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Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.

Abstract Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC.

Cytochrome P450 enzymes (CYP450s) exert mighty catalytic actions in cellular metabolism and detoxication, which play pivotal roles in cell fate determination. Preliminary data shows differential expression levels of CYP27C1, one of the “orphan P450s” in human lung cancer cell lines. Here, we study the functions of CYP27C1 in lung cancer progression and drug endurance, and explore its potential to be a diagnostic and therapeutic target for lung cancer management. Quantitative real-time PCR and immunoblot assays were conducted to estimate the transcription and protein expression level of CYP27C1 in human lung cancer cell lines, which was relatively higher in A549 and H1975 cells, but was lower in H460 cells. Stable CYP27C1-knockdown A549 and H1975 cell lines were established, in which these cells showed enhancement in cell proliferation, colony formation, and migration. In addition, aberrant IGF-1R/Akt/p53 signal transduction was also detected in stable CYP27C1-knockdown human lung cancer cells, which exhibited greater tolerance towards the treatments of anticancer agents (including vinorelbine, picropodophyllin, pacritinib, and SKLB610). This work, for the first time, reveals that CYP27C1 impacts lung cancer cell development by participating in the regulation of the IGF-1R/Akt/p53 signaling pathway, and the level of CYP27C1 plays indispensable roles in dictating the cellular sensitivity towards multiple anticancer agents.

Simple SummaryMicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p.MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AG02-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19(+) B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AG02-IP enrichment between BL and normal B cells. A comparison of AG02-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.

Cigarette smoking causes lung inflammation and tissue damage. Lung fibroblasts play a major role in tissue repair. Previous studies have reported smoking-associated changes in fibroblast responses and methylation patterns. Our aim was to identify the effect of current smoking on miRNA expression in primary lung fibroblasts. Small RNA sequencing was performed on lung fibroblasts from nine current and six ex-smokers with normal lung function. MiR-335-5p and miR-335-3p were significantly downregulated in lung fibroblasts from current compared to ex-smokers (false discovery rate (FDR) &lt, 0.05). Differential miR-335-5p expression was validated with RT-qPCR (p-value = 0.01). The results were validated in lung tissue from current and ex-smokers and in bronchial biopsies from non-diseased smokers and never-smokers (p-value &lt, 0.05). The methylation pattern of the miR-335 host gene, determined by methylation-specific qPCR, did not differ between current and ex-smokers. To obtain insights into the genes regulated by miR-335-5p in fibroblasts, we overlapped all proven miR-335-5p targets with our previously published miRNA targetome data in lung fibroblasts. This revealed Rb1, CARF, and SGK3 as likely targets of miR-335-5p in lung fibroblasts. Our study indicates that miR-335-5p downregulation due to current smoking may affect its function in lung fibroblasts by targeting Rb1, CARF and SGK3.

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression.Methods: RNA isolated from FACS-sorted memory ((M)) Tregs (CD4(+)CD45RO(+)CD25(+)CD127(-)) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in (M)Tregs from patients and HCs. cAMP levels were measured using flow cytometry.Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in (M)Tregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of (M)Tregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, (M)Tregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed (M)Tregs by forskolin restored their suppressive function in vitro.Conclusion: Overexpression of miR-142-3p in (M)Tregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity. Electronic supplementary material The online version of this article (10.1007/s00428-019-02555-3) contains supplementary material, which is available to authorized users.

Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied LncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between I-IL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large 6-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the pC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell specific expression pattern in HL tissues and might therefore be of value as a biomarker.

SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no beta-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.

The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient‐sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC‐driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR‐15a. TSC1 knockdown results in elevated mTORC1‐dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC‐driven cancers.

