, cisplatin and olaparib. Furthermore, we aimed to find possible correlations between response and gene expression patterns associated with BRCAness phenotype. Therefore, 91 clinical MPM samples were digitally screened for gene expression patterns of HR members. Results A BRCAness-dependent increase of apoptosis and senescence during olaparib-based treatment of BRCA-associated-protein 1 (BAP1)-mutated cell lines was observed. The gene expression pattern identified could be found in approx. 10% of patient samples. Against this background, patients could be grouped according to their defects in the HR system. Gene expression levels of Aurora Kinase A (AURKA), RAD50 as well as DNA damage-binding protein 2 (DDB2) could be identified as prognostic markers in MPM. Conclusions Defects in HR compiled under the term BRCAness are a common event in MPM. The present data can lead to a better understanding of the underlaying cellular mechanisms and leave the door wide open for new therapeutic approaches for this severe disease with infaust prognosis. Response to Poly (ADP-ribose)-Polymerase (PARP)-Inhibition could be demonstrated in the BAP1-mutated NCI-H2452 cells, especially when combined with cisplatin. Thus, this combination therapy might be effective for up to 2/3 of patients, promising to enhance patients’ clinical management and outcome. Electronic supplementary material The online version of this article (10.1186/s12885-019-5314-0) contains supplementary material, which is available to authorized users.

ObjectivesRectal infections with Chlamydia trachomatis and/or Neisseria gonorrhoeae (CT/NG) are common in men who have sex with men (MSM) and are linked to HIV transmission. However, rectal CT/NG infections are often asymptomatic and it is not known how they contribute to HIV transmission. We assessed clinical and cytological signs of inflammation as well as rectal HIV-RNA in HIV-infected MSM with and without CT/NG infection.Methods112 HIV-positive MSM with or without rectal symptoms and with or without antiretroviral therapy who underwent high-resolution anoscopy (HRA) at the proctological outpatient centre of the University Hospital Essen, Germany, between November 2013 and February 2014 were included in this cross-sectional study. During the examination, rectal swabs for the assessment of CT/NG, HIV-RNA and inflammatory cells (granulocytes, lymphocytes, histiocytes) were collected. 110 patients were assessed according to the study protocol, and no imputation of missing data was performed.ResultsRectal infections with CT or NG were detected in 17 participants, and 4 participants were coinfected. Only symptomatic CT/NG infections (8/17) showed signs of inflammation in HRA. Symptomatic CT/NG infections were also associated with the detection of lymphocytes and histiocytes in rectal cytology (both PConclusionsOnly symptomatic but not asymptomatic rectal infections with CT and/or NG were associated with clinical and cytological signs of inflammation. Rectal HIV shedding was not promoted by CT/NG infections in patients receiving ART with suppressed plasma HIV-RNA.Trial registration numberUTN: U1111-1150-4804. German Clinical Trials Register (DRKS): DRKS00005468.

CA Wessolly Background: Immunotherapy, especially immune checkpoint inhibition, is one of the most sophisticated approaches in cancer therapy. Immune checkpoint inhibition has already been successfully applied for treatment of non-small cell lung cancer and various other entities. Unfortunately, 60% of the cases show signs of therapy resistance. Additionally, a proportion of cases shows initial insensitivity to immune checkpoint inhibition. We consider a novel escape mechanism in association with deficient proteasomal epitope processing to be one prominent reason for initial insensitivity and therapy resistance. Therefore, we aim to identify mutations in association with these so-called processing escapes, in a highly diverse collective of pulmonary neuroendocrine lung tumors. Materials and Methods: Seventy representative tumor specimens of pulmonary neuroendocrine lung tumors were analyzed retrospectively via immunohistochemical detection of CD4, CD8, CD68, and CD20 as well as programmed cell death protein 1 and programmed cell death 1 ligand 1 for tumor immune infiltration and composition. Afterward, samples were screened for alterations in 48 genes, including 221 known mutational hotspots by massive parallel sequencing using the Illumina TruSeq Amplicon-Cancer Panel. For prediction of proteasomal cleavage probabilities, an R implementation of the machine learning tool NetChop 3.1 was utilized. Results: Immune cell infiltration of different compositions could be found in the majority of tumors. Deficient epitope processing was revealed to be a common event in those with steady distribution across all different subtypes. Despite immune infiltration, no significant antitumor response could be detected. Conclusion: Since it is widely acknowledged that tumors need to avoid the immune system to ensure their survival, processing escapes should already be present during primary tumor development. In line, processing escapes can be found in all tumors, regardless of subtype and mutational burden. Furthermore, there is solid evidence that processing escapes have a negative impact on the antitumor activity of tumor infiltrating immune cells.

