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MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines.

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology.

Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported in classical Hodgkin Lymphoma (cHL) that a member of the THE1B class of LTR elements acted as a promoter for the proto-oncogene and growth factor receptor gene CSF1R and that expression of this gene is required for cHL tumour survival. However, to which extent and how such elements participate in globally shaping the unique cHL gene expression programme is unknown. To address this question we mapped the genome-wide activation of THE1-LTRs in cHL cells using a targeted next generation sequencing approach (RACE-Seq). Integration of these data with global gene expression data from cHL and control B cell lines showed a unique pattern of LTR activation impacting on gene expression, including genes associated with the cHL phenotype. We also show that global LTR activation is induced by strong inflammatory stimuli. Together these results demonstrate that LTR activation provides an additional layer of gene deregulation in classical Hodgkin lymphoma and highlight the potential impact of genome-wide LTR activation in other inflammatory diseases.

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (cHL) are lymphomas that contain CD30-expressing tumor cells and have numerous pathological similarities. Whereas ALCL is usually diagnosed at an advanced stage, cHL more frequently presents with localized disease. The aim of the present study was to elucidate the mechanisms underlying the different clinical presentation of ALCL and cHL. Chemokine and chemokine receptor expression were similar in primary ALCL and cHL cases apart from the known overexpression of the chemokines CCL17 and CCL22 in the Hodgkin and Reed-Sternberg (HRS) cells of cHL. Consistent with the overexpression of these chemokines, primary cHL cases encountered a significantly denser T cell microenvironment than ALCL. Additionally to differences in the interaction with their microenvironment, cHL cell lines presented a lower and less efficient intrinsic cell motility than ALCL cell lines, as assessed by time-lapse microscopy in a collagen gel and transwell migration assays. We thus propose that the combination of impaired basal cell motility and differences in the interaction with the microenvironment hamper the dissemination of HRS cells in cHL when compared with the tumor cells of ALCL. CA extern

Fragestellung: Das Patient Reported Outcome (PRO) spielt eine zunehmende Rolle in der Einschätzung der Effizienz von Behandlungsmethoden. Dabei ist es das Ziel, mit einem möglichst einfachen Tool die Funktion und Beschwerden aus der Sicht des Patienten zu erfassen. Im Bereich der Hüfte[zum vollständigen Text gelangen Sie über die oben angegebene URL], Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018)

Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.

Background: Definite diagnosis and therapeutic management of cholangiocarcinoma (CCA) remains a challenge. The aim of the current study was to investigate feasibility and potential impact on clinical management of targeted sequencing of intraductal biopsies. Methods: Intraductal biopsies with suspicious findings from 16 patients with CCA in later clinical course were analyzed with targeted sequencing including tumor and control benign tissue (n = 55 samples). A CCA-specific sequencing panel containing 41 genes was designed and a dual strand targeted enrichment was applied. Results: Sequencing was successfully performed for all samples. In total, 79 mutations were identified and a mean of 1.7 mutations per tumor sample (range 0–4) as well as 2.3 per biopsy (0–6) were detected and potentially therapeutically relevant genes were identified in 6/16 cases. In 14/18 (78%) biopsies with dysplasia or inconclusive findings at least one mutation was detected. The majority of mutations were found in both surgical specimen and biopsy (68%), while 28% were only present in biopsies in contrast to 4% being only present in the surgical tumor specimen. Conclusion: Targeted sequencing from intraductal biopsies is feasible and potentially improves the diagnostic yield. A profound genetic heterogeneity in biliary dysplasia needs to be considered in clinical management and warrants further investigation. Translational impact: The current study is the first to demonstrate the feasibility of sequencing of intraductal biopsies which holds the potential to impact diagnostic and therapeutical management of patients with biliary dysplasia and neoplasia.

We describe the case of a patient who underwent patent foramen ovale (PFO) closure for secondary stroke prevention. On routine follow-up transesophageal echocardiography (TEE), a papillary fibroelastoma was identified and removed successfully surgically. The fibroelastoma had not been recognized during preprocedural imaging and escaped our attention during limited intraprocedural transesophageal echocardiographic imaging (focusing on the interatrial septum) given the small size and visualization requiring thorough imaging in multiple planes and modified views. Our case illustrates the importance of detailed echocardiographic imaging prior to PFO device implantation; otherwise, subtle concomitant and at least equally probable causes of stroke can be overlooked.