The Epstein–Barr virus (EBV) can cause a wide variety of cancers upon infection of different cell types and induces a highly variable composition of the tumor microenvironment (TME). This TME consists of both innate and adaptive immune cells and is not merely an aspecific reaction to the tumor cells. In fact, latent EBV-infected tumor cells utilize several specific mechanisms to form and shape the TME to their own benefit. These mechanisms have been studied largely in the context of EBV+ Hodgkin lymphoma, undifferentiated nasopharyngeal carcinoma, and EBV+ gastric cancer. This review describes the composition, immune escape mechanisms, and tumor cell promoting properties of the TME in these three malignancies. Mechanisms of susceptibility which regularly involve genes related to immune system function are also discussed, as only a small proportion of EBV-infected individuals develops an EBV-associated malignancy.

Supplemental Digital Content is available in the text., BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In this study, the performance of three rapid BRAF mutation tests was compared. Thirty-nine formalin-fixed paraffin-embedded melanoma tissue samples collected between 2007 and 2014 at a single center were included. These samples were analyzed by immunohistochemistry using the anti-BRAF-V600E (VE1) mouse monocolonal antibody (BRAF-VE1 IHC), a V600E-specific Droplet Digital PCR Test, and the Idylla BRAF- Mutation Test (Idylla). Results were compared with the results of conventional BRAF mutation testing, performed using high-resolution melting analysis followed by Sanger sequencing. Next-generation sequencing was performed on samples with discordant results. The Idylla test and Droplet Digital PCR Test correctly identified all mutated and wild-type samples. BRAF-VE1 IHC showed one discordant result. The Idylla test could identify BRAF-V600 mutations other than BRAF-V600E and was the fastest and least laborious test. The Idylla Mutation Test is the most suitable test for rapid BRAF testing in clinical situations on the basis of the broad coverage of treatment-responsive mutations and the fast procedure without the need to perform a DNA isolation step.

Myc is a well-known transcription factor with important roles in cell cycle, apoptosis, and cellular transformation. Long noncoding RNAs (lncRNAs) have recently emerged as an important class of regulatory RNAs. Here, we show that lncRNAs are a main component of the Myc-regulated transcriptional program using the P493-6 tetracycline-repressible myc model. We demonstrate that both Myc-induced mRNAs and lncRNAs are significantly enriched for Myc binding sites. In contrast to Myc-repressed mRNAs, Myc-repressed lncRNAs are significantly enriched for Myc binding sites. Subcellular localization analysis revealed that compared to mRNAs, lncRNAs more often have a specific subcellular localization with a markedly higher percentage of nuclear enrichment within the Myc-repressed lncRNA set. Parallel analysis of differentially expressed lncRNAs and mRNAs identified 105 juxtaposed lncRNA-mRNA pairs, indicative for regulation in cis. To support the potential relevance of the Myc-regulated lncRNAs in cellular transformation, we analyzed their expression in primary Myc-high and Myc-low B-cell lymphomas. In total, 54% of the lncRNAs differentially expressed between the lymphoma subsets were identified as Myc-regulated in P493-6 cells. This study is the first to show that lncRNAs are an important factor within the Myc-regulated transcriptional program and indicates a marked difference between Myc-repressed lncRNAs and mRNAs.

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth /apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth-and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. (C) 2018 The Author(s) Published by S. Karger AG, Basel

T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T-cells in response to antigen priming.

Author(s): Cornish-Bowden, Athel; Cornish-Bowden, Athel; Rasnick, David; Heng, Henry H; Horne, Steven; Abdallah, Batoul; Liu, Guo; Ye, Christine J; Bloomfield, Mathew; Vincent, Mark D; Aldaz, C Marcelo; Karlsson, Jenny; Valind, Anders; Jansson, Caroline; Gisselsson, David; Graves, Jennifer A Marshall; Stepanenko, Aleksei A; Andreieva, Svitlana V; Korets, Kateryna V; Mykytenko, Dmytro O; Huleyuk, Nataliya L; Baklaushev, Vladimir P; Kovaleva, Oksana A; Chekhonin, Vladimir P; Vassetzky, Yegor S; Avdieiev, Stanislav S; Bakker, Bjorn; Taudt, Aaron S; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana CJ; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline SJM; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Tatche, Maria Colome; Foijer, Floris; Liehr, Thomas; Baudoin, Nicolaas C; Nicholson, Joshua M; Soto, Kimberly; Quintanilla, Isabel; Camps, Jordi; Cimini, Daniela; Durrbaum, M; Donnelly, N; Passerini, V; Kruse, C; Habermann, B; Storchova, Z; Mandrioli, Daniele; Belpoggi, Fiorella; Silbergeld, Ellen K; Perry, Melissa J; Skotheim, Rolf I; Lovf, Marthe; Johannessen, Bjarne; Hoff, Andreas M; Zhao, Sen; SveeStromme, Jonas M; Sveen, Anita; Lothe, Ragnhild A; Hehlmann, R; Voskanyan, A; Fabarius, A; Bocking, Alfred; Biesterfeld, Stefan; Berynskyy, Leonid; Borgermann, Christof; Engers, Rainer; Dietz, Josef; Fritz, A; Sehgal, N; Vecerova, J; Stojkovicz, B; Ding, H; Page, N; Tye, C; Bhattacharya, S; Xu, J