Simple Summary Malignant pleural mesothelioma (MPM) is a rare, biologically extremely aggressive tumor with an infaust prognosis. In this retrospective study, we aimed to assess the role of tumor-infiltrating immune cells and their activity in the respective histologic subtypes. We confirmed a substantial difference between epithelioid and sarcomatoid mesothelioma regarding the host’s anti-cancer immune reaction. Whereas antigen processing and presentation to resident cytotoxic T cells as well as phagocytosis is highly affected in sarcomatoid mesothelioma, cell–cell interaction via cytokines seems to be of greater importance in epithelioid cases. Our work reveals the specific role of the immune system within the different histologic subtypes of MPM, providing a more detailed background of their immunogenic potential. This is of great interest regarding therapeutic strategies addressing immunotherapy in mesothelioma. Abstract Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with asbestos exposure. Median survival ranges from 14 to 20 months after initial diagnosis. As of November 2020, the FDA approved a combination of immune checkpoint inhibitors after promising intermediate results. Nonetheless, responses remain unsatisfying. Adequate patient stratification to improve response rates is still lacking. This retrospective study analyzed formalin fixed paraffin embedded specimens from a cohort of 22 MPM. Twelve of those samples showed sarcomatoid, ten epithelioid differentiation. Complete follow-up, including radiological assessment of response by modRECIST and time to death, was available with reported deaths of all patients. RNA of all samples was isolated and subjected to digital gene expression pattern analysis. Our study revealed a notable difference between epithelioid and sarcomatoid mesothelioma, showing differential gene expression for 304/698 expressed genes. Whereas antigen processing and presentation to resident cytotoxic T cells as well as phagocytosis is highly affected in sarcomatoid mesothelioma, cell–cell interaction via cytokines seems to be of greater importance in epithelioid cases. Our work reveals the specific role of the immune system within the different histologic subtypes of MPM, providing a more detailed background of their immunogenic potential. This is of great interest regarding therapeutic strategies including immunotherapy in mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. Material and Methods: For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. Results: CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. Conclusion: DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management.

// Robert Fred Henry Walter 1, 2, * , Fabian Dominik Mairinger 2, 3, * , Robert Werner 4 , Claudia Vollbrecht 3 , Thomas Hager 2 , Kurt Werner Schmid 2 , Jeremias Wohlschlaeger 2, 5 , Daniel Christian Christoph 6 1 Ruhrlandklinik Essen, West German Lung Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 2 Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 3 Institute of Pathology, Division of Molecular Pathology, Charite, Berlin, Germany 4 Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany 5 Institute of Pathology, Ev.-Luth. Diakonissenkrankenhaus Flensburg, Flensburg, Germany 6 Department of Medical Oncology, West German Cancer Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany * These authors have contributed equally to this work Correspondence to: Robert Fred Henry Walter, e-mail: robert.walter@ruhrlandklinik.uk-essen.de Keywords: lung cancer, neuroendocrine pulmonary tumors, folic acid metabolism, DNA repair, NanoString nCounter Received: January 29, 2016 Accepted: January 30, 2016 Published: February 26, 2016 ABSTRACT Purpose: 25% of all lung cancer cases are neuroendocrine (NELC) including typical (TC) and atypical carcinoid (AC), large-cell neuroendocrine (LCNEC) and small cell lung cancer (SCLC). Prognostic and predictive biomarkers are lacking. Experimental Design: Sixty patients were used for nCounter mRNA expression analysis of the folic-acid metabolism ( ATIC , DHFR , FOLR1 , FPGS , GART , GGT1 , SLC19A1 , TYMS ) and DNA-repair ( ERCC1 , MLH1 , MSH2 , MSH6 , XRCC1 ). Phenotypic classification classified tumors (either below or above the median expression level) with respect to the folic acid metabolism or DNA repair. Results: Expression of FOLR1 , FPGS , MLH1 and TYMS (each p

Introduction The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis. Design FFPE-derived RNA from sections of 23 patients with spontaneous pneumothoraxes was used. Unstained sections of FFPE tissue were stored at various temperatures (−80 °C, −20 °C, 4 °C, 24 °C) prior to RNA extraction. The potential impact on RNA quality of semi-automatic and manual RNA isolation and three different deparaffinization agents (mineral oil, xylene and d-limonene) were compared. Results The storage temperature of FFPE sections affects RNA concentration and fragmentation, with the optimal storage temperature below -20 °C. The RNA extracted with d-limonene shows equivalent quality to the RNA extracted using more toxic standard agents. The manual isolation provides a higher RNA yield compared to the semi-automatic isolation. However, no differences in the amount of longer RNA fragments were observed. Furthermore, the semi-automatic isolation showed an enhanced RNA quality. Conclusion FFPE sections not directly used for RNA extraction should be stored below -20 °C to increase quality and yield of the RNA. Usage of semi-automatic isolation produces superior results and simplifies routine processes by having less hands-on-time. Replacement of toxic xylene by d-limonene may contribute to improved occupational safety while not influencing analytical results.