Here we present our current work on the analysis of a database of HL whole slide images. We performed pre-processing and identified regions of interest (ROI) that contain CD30-positive cells in the images as described before. A CellProfiler pipeline was used to detect and measure cells in the ROI. The cells were then classified using shape descriptors and stored in a database with their 2D coordinates. This allowed for a statistical analysis of the spatial distribution of HRS cells within the tissue.

Hodgkin lymphoma is an unusual type of lymphoma, arising from malignant B-cells. Morphological and immunohistochemical featuresof malignant cells and their distribution differ from other cancer types. Based on systematic tissue image analysis, computer-aided explorationcan provide new insights into Hodgkin lymphoma pathology. Here, we report results from an image analysis of CD30 immunostained classical Hodgkin lymphoma (cHL) tissue section images. We have imple-mented an automatic procedure to handle and explore image data in Aperio's SVS format. We use pre-processing approaches to separate the imageobjects from the background, then select regions of interest and split the large images into tiles. Then, we use a CellProfiler pipeline to detect primary objects. Therefore, the images are split into their color stains using a color deconvolution approach. By setting a threshold in the CD30 stain image we identify CD30 positive cells and compute their shape descriptors. We label the cells based on size, elongation and compactness. We present results for a small set of nodular sclerosis, mixed type and non-lymphoma images.

Hodgkin lymphoma is an unusual type of lymphoma, arising from malignant B-cells. Morphological and immunohistochemical features of malignant cells and their distribution differ from other cancer types. Based on systematic tissue image analysis, computer-aided exploration can provide new insights into Hodgkin lymphoma pathology. In this paper, we report results from an image analysis of CD30 immunostained Hodgkin lymphoma tissue section images. To the best of our knowledge, this is the first systematic application of image analysis to a set of tissue sections of Hodgkin lymphoma. We have implemented an automatic procedure to handle and explore image data in Aperio's SVS format. We use pre-processing approaches on a down-scaled image to separate the image objects from the background. Then, we apply a supervised classification method to assign pixels to predefined classes. Our pre-processing method is able to separate the tissue content of images from the image background. We analyzed three immunohistologically defined groups, non-lymphoma and the two most common forms of Hodgkin lymphoma, nodular sclerosis and mixed cellularity type. We found that nodular sclerosis and non-lymphoma images exhibit different amounts of CD30 stain, whereas mixed cellularity type exhibits a large variance and overlaps with the other groups. The results can be seen as a first step to computationally identify tumor regions in the images. This allows us to focus on these regions when performing computationally expensive tasks like object detection in the high-resolution image., 12 papes, 5 figures, Dagstuhl seminar, Dagstuhl Seminar 12291 "Structure Discovery in Biology: Motifs, Networks & Phylogenies"

Einleitung: Ein 103-jähriger Patient stellte sich in unserer Ambulanz mit einer seit vier Wochen zunehmenden Heiserkeit vor. Ebenso berichtete er über Dysphagie und Globusgefühl. Der HNO-ärztliche Spiegelbefund zeigte eine glatt berandete, prall-elastische, 5,5 x 4,5 x 3,5 [for full text, please go to the a.m. URL], 83. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor {kappa}B (NF-{kappa}B). NF-{kappa}B activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus–positive (EBV+) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV– cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-{kappa}B target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-{kappa}B activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-{kappa}B activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV– than EBV+ cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.

Follicular T cell lymphoma is derived from follicular T-helper cells. In many cases, neoplastic T cells form rosettes around Hodgkin-Reed-Sternberg-like cells, which can lead to the misdiagnosis of classical Hodgkin lymphoma. The aim of the present study was to obtain a better understanding of this rosetting phenomenon and to recognize features that are helpful in the differential diagnosis of classical Hodgkin lymphoma. Sixteen mostly elderly follicular T cell lymphoma patients (mean 66 years) were analyzed. Fifteen of the 16 follicular T cell lymphoma cases presented with Hodgkin-Reed-Sternberg-like cells, which were CD20-positive in 27% of the cases and Epstein-Barr virus-infected in nearly all cases. Frequently, the immunophenotype of rosetting neoplastic T cells differed from the bulk neoplastic cells with less numerous T-follicular helper cell markers expressed, suggesting a modulation of T-follicular helper cell marker expression in the neoplastic T cells. In 75% of the cases, variable CD30 expression was encountered in the neoplastic T cells, likely reflecting an activation state in these cells. Hodgkin-Reed-Sternberg-like cells were positive for CCL17, and follicular T cell lymphoma tumor cells expressed its receptor CCR4 at variable intensity, thus potentially explaining the phenomenon of the tumor cells' rosetting around Hodgkin-Reed-Sternberg-like cells. In summary, this study confirms the presence of Hodgkin-Reed-Sternberg-like cells in a high number of cases of follicular T cell lymphoma, suggesting that Hodgkin-Reed-Sternberg-like cells may contribute to the development of this lymphoma. Hodgkin-Reed-Sternberg-like cells in follicular T cell lymphoma cannot reliably be differentiated from the Hodgkin-Reed-Sternberg cells of classical Hodgkin lymphoma based on their immunophenotype. In contrast, demonstration of a T-follicular helper cell phenotype with CD10 and frequent CD30 expression in the neoplastic T cell population can help to establish the diagnosis of follicular T cell lymphoma, and may even indicate CD30 as a therapeutic target for these patients.