A proportion of classical Hodgkin lymphomas harbor the Epstein Barr virus (EBV). We previously demonstrated that associations between Human Leukocyte Antigen (HLA) alleles and susceptibility to EBV+ classical Hodgkin lymphoma differ between European and Chinese populations. Data on Hispanic populations is missing. Here we examined the association between HLA type, tumor cell HLA expression and other characteristics in Hispanic Hodgkin lymphoma patients. Hispanic Hodgkin lymphoma patients diagnosed at the Los Angeles County-University of Southern California Medical Center from 2000-2012 were included (n = 65). Formalin-fixed paraffin-embedded tumor tissue was analyzed for EBV by in situ hybridization and for HLA class I and class II expression by immunohistochemistry. HLA typing was performed by HLA-A specific quantitative PCR of genomic DNA from tissue. Thirty patients (46%) had EBV+ tumors. Expression of HLA class I (p = 0.0006) was significantly associated with EBV+ tumor status in Hispanic patients, similar to Europeans and Chinese. A positive association between HLA class II expression and EBV+ tumor status, as present in large studies in Europeans, was not found (p = 0.06). The prevalences of the specific European HLA-A*01 risk and European HLA-A*02 protective types were not significantly associated with EBV+ tumors among these Hispanic patients, however numbers were too low to draw firm conclusions. The HLA-A*02: 07 allele, that is associated with EBV+ Hodgkin lymphoma in Chinese, was absent. In conclusion, the association between EBV positivity in tumor cells and HLA class I expression appears to be consistent across different populations. Larger studies in Hispanics are needed to evaluate HLA allele susceptibility associations.

miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs-miR-23a-3p, miR-24-3p, and miR-27a-3p-were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at Least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27(kiP1) and MYC, In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27(kiP1) and MYC.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is characterized by a low percentage of neoplastic lymphocyte predominant (LP) cells in a background of lymphocytes. The goal of this study is to characterize the microenvironment in NLPHL. Ten NLPHL cases and seven reactive lymph nodes (RLN) were analyzed by flow cytometry for the main immune cells and multiple specific subpopulations. To discriminate between cells in or outside the tumor cell area, we used CD26. We observed significantly lower levels of CD20+ B-cells and CD56+ NK cells and higher levels of CD4+ T-cells in NLPHL in comparison to RLN. In the subpopulations, we observed increased numbers of PD-1+CD4+ T follicular helper cells (TFH), CD69+CD4+ and CD69+CD8+ T-cells and CCR7-CD45RA-CD4+ effector memory T-cells, while FoxP3+CD4+ T regulatory cells (Tregs) and CCR7-CD45RA+ terminally differentiated CD4+ T-cells were decreased in NLPHL compared to RLN. CD69+ cells were increased in the tumor cell area in CD4+ and CD8+ T-cells, while FoxP3+CD25+CD4+ Tregs and CD25+CD8+ T-cells were significantly increased outside the tumor area. Thus, we show a markedly altered microenvironment in NLPHL, with lower numbers of NK cells and Tregs. PD-1+CD4+ and CD69+ T-cells were located inside, and Tregs and CD25+CD8+ cells outside the tumor cell area.