Previously, our group demonstrated that nuclear expression of E3 ubiquitin ligase (MDM2) in malignant pleural mesothelioma (MPM) is significantly associated with decreased overall survival. A possible explanation may be that overexpression of MDM2 leads to a proteasomal degradation of TP53 that eventually results in a loss of TP53-induced apoptosis and senescence. It is well known from other tumor entities that restoration of TP53 activity, e.g., by MDM2 inhibition, results in an instant TP53-induced stress and/or DNA damage response of cancer cells. Nutlin-3A (acis-imidazoline analogue) has been described as a potent and selective MDM2 inhibitor preventing MDM2-TP53-interaction by specific binding to the hydrophobic TP53-binding pocket of MDM2. In the present study, the effects of MDM2 inhibition in MPM via Nutlin-3A and standard platinum based chemotherapeutic agents were comparatively tested in three MPM cell lines (NCI-H2052, MSTO-211H, and NCI-H2452) showing different expression profiles of TP53, MDM2, and its physiological inhibitor of MDM2—P14/ARF. Ourin vitroexperiments on MPM cell lines revealed that Nutlin-3A in combination with cisplatin resulted in up to 9.75 times higher induction of senescence (p=0.0050) and up to 5 times higher apoptosis rate (p=0.0067) compared to the commonly applied cisplatin and pemetrexed regimens. Thus Nutlin-3A, a potent inhibitor of MDM2, is associated with a significant induction of senescence and apoptosis in MPM cell lines, making Nutlin-3A a promising substance for a targeted therapy in the subgroup of MPM showing MDM2 overexpression.

Arteriosklerose fuhrt durch langsame Entwicklung von Stenosen oder durch plotzliche Okklusion des Gefaslumens durch einen Thrombus zu klinischer Symptomatik. Diese entsteht durch verminderte Perfusion im Myokard (koronare Herzerkrankung, KHK), im zentralen Nervensystem (Apoplexie) oder in den Extremitaten (periphere arterielle Verschlusskrankheit, pAVK). Die KHK stellt die haufigste Manifestation arteriosklerotischer Gefaslasionen dar und umfasst sowohl die stabile Angina pectoris wie auch die akuten Koronarsyndrome. Arteriosklerose ist eine im Wesentlichen durch Lipoproteinakkumulation in der Arterienwand ausgeloste Erkrankung, welche mit der Formation von Plaques an bestimmten Stellen des arteriellen Systems einhergeht. Die entscheidenden Pathomechanismen umfassen Entzundung, Nekrose, Fibrose und Kalzifizierung. Nach jahrzehntelangem indolenten Verlauf kann es plotzlich zu lebensbedrohlichen akuten Koronarsyndromen kommen. In den allermeisten Fallen ist die zugrunde liegende Lasion eine rupturierte Plaque, deren nekrotisches Material ihres „Kerns“ eine hohe Thrombogenitat aufweist. Die arteriosklerotischen Lasionen, die zu einer plotzlichen Thrombosierung mit kompletter oder inkompletter Okklusion fuhren konnen, sind das sog. „thin-cap fibroatheroma“, die Plaque-Erosion und das sog. kalzifizierte Knotchen bei stark verkalkten Arterien alterer Individuen, wobei der jeweilige Pathomechanismus bis dato nicht vollstandig geklart ist. Die vorliegende Ubersichtsarbeit soll einen Uberblick uber die Entstehung der wichtigsten arteriosklerotischen Veranderungen und deren Progression zur Plaque-Ruptur bzw. Thrombusformation geben.

Increasingly frequent, it is clinically indicated to obtain tissue from a peripheral lung lesion (PLL) to yield a pathological diagnosis. The aim of the present study was to evaluate the diagnostic sensitivity of transbronchial needle aspiration (TBNA) and transbronchial catheter aspiration (TBCA) in addition to transbronchial forceps biopsy (TBB) at conventional bronchoscopy. Eligible patients showing a PLL on computed tomography scans were included in the study. In all patients, following TBB, TBNA and TBCA were employed in randomised order under fluoroscopy. Fourty-eight patients were enrolled, of whom 46 patients with 46 PLLs were included in the analysis. The mean ± SD diameter of the PLL was 27.0 ± 13.3 mm. The overall sensitivity for all modalities was 69.6 %; PLL ≤20 or >20 and ≤30 mm in diameter showed a sensitivity of 60.0 and 72.2 %, respectively. For malignant PLL (n = 33), the combined sensitivity of TBNA + TBCA versus TBB was significantly higher (63.6 vs. 33.3 %, p ≤ 0.05), and could not further be improved by TBB. For benign PLL, TBB was superior to TBNA + TBCA (76.9 vs. 38.5 %). TBB, TBNA and TBCA are complementary to one another. Combining the three techniques, even allows transbronchial specimen collection of PLL