Extranodal marginal zone B-cell lymphoma (MZBL) of the mucosa-associated lymphoid tissue is an indolent lymphoma mostly affecting the gastrointestinal tract. The lymphoma initially has small cell morphology (SC-MZBL) and often arises in the background of Helicobacter pylori induced gastritis. In some cases, a clonal malignant progression to large cell morphology (LC-MZBL) is observed. The contribution of epigenetic alterations, including DNA methylation in gastric MZBL has been studied for single genes but not on a genome-wide level. To gain insight into the DNA methylation changes associated with progression of gastric MZBL of MALT, genome-wide DNA methylation profiling was performed for 30 gastric MZBL samples. Genome-wide DNA methylation profiling identified 7698 significantly differentially methylated loci during gastric MZBL progression (��/��max���0.4, q���0.001). LC-MZBL showed a hypermethylation in comparison to SC-MZBL with an enrichment of regions involved in transcriptional regulation including the CpG loci localized in the CpG islands and promoter regions. After filtering the significantly differentially methylated loci between SC- and LC-MZBL for being located in promoter regions (n = 3824 loci), 1630 genes were identified. Gene ontology analysis of these 1630 genes, identified an enrichment of genes of the Wnt signaling pathway. Since promoter DNA methylation is known to cause transcriptional silencing, the extent to which DNA hypermethylation in the LC-MZBL affects the gene expression was examined. On integrated analysis of mRNA expression and DNA methylation, a decreased expression was observed for 3.10% of genes hypermethylated in LC-MZBL in comparison to SC-MZBL, while 96.56% showed no difference in expression. Based on DNA methylation profile analysis, the LC-MZBL could be distinguished from the DLBCL. A supervised analysis identified loci to be significantly differentially methylated between LC-MZBL and DLBCL. The incidence and profile of mutations in MYD88, CARD11, CD79B and BTK genes for the LC-MZBL was distinct from that of DLBCL. Based on these observations, it can be stated that our data support the view that LC-MZBL is distinct from that of DLBCL. In conclusion, this is the first study to analyze DNA methylation profiling of gastric MZBL of MALT type on a genome-wide scale. It was shown that gastric MZBL progression is associated with specific DNA methylation alterations and the differentially methylated loci between SC-and LC-MZBL are enriched for regions involved in transcriptional regulation, whereby significant changes in the RNA expression levels was not observed in the majority of differentially methylated genes. Nevertheless, it was shown that the morphological differences between SC-MZBL and LC-MZBL are reflected by characteristic DNA methylation profiles. Further it was shown, that LC-MZBL is distinct from the DLBCL on the basis of their DNA methylation profiles, indicating that LC-MZBL should be regarded as an entity distinct from the DLBCL.

Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumors with variable clinical, morphologic, and phenotypic characteristics. Transcriptome analysis was performed on multiple FDC sarcomas and compared with other mesenchymal tumors, microdissected Castleman FDCs, and normal fibroblasts. Using unsupervised analysis, FDC sarcomas clustered with microdissected FDCs, distinct from other mesenchymal tumors and fibroblasts. The specific endowment of FDC-related gene expression programs in FDC sarcomas emerged by applying a gene signature of differentially expressed genes (n = 1,289) between microdissected FDCs and fibroblasts. Supervised analysis comparing FDC sarcomas with microdissected FDCs and other mesenchymal tumors identified 370 and 2,927 differentially expressed transcripts, respectively, and on the basis of pathway enrichment analysis ascribed to signal transduction, chromatin organization, and extracellular matrix organization programs. As the transcriptome of FDC sarcomas retained similarity with FDCs, the immune landscape of FDC sarcoma was investigated by applying the CIBERSORT algorithm to FDC sarcomas and non-FDC mesenchymal tumors and demonstrated that FDC sarcomas were enriched in T follicular helper (TFH) and T regulatory (TREG) cell populations, as confirmed in situ by immunohistochemistry. The enrichment in specific T-cell subsets prompted investigating the mRNA expression of the inhibitory immune receptor PD-1 and its ligands PD-L1 and PD-L2, which were found to be significantly upregulated in FDC sarcomas as compared with other mesenchymal tumors, a finding also confirmed in situ. Here, it is demonstrated for the first time the transcriptional relationship of FDC sarcomas with nonmalignant FDCs and their distinction from other mesenchymal tumors. Implications: The current study provides evidence of a peculiar immune microenvironment associated with FDC sarcomas that may have clinical utility. Mol Cancer Res; 15(5); 541–52. ©2017 AACR.