Despite numerous studies reporting deregulated microRNA (miRNA) and gene expression patterns in clear cell renal cell carcinoma (ccRCC), no direct comparisons have been made to its presumed normal counterpart: the renal proximal tubular epithelial cells (PTECs). The aim of this study was to determine the miRNA expression profiles of 10 ccRCC-derived cell lines and short-term cultures of PTEC and to correlate these with their gene expression and copy-number profiles. Using microarray-based methods, a significantly altered expression level in ccRCC cell lines was observed for 23 miRNAs and 1630 genes. The set of miRNAs with significantly decreased expression levels include all members of the miR-200 family known to be involved in the epithelial to mesenchymal transition process. Expression levels of 13 of the 47 validated target genes for the downregulated miRNAs were increased more than twofold. Our data reinforce the importance of the epithelial to mesenchymal transition process in the development of ccRCC.

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with respect to the sequence of the miRNA binding site and the sequences flanking the miRNA binding sites. In-silico validation approaches are presented to test the predicted efficiencies of the sponges in comparison to known target genes. In addition, we describe in vitro validation experiments to confirm the effectiveness of the miRNA sponges. Finally, we describe how the here described procedure can be adapted to easily generate sponges that target multiple miRNAs simultaneously. In summary, our approach allows rapid generation of single or combination miRNA sponges that can be used for long-term miRNA loss-of-function studies. (C) 2012 Elsevier Inc. All rights reserved.

MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. Maturation of miRNAs comprises several regulated steps resulting in ∼22-nucleotide single-stranded mature miRNAs. Regulation of miRNA expression can occur both at the transcriptional level and at the post-transcriptional level during miRNA processing. Recent studies have elucidated specific aspects of the well-regulated nature of miRNA processing involving various regulatory proteins, editing of miRNA transcripts, and cellular location. In addition, single nucleotide polymorphisms in miRNA genes can also affect the processing efficiency of primary miRNA transcripts. In this review we present an overview of the currently known regulatory pathways of miRNA processing and provide a basis to understand how aberrant miRNA processing may arise and may be involved in pathophysiological conditions such as cancer.

Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) secrete factors that interact with inflammatory background cells and may serve as biomarkers for disease activity. To detect new proteins related to pathogenesis, we analyzed the secretome of HRS cells. Proteins in cell culture supernatant of 4 HL cell lines were identified using 1DGE followed by in-gel trypsin digestion and LC-MS/MS. In total, 1290 proteins, including 368 secreted proteins, were identified. Functional grouping of secreted proteins revealed 37 proteins involved in immune response. Sixteen of the 37 proteins (ie, ALCAM, Cathepsin C, Cathepsin S, CD100, CD150, CD26, CD44, CD63, CD71, Fractal-kine, IL1R2, IL25, IP-10, MIF, RANTES, and TARC) were validated in HL cell lines and patient material using immunohistochemistry and/or ELISA. Expression of all 16 proteins was confirmed in HL cell lines, and 15 were also confirmed in HL tissues. Seven proteins (ALCAM, cathepsin S, CD26, CD44, IL1R2, MIF, and TARC) revealed significantly elevated levels in patient plasma compared with healthy controls. Proteomics analyses of HL cell line supernatant allowed detection of new secreted proteins, which may add to our insights in the interaction between HRS cells and infiltrating lymphocytes and in some instances might serve as biomarkers.

Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL.

To identify genes involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we performed serial analysis of gene expression (SAGE) and array-based comparative genomic hybridization (aCGH). Comparison of SAGE libraries of cHL cell lines L428 and L1236 with that of germinal centre B cells revealed consistent overexpression of only 14 genes. In contrast, 141 genes were downregulated in both cHL cell lines, including many B cell and HLA genes. aCGH revealed gain of 2p, 7p, 9p, 11q and Xq and loss of 4q and 11q. Eighteen percent of the differentially expressed genes mapped to regions with loss or gain and a good correlation was observed between underexpression and loss or overexpression and gain of DNA. Remarkably, gain of 2p and 9p did not correlate with increased expression of the proposed target genes c-REL and JAK2. Downregullation of many genes within the HLA region also did not correlate with loss of DNA. FSCN1 and IRAK1 mapping at genomic loci (7p and Xq) that frequently showed gain were overexpressed in cHL cell lines and might be involved in the pathogenesis of cHL. Copyright (c) 2006 John Wiley & Sons, Ltd.

Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1 beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases. (c) 2006 Elsevier Ltd. All rights reserved.

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.

Despite the fact that numerous studies have been performed on diffuse large B-cell lymphoma (DLBCL), only few have concerned extranodal lymphomas occurring in the testis. We performed a cytogenetic and molecular study of 17 testicular non-Hodgkin lymphomas, of which 14 were proven primary DLBCL of the testis. Cytogenetic analysis revealed in 8 out of 11 evaluable cases a structural abnormality of the long arm of chromosome 6, with deletion or addition of material of unknown origin, and with breakpoints spanning the region 6q12-6q23. The cytogenetic findings were confirmed by fluorescent in situ hybridization (FISH) with a chromosome 6 painting probe. Using array based-comparative genomic hybridization on 16 evaluable cases, including 5 cases not tested by cytogenetics or FISH, 14 (88%) showed chromosome 6q deletions. We identified two regions of minimal deletion (RMD), at 104-113 Mb (6q16.3-q21) and 137.5-138.8 Mb (6q23.3), respectively. In one case, we observed a 2.7 Mb homozygous deletion ranging from 135.3 to 138.0 Mb that partly overlapped with the RMD at 6q23.3. Our study indicates that 6q deletions play a major pathogenetic role in DLBCL of the testis and that many of these deletions are part of unbalanced translocations. (c) 2006 Wiley-Liss, Inc.

Little is known about the cytokine profile of nodular lymphocyte predominance Hodgkin's lymphoma (NLPHL) and the significance of the characteristic rosetting CD4(+)/CD57(+) T cells. We analysed the T lymphocyte populations isolated from lymph node suspensions from five patients with NLPHL, two with follicular hyperplasia and progressive transformation of germinal centres (PTGC), three with classical Hodgkin's lymphoma (CHL) and five with hyperplasia of the tonsil. We sorted the T cells based on expression of CD3, CD4 and CD57 by flow cytometry and evaluated the cytokine mRNA profiles of the T cells with quantitative RT-PCR. NLPHL cases were as rich in T cells as the CHL cases, but all NLPHL cases had a much higher frequency of CD4(+)/CD57(+) T cells. In contrast to the CD4(+)/CD57(+) T cells from tonsils, IL2 and IL4 mRNAs were consistently absent from the CD4(+)/CD57(+) T cells of NLPHL. Even after stimulation, no IL4 transcripts could be detected in the CD4(+)/CD57(+) T cells of NLPHL. On the other hand, IFN gamma transcripts were elevated in NLPHL and PTGC T cell subsets as compared to tonsillar T cell subsets. IL13 mRNA was exclusively produced by the T cells of CHL cases, indicating that IL13 may be a key cytokine in CHL. In conclusion, elevated levels of CD4(+)/CD57(+) T cells are characteristic of NLPHL and these T cells display a distinct cytokine mRNA profile. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin’s lymphomas (HLs). It is now known that BIC is a primary microRNA (pri-miRNA) that can be processed to miRNA-155. We analyzed HL and NHL cell lines and tissue samples to determine miR-155 expression levels. High levels of miR-155 could be demonstrated in all HL tissue samples and most cell lines. Most NHL subtypes were negative for BIC as determined by RNA-ISH. However, 8/8 cases of primary mediastinal B cell lymphoma (PMBL) showed BIC staining in 12% of the tumor cells. QRT-PCR analysis confirmed high relative BIC levels (35). 12/18 cases of DLBCL showed a variable BIC staining with on average 7% of positive cells. Interestingly, activated B cell-like (ABC-like) DLBCL showed on average 12% of tumor cells positive for BIC while germinal centre B cell-like (GCB-like) DLBCL only showed on average 4% of cells positive for BIC. Differences in BIC expression levels were confirmed by qRT-PCR analysis revealing average BIC levels of 22 for ABC-like and 12 for GCB-like DLBCL. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analyzed. In summary, we demonstrate expression of pri-miRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL. In DLBCL, BIC expression is most pronounced in the ABC-like phenotype, possibly as a result of NF-κB activation.