// Robert Fred Henry Walter 1,2 , Robert Werner 2 , Saskia Ting 2 , Claudia Vollbrecht 3 , Dirk Theegarten 2 , Daniel Christian Christoph 4 , Kurt Werner Schmid 2 , Jeremias Wohlschlaeger 2 and Fabian Dominik Mairinger 2 1 Ruhrlandklinik, West German Lung Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 2 Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 3 Institute of Pathology, University Hospital Cologne, Cologne, Germany 4 Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany Correspondence to: Robert Fred Henry Walter, email: // Keywords : small-cell lung cancer, large-cell neuroendocrine lung cancer, carcinoids, apoptosis, NanoString nCounter Received : March 11, 2015 Accepted : April 15, 2015 Published : May 04, 2015 Abstract Background: Neuroendocrine tumors of the lung comprise typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine cancer (LCNEC) and small-cell lung cancer (SCLC). Cell cycle and apoptosis are key pathways of multicellular homeostasis and deregulation of these pathways is associated with cancerogenesis. Materials and Methods: Sixty representative FFPE-specimens (16 TC, 13 AC, 16 LCNEC and 15 SCLC) were used for mRNA expression analysis using the NanoString technique. Eight genes related to apoptosis and ten genes regulating key points of cell cycle were investigated. Results: ASCL1 , BCL2 , CASP8 , CCNE1 , CDK1 , CDK2 , CDKN1A and CDKN2A showed lower expression in carcinoids compared to carcinomas. In contrast, CCNE1 and CDK6 showed elevated expression in carcinoids compared to carcinomas. The calculated BCL2/BAX ratio showed increasing values from TC to SCLC. Between SCLC and LCNEC CDK2 , CDKN1B , CDKN2A and PNN expression was significantly different with higher expression in SCLC. Conclusion: Carcinoids have increased CDK4/6 and CCND1 expression controlling RB1 phosphorylation via this signaling cascade. CDK2 and CCNE1 were increased in carcinomas showing that these use the opposite way to control RB1. BAX and BCL2 are antagonists in regulating apoptosis. BCL2 expression increased over BAX expression with increasing malignancy of the tumor from TC to SCLC.

Summary Custodiol-N, a new preservation solution, has been shown particularly suitable for hypothermic machine perfusion preservation (HMP) in isolated porcine kidneys. These preliminary results should be confirmed in an actual transplant model in vivo. Kidney function after 21 h of HMP was studied in an autotransplant model using Landrace pigs (25–30 kg; n = 6 per group). Perfusion was performed with oxygenated perfusate, using either Custodiol-N solution including 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1) as gold standard. Viability of the grafts was followed for 1 week after bilateral nephrectomy in the recipient pigs. HMP with CND resulted in less acute tubular injury, evaluated by levels of fatty acid-binding protein and better clearance function during the first 24 h after Tx than with KPS-1 (P

Background: Malignant pleural mesothelioma (MPM) is a highly aggressive tumour that is first-line treated with a combination of cisplatin and pemetrexed. Until now, predictive and prognostic biomarkers are lacking, making it a non-tailored therapy regimen with unknown outcome. P53 is frequently inactivated in MPM, but mutations are extremely rare. MDM2 and P14/ARF are upstream regulators of P53 that may contribute to P53 inactivation. Methods: A total of 72 MPM patients were investigated. MDM2 immunoexpression was assessed in 65 patients. MDM2 and P14/ARF mRNA expression was analysed in 48 patients of the overall collective. The expression results were correlated to overall survival (OS) and progression-free survival (PFS). Results: OS and PFS correlated highly significantly with MDM2 mRNA and protein expression, showing a dismal prognosis for patients with elevated MDM2 expression (for OS: Score (logrank) test: P⩽0.002, and for PFS: Score (logrank) test; P

Background Activation of the Rho–Rho-kinase pathway has been shown to cause vasoconstriction in renal afferent arterioles. Vascular dysfunction plays a pivotal role in triggering reperfusion injury after kidney transplantation. Therefore, the effect of a Rho-kinase inhibitor, added to the preservation solution, on renal function after 18 h of storage at 4 °C was evaluated. Methods Porcine kidneys were preserved with cold HTK-solution. During preservation, in the study group, HTK was supplemented with the Rho-kinase inhibitor HA1077, whereas the control group received no further treatment ( n = 6, respectively). Kidney function after 18 h of storage at 4 °C was evaluated by 90 min of isolated reperfusion in vitro . Results Rho-kinase inhibition (RKI) was associated with significantly higher renal perfusate flow compared to the control group. Endothelial function, as measured by perfusate levels of nitric oxide and gene expression of eNOS, was significantly increased in the study group. In our model, RKI also significantly improved glomerular function (clearance of creatinine) as well as tubular cell integrity as reflected by reduced fractional sodium excretion and release of fatty acid binding protein, a specific tubular cell marker. Conclusion Our results indicate that blocking the Rho-kinase pathway during cold preservation may lead to a better graft function upon reperfusion.

Rectal infections with112 HIV-positive MSM with or without rectal symptoms and with or without antiretroviral therapy who underwent high-resolution anoscopy (HRA) at the proctological outpatient centre of the University Hospital Essen, Germany, between November 2013 and February 2014 were included in this cross-sectional study. During the examination, rectal swabs for the assessment of CT/NG, HIV-RNA and inflammatory cells (granulocytes, lymphocytes, histiocytes) were collected. 110 patients were assessed according to the study protocol, and no imputation of missing data was performed.Rectal infections with CT or NG were detected in 17 participants, and 4 participants were coinfected. Only symptomatic CT/NG infections (8/17) showed signs of inflammation in HRA. Symptomatic CT/NG infections were also associated with the detection of lymphocytes and histiocytes in rectal cytology (both P0.001). In contrast, asymptomatic CT/NG infections neither resulted in clinical nor cytological signs of inflammation. Rectal HIV-RNA was undetectable in all participants with rectal CT/NG infections who received combined antiretroviral therapy (ART) when plasma HIV-RNA was below the limit of detection (n=13). Besides rectal CT/NG infections, syphilis (n=4) and HPV-associated lesions (n=37) were frequently detected, and proctological symptoms were associated with simultaneous infection with ≥2 STDs.Only symptomatic but not asymptomatic rectal infections with CT and/or NG were associated with clinical and cytological signs of inflammation. Rectal HIV shedding was not promoted by CT/NG infections in patients receiving ART with suppressed plasma HIV-RNA.UTN: U1111-1150-4804. German Clinical Trials Register (DRKS): DRKS00005468.