// Luisa Lorenzi 1 , Claudia Doring 2 , Tobias Rausch 3 , Vladimir Benes 3 , Silvia Lonardi 1 , Mattia Bugatti 1 , Elias Campo 4 , Jose Cabecadas 5 , Ingrid Simonitsch-Klupp 6 , Anita Borges 7 , Jay Mehta 7 , Claudio Agostinelli 8 , Stefano Aldo Pileri 8, 9 , Fabio Facchetti 1 , Martin-Leo Hansmann 2 , Sylvia Hartmann 2 1 Pathology Unit, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy 2 Dr. Senckenberg Institute of Pathology, Goethe University, Frankfurt am Main, Germany 3 Genecore, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 4 Haematopathology Section, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain 5 Department of Pathology, Portuguese Institute of Oncology, Lisbon, Portugal 6 Institute of Pathology, Medical University of Vienna, Vienna, Austria 7 Histopathology, SRL Diagnostics, Mumbai, India 8 Department of Experimental, Diagnostic and Specialty Medicine, Haematopathology Section, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 9 Unit of Diagnostic Haematopathology, European Institute of Oncology, Milan, Italy Correspondence to: Luisa Lorenzi, email: lorenziluisa@gmail.com Keywords: follicular dendritic cell sarcoma, whole transcriptome sequencing, immunohistochemistry, follicular dendritic cell-secreted protein, serglycin Received: September 16, 2016 Accepted: January 17, 2017 Published: January 27, 2017 ABSTRACT Follicular dendritic cell (FDC)-sarcoma is a rare neoplasm with morphologic and phenotypic features of FDCs. It shows an extremely heterogeneous morphology, therefore, its diagnosis relys on the phenotype of tumor cells. Aim of the present study was the identification of new specific markers for FDC-sarcoma by whole transcriptome sequencing (WTS). Candidate markers were selected based on gene expression level and biological function. Immunohistochemistry was performed on reactive tonsils, on 22 cases of FDC-sarcomas and 214 control cases including 114 carcinomas, 87 soft tissue tumors, 5 melanomas, 5 thymomas and 3 interdigitating dendritic cell sarcomas. FDC secreted protein (FDCSP) and Serglycin (SRGN) proved to be specific markers of FDC and related tumor. They showed better specificity and sensitivity values than some well known markers used in FDC sarcoma diagnosis (specificity: 98.6%, and 100%, respectively; sensitivity: 72.73% and 68.18%, respectively). In our cohorts CXCL13, CD21, CD35, FDCSP and SRGN were the best markers for FDC-sarcoma diagnosis and could discriminate 21/22 FDC sarcomas from other mesenchymal tumors by linear discriminant analysis. In summary, by WTS we identified two novel FDC markers and by the analysis of a wide cohort of cases and controls we propose an efficient marker panel for the diagnosis of this rare and enigmatic tumor.

Follicular dendritic cell (FDC) sarcomas are rare mesenchymal tumours, which are fatal in 20% of the patients and usually occur in secondary lymphoid organs or extranodal localizations. Due to the rareness of these tumours, only few studies have been conducted on molecular level. In the present study, we performed microRNA (miRNA) profiling of 31 FDC sarcomas and identified two subgroups, one with high miRNA expression and the other group with low miRNA expression levels. The first group showed a strong similarity to fibroblasts and myopericytomas, whereas the second group was more closely related to FDCs from Castleman's disease. Both groups showed important differences compared with myeloid-derived dendritic cells, confirming mesenchymal origin of FDCs and their derived sarcomas. The two FDC sarcoma groups did not differ on morphological grounds, mitotic activity or BRAF mutation status. However, patients of group I presented a tendency to a shorter overall survival and more frequent podoplanin expression by immunohistochemistry. The importance of these newly recognized FDC sarcoma subgroups in terms of clinical behaviour and therapeutic implications should be assessed in a larger cohort in future studies.