Translocations involving band 3q27, affecting the major breakpoint region (MBR) of BCL6, are common in diffuse large B-cell lymphomas (DLBCLs). Recent data suggest an alternative breakpoint cluster region (ABR) located between 245 and 285 kb 5' of BCL6, which might be associated with Follicular Lymphoma (FL). Ten DLBCLs and 9 FLs grade 3B with cytogenetic rearrangements at 3q27 were studied by fluorescence in situ hybridization (FISH) to discriminate between breakpoints at the ABR and MBR. Eight DLBCLs contained a breakpoint in the MBR, and 6 FL grade 3B (FL3B) cases contained a breakpoint in the ABR. No specific chromosomal partners could be identified in both groups. Previously published data have suggested that FL3B cases with 3q27 aberrations are closely related to the majority of DLBCLs of germinal center cell origin. However, our findings suggest that the mechanism of 3q27 rearrangement in FL3B cases is similar to the mechanism in follicular lymphomas grade 1,2, and 3A cases. (c) 2005 Wiley-Liss, Inc.

Alkaline phosphatase (AP) can be considered as a host defence molecule since this enzyme is able to detoxify bacterial endotoxin at physiological pH. The question emerged whether this anti-endotoxin principle is inducible in the glomerulus and if so, which glomerular cells might be involved in the expression of ectoAP after stimulation with pro-inflammatory agents. Therefore kidneys of rats treated with either lipopolysaccharide (LPS), E. coli bacteria or non-toxic monophosphoryl lipid A (MPLA) were examined for AP activity 6 or 24 h after challenge. In addition cultures of endothelial cells or mesangial cells were evaluated for AP activity after stimulation with either LPS, TNFalpha or IL-6, and mRNA for AP was studied in TNFalpha-stimulated and control mesangial cells. The results show significant up-regulation of glomerular AP in LPS- or E. coli-injected rats compared to rats injected with MPLA. Endothelial and mesangial cells in vitro showed significant up-regulation of AP activity following stimulation with LPS, TNFalpha or IL-6, whereas increased mRNA for AP was observed in mesangial cells after TNFalpha stimulation compared to non-stimulated control cells. Since it appeared that hydrolysis occurred when endotoxin was used as a substrate in the histochemical staining, we concluded that inducible glomerular ectoAP may reflect a local endotoxin detoxifying principle of the kidney.

The histological transformation from a follicular lymphoma (FL) to a diffuse large B-cell lymphoma (DLBL) occurs in 22% to 30% of all cases of FL. The aim of this study was to identify specific chromosomal gains/losses associated with transformation of FL to DLBL, in addition to the well-known mechanisms like p53 mutation and protein expression and c-myc translocation and up-regulation. This is the first study to meet 2 important conditions for such a comparison. First, we demonstrate that the FL and the DLBL were clonally related, based on identical immunoglobulin gene rearrangements in 5 of the 6 cases. Second, we used laser microdissection microscopy to isolate only the neoplastic cells from the initial FL samples. The results indicate that no single chromosomal abnormality seems to be responsible for the transformation of FL to DLBL. P53 protein overexpression was found in 4 and c-myc translocation in 3 of the 6 transformed DLBLs, but not in the initial FL samples. Additional chromosomal abnormalities were detected by comparative genomic hybridization in all 6 cases when the DLBL was compared with the FL. In the 5 cases with transformation of grade 1 or 2 FL to DLBL, gains at chromosomes 7 (5 of 5 cases), 10p1 (3 of 5 cases), 12 (3 of 5 cases), and 20p13 (2 of 5 cases) and loss at 9q (4 of 5 cases) were the most frequently found abnormalities. A gain on chromosome 7p, in combination with a loss on 9q, was found in 4 of the 5 DLBL that transformed from FL grade 1 or 2.