// Fabian D. Mairinger 1, * , Jan Schmeller 1, * , Sabrina Borchert 1 , Michael Wessolly 1 , Elena Mairinger 1 , Jens Kollmeier 2 , Thomas Hager 1 , Thomas Mairinger 3 , Daniel C. Christoph 4, 5 , Robert F.H. Walter 1, 6 , Wilfried E.E. Eberhardt 4, 6 , Till Plones 7 , Jeremias Wohlschlaeger 1, 8 , Bharat Jasani 9 , Kurt Werner Schmid 1 and Agnes Bankfalvi 1 1 Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 2 Department of Pneumology, Helios Klinikum Emil von Behring, Berlin, Germany 3 Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany 4 Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 5 Department of Internistic Oncology, Kliniken Essen Mitte, Essen, Germany 6 Ruhrlandklinik, West German Lung Centre, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 7 Department of Thoracic Surgery and Thoracical Endoscopy, Ruhrlandklinik, University Hospital Essen, University of Duisburg-Essen, Essen, Germany 8 Department of Pathology, Diakonissenkrankenhaus Flensburg, Flensburg, Germany 9 Department of Pathology, Targos Molecular Pathology GmbH, Kassel, Germany * These authors have contributed equally to this work Correspondence to: Fabian D. Mairinger, email: fabian.mairinger@uk-essen.de Keywords: malignant pleural mesothelioma; metallothionein; overall survival; prognostic biomarker; platin-based chemotherapy; Pathology Received: August 18, 2017 Accepted: March 11, 2018 Published: April 27, 2018 ABSTRACT Background: Malignant pleural mesothelioma (MPM) is a biologically highly aggressive tumor arising from the pleura with a dismal prognosis. Cisplatin is the drug of choice for the treatment of MPM, and carboplatin seems to have comparable efficacy. Nevertheless, cisplatin treatment results in a response rate of merely 14% and a median survival of less than seven months. Due to their role in many cellular processes, methallothioneins (MTs) have been widely studied in various cancers. The known heavy metal detoxifying effect of MT-I and MT-II may be the reason for heavy metal drug resistance of various cancers including MPM. Methods: 105 patients were retrospectively analyzed immunohistochemically for their MT expression levels. Survival analysis was done by Cox-regression, and statistical significance determined using likelihood ratio, Wald test and Score (logrank) tests. Results: Cox-regression analyses were done in a linear and logarithmic scale revealing a significant association between expression of MT and shortened overall survival (OS) in a linear (p=0.0009) and logarithmic scale (p=0.0003). Reduced progression free survival (PFS) was also observed for MT expressing tumors (linear: p=0.0134, log: p=0.0152). Conclusion: Since both, overall survival and progression-free survival are negatively correlated with detectable MT expression in MPM, our results indicate a possible resistance to platin-based chemotherapy associated with MT expression upregulation, found exclusively in progressive MPM samples. Initial cell culture studies suggest promoter DNA hypomethylation and expression of miRNA-566 a direct regulator of copper transporter SLC31A1 and a putative regulator of MT1A and MT2A gene expression, to be responsible for the drug resistance.

Platin-containing regimes are currently considered as state-of-the-art therapies in malignant pleural mesotheliomas (MPM) but show dissatisfying response rates ranging from 6 to 16% only. Still, the reasons for the rather poor efficacy remain largely unknown. A clear stratification of patients based on new biomarkers seems to be a promising approach to enhance clinical management, which would be a long-needed improvement for MPM patients but does not seem likely soon unless new biomarkers can be validated. Twenty-four formalin-fixed, paraffin-embedded (FFPE) tumour specimens were subjected to a miRNA expression screening of 800 important miRNAs using digital quantification via the nCounter technique (NanoString). We defined a small subset of miRNAs regulating the key enzymes involved in the repair of platin-associated DNA damage. Particularly, the TP53 pathway network for DNA damage recognition as well as genes related to the term “BRCAness” are the main miRNA targets within this context. The TP53 pathway network for DNA damage recognition as well as genes related to the term “BRCAness” are the main players for risk stratification in patients suffering from this severe disease. Taking the specific molecular profile of the tumour into account can help to enhance the clinical management prospectively and to smooth the way to better response prediction.

The aim of this study was to analyze the diagnostic role of PTEN and ARID1A in effusion cytology. Effusions (n = 279), consisting of 226 carcinomas (70 ovarian, 64 breast, 36 lung, and 15 uterine corpus carcinomas; 41 carcinomas of other origin) and 53 malignant mesotheliomas, were analyzed for PTEN and ARID1A expression using immunohistochemistry. PTEN was preserved in 166 (59%) tumors, partially lost in 38 (14%), and absent in 75 (27%), with lower expression in malignant mesotheliomas compared to carcinomas, though not significantly (p = 0.084). ARID1A was preserved in 243 (88%) tumors, partially lost in 18 (6%), and absent in 18 (6%). The majority of tumors with absent ARID1A were ovarian carcinomas, predominantly of clear cell or low-grade serous type. Reactive mesothelial cells in carcinoma specimens were uniformly positive for both proteins. ARID1A mutation analysis showed no mutations in eight analyzed specimens negative by immunohistochemistry. Loss of PTEN and ARID1A expression is highly specific for malignancy in effusion pathology. Loss of PTEN is not informative of organ of origin, whereas absence of ARID1A should raise suspicion of an ovarian primary.

Background Hypothermic machine perfusion (HMP) for preservation led to compelling success for outcomes of renal transplantation. Application of different concentrations of oxygen during renal HMP has not been systematically analyzed. This study investigates the aspects of renal function and morphology in dependence of oxygen concentrations during HMP in a porcine donation after circulatory death model. Methods After 30 min of warm ischemia, porcine kidneys were randomly assigned to preservation for 21 hr by HMP without oxygenation (HMPnoox), oxygenated HMP with air (HMPair), or 100% oxygen (HMPox100%). Afterward, kidneys were reperfused for 2 hr in an ex vivo model for assessment of function and integrity. Results Application of HMPox100% led to significantly increased blood flow during reperfusion compared to HMPnoox. Preservation by HMPox100% led to a doubling of creatinine clearance after 90 and 120 min of reperfusion (13.4 and 12.0 mL/min) compared to preservation by HMPnoox (7.3 and 7.7 mL/min; P=0.01). Oxygenated HMP with air led to results between the two other groups. Fractional excretion of sodium demonstrated a strong tendency of higher values after HMPnoox compared to HMPox100% (P=0.096) and HMPair (P=0.09). Analysis of structural integrity during reperfusion demonstrated significantly higher values of lactate dehydrogenase resembling cell damage (P=0.02), higher values of gamma-glutamyl-transferase (gGT) resembling tubulus injury (P=0.048), and more pronounced tubular dilatation (P=0.02) after HMPnoox compared to HMPox100%. Conclusion The present study demonstrates that application of HMPox100% for kidneys from donations after circulatory death results in better renal function during early reperfusion compared to HMPnoox.

Summary Background In sarcoidosis, an antigen specific immune response is a putative mechanism, resulting in granulomatous inflammation. Since the proteasome is involved in antigen presentation, alterations in the alveolar and parenchymal proteasomal system may be a feature of sarcoidosis. Objectives To explore the role of proteasomes and immunoproteasomes in sarcoidosis. Methods Total proteasome concentration and activity was assessed in bronchoalveolar lavage (BAL) supernatant obtained from sarcoidosis patients ( n = 67) and healthy controls ( n = 18) using ELISA and cleavage of specific fluorogenic substrates (±epoxomicin), respectively. Immunohistochemistry of lung tissue sections and immunocytochemistry of BAL macrophages for immunoproteasome was performed in sarcoidosis patients and controls. Results Proteasome was present in BAL supernatants of all sarcoidosis patients. In sarcoidosis, abundant immunoproteasome staining was seen in pneumocytes type II and granulomas. Total proteasome concentration was greater in active sarcoidosis, stages II (101 ng/ml ± 79; p = 0.009) and III (119 ng/ml ± 66; p = 0.012), than in inactive sarcoidosis or in healthy controls (35 ng/ml ± 34). In the absence of epoxomicin, all fluorogenic substrates were hydrolyzed by BAL supernatant of sarcoidosis patients and controls. Conclusions Patients with active sarcoidosis but not healthy controls demonstrate immunoproteasome in the lung tissue and in granulomas. Thus, the putative immune response in sarcoidosis may be mediated or sustained by the proteasomal system.

Background: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. Material and methods: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. Results: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). Conclusion: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation.

Background: Lung cancer still remains the leading cause of cancer for men after prostate cancer and breast cancer for women. Angiogenesis is considered a major microenvironment modifier. Material and Methods: Demographic data and study design; The study is based on a collective of twenty representative specimens of each tumour entity (Typical Carcinoid, Atypical Carcinoid, Large-Cell Neuroendocrine Carcinoma , Small Cell Lung Cancer) for mRNA expression analysis. The following methods were performed: RNA Extraction and RNA Integrity Assessment, NanoString CodeSet Design and Expression Quantification, NanoString Data Processing and Statistical Analysis. Results: KDR rendered significant association to aggressiveness of the tumour and decreases with increasing malignancy (p=0.049). A decreased expression of HIF1A and KDR mRNA as associated with a higher risk of tumour invasion in vessels (HIF1A: p=0.034; KDR: p=0.029). FIGF and HIF1A expression levels are significantly associated with progression-free survival (FIGF: p= 0.021; HIF1A: p= 0.049). CRHR2 and FLT4 are stronger expressed in female than in male patients (CRHR2: p=0.024, FLT4: p=0.004). FIGF expression is still significant between LCNEC and SCLC (p=0.023). FLT4 and KDR show highly significant association to one of the analysed groups (FLT4: p=0.001; KDR: p=0.006). Additionally, HIF1A expression differs significantly between these focus cohorts (p=0.018). Conclusion: We should consider for clinical practice application which factors affect most the tumour growth and distal metastasis, thereafter investigate easy to administer drugs with low side effects. Probably a cluster system of therapy should be established where a drug targets simultaneously different pathways of the same origin.

BACKGROUND Endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA) is a well-established method to assess mediastinal lymph nodes for lung cancer. However, a proportion of patients require further investigation, due to the low negative predictive value (NPV). The objective of this study was to determine whether the assessment of short stature homeobox 2 (SHOX2) DNA methylation level in lymph node tissue obtained by EBUS-TBNA improves the accuracy of mediastinal staging. PATIENTS AND METHODS EBUS-TBNA was carried out for suspicious lymph nodes of 154 patients. Negative or ambiguous histological results were confirmed by surgical means and clinical follow-up over 6 months. EBUS-TBNA was assessed on 80 positive and 85 negative classified lymph nodes and compared with the result of the SHOX2 DNA methylation real-time PCR analysis. Relative methylation measured by delta-delta cycle threshold (ΔΔCt) was used to classify the samples. Clinical performance of the EBUS-TBNA procedure with and without the additional SHOX2 assessment was calculated against the final classification according to the gold standard. RESULTS Based on data from 105 patients, an average 80-fold increase in the SHOX2 methylation level was measured for positive compared with negative lymph nodes. SHOX2 results with a ΔΔCt value of

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl2 and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte-microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes. © 2013 The Authors.

The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 20°C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

The chromosomal passenger complex (CPC) acts as a key regulator of mitosis, preventing asymmetric segregation of chromosomal material into daughter cells. The CPC is composed of three non-enzymatic components termed Survivin, the inner centromere protein (INCENP) and Borealin, and an enzymatic component, Aurora B kinase. Survivin is necessary for the appropriate separation of sister chromatids during mitosis and is involved in liver regeneration, but its role in regenerative processes is incompletely elucidated. Whether Survivin, which is classified as an inhibitor of apoptosis protein (IAP) based on domain composition, also has a role in apoptosis is controversial. The present study examined the in vivo effects of Survivin ablation in the liver and during liver regeneration after 70% hepatectomy in a hepatocyte-specific knockout mouse model. The absence of Survivin caused a reduction in the number of hepatocytes in the liver, together with an increase in cell volume, macronucleation and polyploidy, but no changes in apoptosis. During liver regeneration, mitosis of hepatocytes was associated with mislocalization of the members of the CPC, which were no longer detectable at the centromere despite an unchanged protein amount. Furthermore, the loss of survivin in regenerating hepatocytes was associated with reduced levels of phosphorylated Histone H3 at serine 28 and abolished phosphorylation of CENP-A and Hec1 at serine 55, which is a consequence of decreased Aurora B kinase activity. These data indicate that Survivin expression determines hepatocyte number during liver development and liver regeneration. Lack of Survivin causes mislocalization of the CPC members in combination with reduced Aurora B activity, leading to impaired phosphorylation of its centromeric target proteins and inappropriate cytokinesis.

Introduction: Folate receptor alpha (FRA) regulates cellular uptake of folates and antifolates. Information about FRA protein expression in metastatic non–small-cell lung cancer (NSCLC) is limited. We investigated FRA as a biomarker for pemetrexed-based chemotherapy and compared it with thymidylate synthase (TS), the main target of pemetrexed. Methods: Pretreatment tumor specimens from 207 patients with advanced NSCLC were assessed for FRA and TS protein expression by immunohistochemistry using the H-score (range, 0–300) and correlated to patients’ clinicopathological data, radiographic response, progression-free survival (PFS), and overall survival (OS). Results: Low total (cytoplasmic and nuclear) TS protein expression (H-score < 210) was associated with improved PFS (median: 5.6 versus 3.5 months; hazard ratio [HR] = 0.6379, p = 0.0131) and prolonged OS (median: 22.5 versus 11.5 months; HR = 0.5680, p = 0.0107). An association between lower TS levels and response to pemetrexed-based therapy was found—mean H-score 187 ± 5, median 180 for responders versus mean H-score 201 ± 4, median 210, for non-responders, p = 0.0244. High intracellular FRA expression (H-score ≥110) was associated with prolonged OS (28.9 versus 11.7 months, HR = 0.5316, p = 0.0040) and a trend for association with PFS (5.6 versus 4.1 months, HR = 0.7395, p = 0.0801) was noted. Membranous FRA expression was seen in 83% of patients, moreover, high membranous expression (H-score ≥20) was associated with improved PFS (5.6 versus 3.7 months, HR = 0.6445, p = 0.0306) and OS (22.1 versus 11.5 months, HR = 0.5378, p = 0.0131). Conclusions: A large number of NSCLC patients have high expression of FRA and/or a low level of TS expression. Expression levels of FRA and TS were associated with clinical benefit from pemetrexed therapy.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. 25% show neuroendocrine differentiation (typical/atypical carcinoids, large-/small-cell neuroendocrine carcinomas). Carcinoids present with long survival rates, but metastatic carcinoids correlate with decreased survival and are commonly insensitive to standard chemotherapy or radiation. Therefore, novel therapeutic strategies are urgently needed. Material and methods: 70 representative tumor specimens were used for next-generation sequencing analysis of 14 genes related to therapy response. Additionally, mRNA-expression profiles of 60 matching samples were determined for 13 selected drug targets by using the NanoString nCounter technology. Results: A number of features known to sensitize tumors for different targeted therapies could be identified, which hopefully improve the clinical management of this subgroup of lung neoplasias. In particular, EGFR expression was observed in the investigated tumors in a noteworthy manner. Additionally, MDM2 was strongly expressed in the majority of all samples whereas the expression of its physiological inhibitor, CDKN2A, was nearly absent in all low-grade tumors. TP53 showed a high frequency of variants in high-grade tumors but mutations were rare in carcinoids. Conclusion: Based on our results, therapeutic approaches with MDM2-inhibitors and monoclonal anti-EGFR antibodies may be promising in pulmonary carcinoid tumors. CA extern

OA embargo BACKGROUND:: Pemetrexed-based chemotherapy represents the standard of care in first-line treatment of advanced malignant pleural mesothelioma (MPM). However, there are no established predictors of clinical benefit. Pemetrexed inhibits multiple enzymes involved in pyrimidine and purine synthesis, but the main target is thymidylate synthase (TS). After cellular uptake pemetrexed is converted into more effective polyglutamated forms by folylpoly-γ- glutamate synthetase (FPGS). We hypothesized that FPGS and TS protein expressions are associated with clinical outcome after pemetrexed-based chemotherapy. METHODS:: Pretreatment tumor samples from 84 patients with histologically confirmed MPM, who received pemetrexed combined with platinum (79 of 84) or single-agent pemetrexed (5 of 84) as first-line treatment, were retrospectively analyzed. FPGS and TS protein expressions were semiquantitatively assessed by using the Hybrid (H)-scoring system (range, 0-300). H-scores were correlated with radiological response according to modified Response Evaluation Criteria in Solid Tumors, progression-free survival (PFS) and overall survival (OS). RESULTS:: Median H-score of the entire cohort was 230 for FPGS (range, 100-300), and 210 for TS (range, 100-300). High FPGS protein expression was significantly associated with longer PFS (pCOX = 0.0337), better objective tumor response (partial response versus stable disease + progressive disease; pKW = 0.003), and improved disease-control rate (partial response + stable disease versus progressive disease; pKW = 0.0208), but not with OS. In addition, high TS protein expression was associated with progressive disease under pemetrexed-based therapy (p = 0.0383), and shorter OS (pCOX = 0.0071), but no association with PFS was observed. CONCLUSION:: FPGS and TS expressions were associated with clinical response and outcome to pemetrexed-based first-line chemotherapy in MPM. Prospective evaluation of FPGS and TS expressions and their prognostic/predictive power in MPM patients is warranted. © 2012 by the International Association for the Study of Lung.

Background:Tubulin-binding agents (TBAs) are effective in non-small cell lung cancer (NSCLC) treatment. Both ΒIII-and ΒV-tubulins are expressed by cancer cells and may lead to resistance against TBAs.Methods:Pre-treatment samples from 65 locally advanced or oligometastatic NSCLC patients, who underwent uniform induction chemotherapy with paclitaxel and platinum followed by radiochemotherapy with vinorelbine and platinum were retrospectively analysed by immunohistochemistry. Protein expression of ΒIII-and ΒV-tubulin was morphometrically quantified.Results:Median pre-treatment H-score for ΒIII-tubulin was 110 (range: 0-290), and 160 for ΒV-tubulin (range: 0-290). Low ΒIII-tubulin expression was associated with improved overall survival (OS) (P=0.0127, hazard ratio (HR): 0.328). An association between high ΒV-tubulin expression and prolonged progression-free survival (PFS, median 19.2 vs 9.4 months in high vs low expressors; P=0.0315, HR: 1.899) was found. Further, high ΒV-tubulin expression was associated with objective response (median H-score 172.5 for CR+PR vs 120 for SD+PD patients, P=0.0104) or disease control following induction chemotherapy (170 for CRPRSD vs 100 for PD patients, P=0.0081), but not radiochemotherapy.Conclusion:Expression of ΒV-tubulin was associated with treatment response and PFS following paclitaxel-based chemotherapy of locally advanced and oligometastatic NSCLC patients. Prolonged OS was associated with low levels of ΒIII-tubulin. Prospective evaluation of ΒIII/ΒV-tubulin expression in NSCLC is warranted. © 2012 Cancer Research UK.