Primary central nervous system lymphoma (PCNSL) is a rare disease. A small number of cytogenetic studies of PCNSL have been conducted and several reports have been published on associated molecular and protein expression data. We combined these approaches in a series of eight PCNSL cases, analyzing the chromosomal abnormalities using comparative genomic hybridization (CGH), testing for Epstein Barr virus (EBV) involvement by in situ hybridization for EBER, assessing expression of p53, Bcl-2, Bcl-6 and CD10 by means of immunohistochemistry, and screening for mutations of the TP53 gene by DGGE. TP53 gene mutations and EBV expression were not detected. Most of the cases showed p53, Bcl-6 and Bcl-2 protein expression. CGH revealed DNA copy number changes in all eight cases. The most frequent changes were gains of chromosome 12 (63%), chromosome 18 (50%) and 20q (38%), and loss of chromosome arm 6q (75%). No correlation between protein expression and chromosomal abnormalities was found in these eight cases. Although gains of chromosome 12, 18 and 20q and loss of 6q have also been reported in systemic diffuse large B-cell lymphomas, the frequency of 6q deletion is clearly higher in PCNSL. This creates a similarity to primary lymphomas of the testes that also frequently have deletions of 6q. This suggests that suppressor genes located on chromosome 6q may play a role in the development of lymphomas at immunoprivileged sites, like the CNS.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the toss of a chromosome 13 region between RB1 gene and locus D13S25, A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the Loci D13S1168 (W19598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb, Three cDNAs likely corresponding to three different genes hare been found in the minimally deleted region, sequenced and mapped against the contigged cosmids, cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5, This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements. (C) 1998 Federation of European Biochemical Societies.

The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines.

The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer. Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer. We have analyzed the gene and its transcripts in lung cancers and renal cell cancer-derived cell lines. A lung adenocarcinoma cell line, GLC-A2, appeared to have a homozygous deletion in intron 5 of FHIT. RT-PCR analysis revealed a normal-sized PCR product in all of the cell lines, including GLC-A2. A number- of them had an additional aberrant product Analysis of a great number of control fell lines and tissues showed that the majority of these also had abet-rant PCR products in addition to a normal-sized PCR product. Different specimens of the same cell type showed variable additional RT-PCR products. Normal-sized PCR products had a sequence identical to the FHIT sequence. PCR products longer than normal had insertions of different sizes at different positions. With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons. Thus, the presence of aberrant transcripts is not cancer-specific. Conceivably, sequences responsible for the instability of the FRA3B region are being transcribed into FHIT pre-mRNA and may cause the abnormal splicing and processing of the transcripts. (C) 1997 Wiley-Liss, Inc.

The chromosomal pattern of 31 specimens of chromophilic renal-cell cancer (RCC), selected according to the criteria mentioned in the classification of Thoenes and Storkel, is presented. A high male preponderance was found (8.7:1). Cytogenetic analysis revealed a typical pattern of numeric alterations specific for this sub-type in the majority of cases (i.e., -Y, +7, +12, +16, +17, and/or +20), which is different from the chromosomal patterns found in other sub-types of RCC. Gain of chromosome 20, as well as loss of the extra copy of chromosome 17 or loss of 17p, was found to be related to the higher-grade chromophilic carcinomas. None of the 14 cases examined by SSCP analysis revealed mutations of the pP53 gene, indicating that other genes at 17(p) might be important in the progression of this sub-type of RCC. (C) 1996 Wiley-Liss, Inc.

Small cell lung cancer (SCLC) has been correlated with a deletion in the short arm of chromosome 3, with the region 3p21 being lost from one homolog in almost all cases. Two SCLC cell lines have homozygous deletions in 3p21, and these deletions overlap with a fragment of chromosome 3 that has tumor suppression activity in vivo. We have isolated some cDNA clones from this region that are homologous to the genes constituting the semaphorin family. They represent a novel human semaphorin, termed sema III/F (HGMW-approved symbol SEMA3F), which is expressed as a 3.8-kb transcript in a variety of cell lines and tissues; it is detected as early as Embryonic Day 10 in mouse development. There is high expression in mammary gland, kidney, fetal brain, and lung and lower expression in heart and liver. Although there is reduced expression of this gene in several SCLC lines, no mutations were found. This semaphorin homolog has characteristics of a secreted member of the semaphorin III family, with 52% identity with mouse semaphorin E and 49% identity with chicken collapsin/semaphorin D. (C) 1996 Academic Press, Inc.

Cytotoxic CD8 T cells have pivotal effector and regulatory immune-functions and are, as such, involved in the host-immune response following infection. Dysfunction of CD8 T cells, on the other hand, may contribute to autoimmunity. CD8 T cells exert their function by releasing cytotoxic mediators and cytokines. Acute infection triggers a response to effectively clear the pathogen and develop long-lasting protective memory, while chronic infections may lead to exhaustion and dysfunction of CD8 T cells. The latter phenomenon relates to the tunable balance of the immune system to preserve tolerance and prevent development of autoimmunity. CD8 T cells also play a crucial role in preventing development of cancer cells. Tumor cells may evade an effective anti-tumor immune response and clinical exploitation of immune checkpoints to break this tolerance and induce anti-tumor immunity clearly illustrates the two-edged characteristics of the immune response. Vice versa, autoimmune disorders and chronic infections may lead to the onset of cancer, which suggests that these pathological conditions may share mutual pathways. Understanding the molecular basis of autoreactive and defective CD8 T cell responses provides a basis for the rational design of immunotherapies aiming to restore CD8 T cell function and combat chronic viral infections and cancer.

Tumor cells of classical Hodgkin lymphoma (cHL) are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I is expressed in most EBV+ cHL cases, with an even enhanced expression in a proportion of the cases. Promyelocytic leukemia protein (PML) and special AT-rich region binding protein 1 (SATB1) are two global chromatin organizing proteins that have been shown to regulate HLA class I expression in Jurkat cells. We analyzed HLA class I, number of PML nuclear bodies (NBs) and SATB1 expression in tumor cells of 54 EBV+ cHL cases and used 27 EBV- cHL cases as controls. There was a significant difference in presence of HLA class I staining between EBV+ and EBV2 cases (p

In a family with a constitutional translocation t(3;6), the oldest member carrying the translocation had developed multiple nonpapillary renal cell carcinomas (RCCs). The translocation breakpoint was positioned between 3p13 and 3p14.1. This is close to the region in which a t(3;8) breakpoint has been reported in a family with hereditary RCC. We defined the location of the t(3;6) and t(3;8) breakpoints by fluorescence in situ hybridization (FISH) analysis with yeast artificial chromosomes (YACs) from the 3p14-13 region. Both interphase nuclei and metaphase cells from translocation-carrying members of both families have been used, allowing the definition of flanking YACs for each breakpoint. We could thereby clearly confirm that the breakpoints are different, the t(3;8) breakpoint being most distal. In addition, we have shown that both translocation breakpoints are located distal to the homozygously deleted region in the U2020 lung cancer cell line. (C) 1995 Wiley-Liss, Inc.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17 similar to 92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that similar to 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.

MicroRNAs ( miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naive, germinal center ( GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization ( ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naive, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone. Laboratory Investigation (2009) 89, 708-716; doi:10.1038/labinvest.2009.26; published online 6 April 2009

Little is known about the gene expression profile and significance of the rosetting CD4+CD26- T cells in classical Hodgkin's lymphoma (cHL). To characterize these T cells, CD4+CD26- and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26- T-cells in NSHL than in reactive LNs. The resting CD4+CD26-T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2- related cytokines IL-2, IFN-gamma, IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro, lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26- T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.

Background Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe – poly(A)-tail distances. Results Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe – poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe – poly(A)-tail distance >500 nucleotides appearing only after one round of amplification. Conclusion Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA.