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Abstract We previously showed that MYC promoted Burkitt lymphoma (BL) growth by inhibiting the tumor suppressor miR-150, resulting in release of miR-150 targets MYB and ZDHHC11. The ZDHHC11 gene encodes three different transcripts including a mRNA (pcZDHHC11), a linear long non-coding RNA (lncZDHHC11) and a circular RNA (circZDHHC11). All transcripts contain the same region with 18 miR-150 binding sites. Here we studied the relevance of circZDHHC11, including this miR-150 binding site region, for growth of BL cells. CircZDHHC11 was mainly present in the cytoplasmic fraction in BL cells and its localization was not altered upon miR-150 overexpression. Knockdown of circZDHHC11 caused a strong inhibition of BL growth without affecting the expression levels of MYC, MYB, miR-150 and other genes. Overexpression of circZDHHC11 neither affected cell growth, nor rescued the phenotype induced by miR-150 overexpression. Genomic deletion of the miR-150 binding site region did not affect growth, nor did it change the effect of circZDHHC11 knockdown. This indicated that the miR-150 binding site region is dispensable for the growth promoting role of circZDHHC11. To conclude, our results show that circZDHHC11 is a crucial factor supporting BL cell growth independent of its ability to sponge miR-150.

Abstract Background Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL. Methods We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively. Results MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196. Conclusion The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Abstract Diagnosing post-transplant lymphoproliferative disorder (PTLD) is challenging and often requires invasive procedures. Analyses of cell-free DNA (cfDNA) isolated from plasma is minimally invasive and highly effective for genomic profiling of tumors. We studied the feasibility of using cfDNA to profile PTLD and explore its potential to serve as a screening tool. We included seventeen patients with monomorphic PTLD after solid organ transplantation in this multi-center observational cohort study. We used low-coverage whole genome sequencing (lcWGS) to detect copy number variations (CNVs) and targeted next-generation sequencing (NGS) to identify Epstein-Barr virus (EBV) DNA load and somatic single nucleotide variants (SNVs) in cfDNA from plasma. Seven out of seventeen (41%) patients had EBV-positive tumors, and 13/17 (76%) had stage IV disease. Nine out of seventeen (56%) patients showed CNVs in cfDNA, with more CNVs in EBV-negative cases. Recurrent gains were detected for 3q, 11q, and 18q. Recurrent losses were observed at 6q. The fraction of EBV reads in cfDNA from EBV-positive patients was 3-log higher compared to controls and EBV-negative patients. 289 SNVs were identified, with a median of 19 per sample. SNV burden correlated significantly with lactate dehydrogenase levels. Similar SNV burdens were observed in EBV-negative and EBV-positive PTLD. The most commonly mutated genes were TP53 and KMT2D (41%), followed by SPEN, TET2 (35%), and ARID1A, IGLL5, and PIM1 (29%), indicating DNA damage response, epigenetic regulation, and B-cell signaling/NFkB pathways as drivers of PTLD. Overall, CNVs were more prevalent in EBV-negative lymphoma, while no difference was observed in the number of SNVs. Our data indicated the potential of analyzing cfDNA as a tool for PTLD screening and response monitoring.

Abstract Histological transformation of marginal zone lymphoma (tMZL) into diffuse large B-cell lymphoma is associated with poor outcomes. Clinical characteristics associated with transformation risk and outcome after transformation are largely unknown due to scarcity of data. In this population-based study, competing risk analyses were performed to elucidate clinical characteristics associated with developing transformation among 1793 MZL patients using the Netherlands Cancer Registry. Cox regression analyses were performed to elucidate clinical characteristics associated with risk of relapse and mortality after transformation. Transformation occurred in 75 (4%) out of 1793 MZL patients. Elevated LDH and nodal MZL subtype at MZL diagnosis were associated with an increased risk, and radiotherapy with a reduced risk of developing tMZL. Most tMZL patients received R-(mini)CHOP (n = 53, 71%). Age >60 years and (immuno)chemotherapy before transformation were associated with an increased risk of relapse and mortality after transformation. Two-year progression-free survival (PFS) and overall survival (OS) were 66% (95% CI 52–77%) and 75% (95% CI 62–85%) for R-(mini)CHOP-treated tMZL patients, as compared to a PFS and OS both of 41% (95% CI 19–63%) for patients treated otherwise. Our study offers comprehensive insights into characteristics associated with transformation and survival after transformation, thereby optimizing guidelines and patient counseling.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERβ) as a new target in DLBCL. Here, we demonstrate that ERβ is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERβ plays a role in the protection against apoptosis in DLBCL. Targeting of the ERβ with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERβ knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients.

IntroductionTransformation from lung adenocarcinoma (LUAD) to small cell lung cancer (SCLC) is one of the mechanisms responsible for acquired EGFR-TKIs resistance. Although it rarely happens this event determines a rapid disease deterioration and needs specific treatment.Patient and methodWe report a case of 75-year-old LUAD female with a p.L858R mutation in Epidermal Growth Factor Receptor (EGFR) who presented with SCLC transformation after responding to first line osimertinib treatment for only 6 months. To understand the underlying molecular mechanism, we retrospectively sequenced the first (LUAD) and the second (SCLC) biopsy using a 56 multi-gene panel. Immunohistochemistry (IHC) staining and Fluorescence In Situ Hybridization (FISH) was applied to confirm the genetic aberrations identified.ResultsEGFR p.E709A and p.L858R, Tumor Protein p53 (TP53) p.A159D and Retinoblastoma 1 (RB1) c.365-1G>A were detected in both the diagnostic LUAD and transformed SCLC samples. A high copy number gain for Proto-Oncogene C-Myc (MYC) and a Phosphoinositide 3-Kinase Alpha (PIK3CA) p.E545K mutation were found in the transformed sample specifically. Strong TP53 staining and negative RB1 staining were observed in both LUAD and SCLC samples, but FISH only identified MYC amplification in SCLC tissue.ConclusionWe consider the combined presence of MYC amplification with mutations in TP53 and RB1 as drivers of SCLC transformation. Our results highlight the need to systematically evaluate TP53 and RB1 status in LUAD patients to offer a different therapeutic strategy.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Tumor cells of classic Hodgkin lymphoma (cHL) are derived from antigen presenting B cells that are infected by Epstein Barr virus (EBV) in ~30% of patients. Polymorphic Killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells interact with human leukocyte antigen (HLA) class I and play a key role in immune surveillance against virally infected cells and tumor cells. We investigated the effect of KIR types on cHL susceptibility overall (n=211) and in EBV-stratified subgroups using the Dutch GoNL cohort as controls (n=498). The frequency of the KIR haplotype B subgroup was significantly different between EBV+ and EBV− cHL patients (62% vs. 77%, p=0.04) and this difference was more pronounced in nodular sclerosis (NS) cHL (49% vs. 79%, p=0.0003). The frequency of KIR haplotype B subgroup was significantly lower in EBV+ NS cHL compared to controls (49% vs. 67%, p=0.01). Analyses of known KIR – HLA interaction pairs revealed lower carrier frequencies of KIR2DS2 – HLA-C1 (29% vs. 46%, p=0.03) and KIR2DL2 – HLA-C1 (29% vs. 45%, p=0.04) in EBV+ NS cHL patients compared to controls. Carriers of the KIR haplotype B subgroup are less likely to develop EBV+ NS cHL, probably because of a more efficient control over EBV-infected B cells.

Abstract Background The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a disease biomarker in oesophageal squamous cell carcinoma (ESCC) that can be used for treatment response evaluation and early detection of tumour recurrence. Methods Matched tumour tissue, pre- and post-surgery plasma and WBCs obtained from 17 ESCC patients were sequenced using a panel of 483 cancer-related genes. Results Somatic mutations were detected in 14 of 17 tumour tissues. Putative harmful mutations were observed in genes involved in well-known cancer-related pathways, including PI3K-Akt/mTOR signalling, Proteoglycans in cancer, FoxO signalling, Jak-STAT signalling, Chemokine signalling and Focal adhesion. Forty-six somatic mutations were found in pre-surgery cfDNA in 8 of 12 patients, with mutant allele frequencies (MAF) ranging from 0.24 to 4.91%. Three of the 8 patients with detectable circulating tumour DNA (ctDNA) had stage IIA disease, whereas the others had stage IIB-IIIB disease. Post-surgery cfDNA somatic mutations were detected in only 2 of 14 patients, with mutant allele frequencies of 0.28 and 0.36%. All other somatic mutations were undetectable in post-surgery cfDNA, even in samples collected within 3–4 h after surgery. Conclusion Our study shows that somatic mutations can be detected in pre-surgery cfDNA in stage IIA to IIIB patients, and at a lower frequency in post-surgery cfDNA. This indicates that cfDNA could potentially be used to monitor disease load, even in low disease-stage patients.

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.

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteome

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, characterized by high levels of genomic instability and the activation of DNA damage repair pathways. We previously found high expression of the cell cycle regulator WEE1 in DLBCL cell lines. Here, we investigated the combination of the WEE1 inhibitor, AZD1775, with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and radiation therapy (RT), with the aim of improving first-line treatment. Methods: Cell viability experiments were performed to determine synergistic combinations. Levels of DNA damage were established using flow cytometry for γH2AX and protein analysis for DNA damage response proteins CHK1 and CHK2. Flow cytometry analysis for cell cycle and pH3 were performed to determine cell cycle distribution and premature mitotic entry. Results: Treatment with either RT or CHOP led to enhanced sensitivity to AZD1775 in several DLBCL cell lines. Treatment of cells with AZD1775 induced unscheduled mitotic progression, resulting in abnormal cell cycle distribution in combination with RT or CHOP treatment. In addition, a significant increase in DNA damage was observed compared with CHOP or RT alone. Of the single CHOP components, doxorubicin showed the strongest effect together with AZD1775, reducing viability and increasing DNA damage. Conclusion: In conclusion, the combination of RT or CHOP with AZD1775 enhances sensitivity to WEE1 inhibition through unscheduled G2/M progression, leading to increased DNA damage. Based on these results, WEE1 inhibition has great potential together with other G2/M arresting or DNA damaging (chemo) therapeutic compounds and should be further explored in clinical trials.

Patients with MYC-rearrangement positive large B-cell lymphoma (MYC+ LBCL) have an inferior prognosis following standard first-line therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) as compared to patients without MYC rearrangement. Although intensive chemotherapy regimens yield higher remission rates, toxicity remains a concern. Lenalidomide is an oral immunomodulatory drug which downregulates MYC and its target genes thereby providing support using lenalidomide as additional therapeutic option for MYC+ LBCL. A phase II trial was conducted evaluating the efficacy of lenalidomide (15 mg day 1-14) in combination with R-CHOP (R2CHOP) in newly diagnosed MYC+ LBCL patients identified through a nationwide MYC-FISH screening program. The primary endpoint was complete metabolic response (CMR) on centrally reviewed 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)-computer tomography (CT)-scan at end-of-treatment. Secondary endpoints were overall survival (OS), disease-free survival (DFS) and event-free survival (EFS). Eighty-two patients with stage II-IV MYC+ LBCL were treated with 6 cycles of R2CHOP. At EOT, 67% (confidence interval (CI) 58-75%) of the patients reached CMR. With a median follow-up of 25.4 months, 2-year estimates (95% CI) for OS, DFS, EFS were 73% (62-82%), 75% (63-84%) and 63% (52-73%) respectively. In this prospective trial for newly diagnosed MYC+ LBCL patients, we found that administering R2CHOP was safe, and yields comparable CMR and survival rates as in studies applying more intensive chemotherapy regimens. Hence, these findings offer new prospects for MYC+ LBCL patients and warrant comparison in prospective randomized clinical trials. This trial was registered at www.clinicaltrialsregister.eu (#2014-002654-39).

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 MTregs 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 MTregs 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 MTregs (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, MTregs 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 MTregs by forskolin restored their suppressive function in vitro.Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

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 β-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.

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28− T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3′UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3′UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3′UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

Forty percent of patients with diffuse large B-cell lymphoma (DLBCL) show resistant disease to standard chemotherapy (CHOP) in combination with the anti-CD20 monoclonal antibody rituximab (R). Although many new anti-cancer drugs were developed in the last years, it is unclear which of these drugs can be safely combined to improve standard therapy without antagonizing anti-CD20 efficacy. In this study, we aimed to identify rituximab compatible drug-target combinations for DLBCL. For this, we collected gene expression profiles of 1,804 DLBCL patient samples. Subsequently, we performed a guilt-by-association analysis with MS4A1 (CD20) and prioritized the 500 top-ranked CD20-associated gene probes for drug-target interactions. This analysis showed the well-known genes involved in DLBCL pathobiology, but also revealed several genes that are relatively unknown in DLBCL, such as WEE1 and PARP1. To demonstrate potential clinical relevance of these targets, we confirmed high protein expression of WEE1 and PARP1 in patient samples. Using clinically approved WEE1 and PARP1 inhibiting drugs in combination with rituximab, we demonstrated significantly improved DLBCL cell killing, also in rituximab-insensitive cell lines. In conclusion, as exemplified by WEE1 and PARP1, our CD20-based genome-wide analysis can be used as an approach to identify biological relevant drug-targets that are rituximab compatible and may be implemented in phase 1/2 clinical trials to improve DLBCL treatment.

BACKGROUND:Due to the high burden of dengue disease worldwide, a better understanding of the interactions between dengue virus (DENV) and its human host cells is of the utmost importance. Although microRNAs modulate the outcome of several viral infections, their contribution to DENV replication is poorly understood. METHODS AND PRINCIPAL FINDINGS:We investigated the microRNA expression profile of primary human macrophages challenged with DENV and deciphered the contribution of microRNAs to infection. To this end, human primary macrophages were challenged with GFP-expressing DENV and sorted to differentiate between truly infected cells (DENV-positive) and DENV-exposed but non-infected cells (DENV-negative cells). The miRNAome was determined by small RNA-Seq analysis and the effect of differentially expressed microRNAs on DENV yield was examined. Five microRNAs were differentially expressed in human macrophages challenged with DENV. Of these, miR-3614-5p was found upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. The cellular targets of miR-3614-5p were identified by liquid chromatography/mass spectrometry and western blot. Adenosine deaminase acting on RNA 1 (ADAR1) was identified as one of the targets of miR-3614-5p and was shown to promote DENV infectivity at early time points post-infection. CONCLUSION/SIGNIFICANCE:Overall, miRNAs appear to play a limited role in DENV replication in primary human macrophages. The miRNAs that were found upregulated in DENV-infected cells did not control the production of infectious virus particles. On the other hand, miR-3614-5p, which was upregulated in DENV-negative macrophages, reduced DENV infectivity and regulated ADAR1 expression, a protein that facilitates viral replication.

Antigen presentation by tumor cells in the context of Human Leukocyte Antigen (HLA) is generally considered to be a prerequisite for effective immune checkpoint inhibitor therapy. We evaluated cell surface HLA class I, HLA class II and cytoplasmic HLA-DM staining by immunohistochemistry (IHC) in 389 classical Hodgkin lymphomas (cHL), 22 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL), 137 diffuse large B-cell lymphomas (DLBCL), 39 primary central nervous system lymphomas (PCNSL) and 19 testicular lymphomas. We describe a novel mechanism of immune escape in which loss of HLA-DM expression results in aberrant membranous invariant chain peptide (CLIP) expression in HLA class II cell surface positive lymphoma cells, preventing presentation of antigenic peptides. In HLA class II positive cases, HLA-DM expression was lost in 49% of cHL, 0% of NLPHL, 14% of DLBCL, 3% of PCNSL and 0% of testicular lymphomas. Considering HLA class I, HLA class II and HLA-DM together, 88% of cHL, 10% of NLPHL, 62% of DLBCL, 77% of PCNSL and 87% of testicular lymphoma cases had abnormal HLA expression patterns. In conclusion, an HLA expression pattern incompatible with normal antigen presentation is common in cHL, DLBCL, PCNSL and testicular lymphoma. Retention of CLIP in HLA class II caused by loss of HLA-DM is a novel immune escape mechanism, especially prevalent in cHL. Aberrant HLA expression should be taken into account when evaluating efficacy of checkpoint inhibitors in B-cell lymphomas.

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.

BACKGROUND:Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function. MATERIALS AND METHODS:We investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-β1-induced miRNA expression changes were validated and replicated in an independent set of lung fibroblasts composted of 10 controls and 15 COPD patients using qRT-PCR. Ago2-immunoprecipitation followed by mRNA expression profiling was used to identify the miRNA-targetomes of unstimulated and TGF-β1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-β1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-β1-stimulated fibroblasts. RESULTS:Twenty-nine miRNAs were significantly differentially expressed after TGF-β1 stimulation (FDR

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.

Hodgkin lymphoma (HL) is derived from preapoptotic germinal center B cells, although a general loss of B cell phenotype is noted. Using quantitative reverse transcription-polymerase chain reaction and miRNA microarray, we determined the microRNA (miRNA) profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas. The two methods showed a strong correlation for the detection of miRNA expression levels. The HL-specific miRNA included miR-17-92 cluster members, miR-16, miR-21, miR-24, and miR-155. Using a large panel of cell lines, we found differential expression between HL and other B-cell lymphoma-derived cell lines for 27 miRNA. A significant down-regulation in HL compared to non-Hodgkin lymphoma was observed only for miR-150. Next, we performed target gene validation of predicted target genes for miR-155, which is highly expressed in HL and is differentially expressed between HL and Burkitt lymphoma. Using luciferase reporter assays, we validated 11 predicted miR-155 target genes in three different HL cell lines. We demonstrated that AGTR1, FGF7, ZNF537, ZIC3, and IKBKE are true miR-155 target genes in HL.

Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; pT and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.

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.

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.

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.

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.

BackgroundHepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.MethodsTissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.ResultsCOX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.ConclusionsIn human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.

Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC,

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 EBV- cases (p

Background The c-Met signaling pathway regulates a variety of biological processes, including proliferation, survival and migration. Deregulated c-Met activation has been implicated in the pathogenesis and prognosis of many human malignancies. We studied the function and prognostic significance of c-Met and hepatocyte growth factor protein expression in patients with classical Hodgkin’s lymphoma.Design and Methods Expression of c-Met and its ligand, hepatocyte growth factor, were determined by immunohistochemistry. Prognostic values were defined in cohorts of German and Dutch patients with classical Hodgkin’s lymphoma. Functional studies were performed on Hodgkin’s lymphoma cell lines.Results Expression of c-Met was detected in the tumor cells of 52% (80/153) of the patients and expression of its ligand, hepatocyte growth factor, in 8% (10/121) of the patients. c-Met expression correlated with a 5-year freedom from tumor progression of 94%, whereas lack of expression correlated with a 5-year freedom from tumor progression of 73% (P

Background Plasma thymus and activation-regulated chemokine is a potential biomarker for classical Hodgkin’s lymphoma. To define its value as a marker to monitor treatment response, we correlated serial plasma thymus and activation-regulated chemokine levels with clinical response in newly diagnosed and relapsed classical Hodgkin’s lymphoma patients.Design and Methods Plasma was collected from 60 (39 early stage and 21 advanced stage) newly diagnosed classical Hodgkin’s lymphoma patients before, during, and after treatment, and from 12 relapsed patients before and after treatment. Plasma thymus and activation-regulated chemokine levels were determined by enzyme-linked immunosorbent assay and were related to pre-treatment metabolic tumor volume, as measured by quantification of 2-[18F]fluoro-2-deoxyglucose positron emission tomography images, and to treatment response.Results Baseline plasma thymus and activation-regulated chemokine levels correlated with stage of disease and bulky disease, and more closely with metabolic tumor volume. Response to treatment was observed among 38 of 39 early stage and 19 of 21 advanced stage patients. Reduction in plasma thymus and activation-regulated chemokine to normal range levels could be observed as early as after one cycle of chemotherapy in all responsive patients, while plasma levels remained elevated during and after treatment in the 3 non-responsive patients. Plasma thymus and activation-regulated chemokine was elevated in all 12 relapsed patients at time of relapse and remained elevated after salvage treatment in the 4 non-responsive patients.Conclusions Baseline plasma thymus and activation-regulated chemokine levels correlate with classical Hodgkin’s lymphoma tumor burden and serial levels correlate with response to treatment in patients with classical Hodgkin’s lymphoma.

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∼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.

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV- cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV- cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV- cHL vs controls (p = 0.045) and EBV+ vs EBV- cHL cases (p = 2×10(-5)). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV- cHL in the northern Chinese population.

The pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients using a PCR-based sequence-specific oligonucleotide probe (SSOP) hybridization approach. The allele frequencies were compared to HLA typings of more than 6,000 controls. The age of the cHL patients varied between 13 and 81 years with a median of 35 years. Nodular sclerosis subtype was the most common subtype (87%) and EBV was detected in 25% of the cHL patients. HLA-B5 was significantly increased and HLA-DR7 significantly decreased in the total cHL patient population as compared to controls. Two class II associations were observed to be specific for the EBV- cHL population with an increase of HLA-DR2 and HLA-DR5. Allele frequencies of HLA-A1, HLA-B37 and HLA-DR10 were significantly increased in the EBV+ cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in Caucasians. The allele frequency of HLA-A2 was significantly decreased in the EBV+ cHL population. Analysis of haplotypes with a frequency of >1% revealed a significant increase of HLA-A2-B7-DR2 in EBV- cHL as compared to controls. SSOP association analysis revealed significant differences between EBV+ and EBV- cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV+ cHL. Furthermore several new protective and predisposing HLA class I and II associations for the EBV+, the EBV- and the entire cHL population were identified.

BackgroundThe incidence of classical Hodgkin lymphoma (cHL) and its association with Epstein-Barr virus (EBV) varies significantly with age, sex, ethnicity and geographic location. This is the first report on epidemiological features of cHL patients from Northern regions of China. These features are compared to data from a previously published Dutch cHL population.Methodology/principal findings157 cHL patients diagnosed between 1997 and 2008 in the North of China were included after histopathological re-evaluation. The Dutch population-based cohort consisted of 515 cHL patients diagnosed between 1987 and 2000. EBV status was determined by in situ hybridization of EBV- encoded small RNAs. In the Chinese population, tumor cells of 39% of the cHL patients were EBV+ and this was significantly associated with male sex, mixed cellularity subtype and young age (Conclusion/significanceCHL patients from Northern regions of China show a distinctive age distribution pattern with a striking incidence peak of EBV+ mixed cellularity cases among children and adolescents and another high incidence peak of EBV- nodular sclerosis cases in young adults. In comparison to Dutch cHL patients there are pronounced differences in age distribution, subtype and EBV status, presumably caused by complex gene-environmental interactions.

BackgroundIn Caucasian populations, the tumor cells of Epstein Barr virus (EBV)-positive classical Hodgkin Lymphomas (cHL) patients more frequently express HLA class I and HLA class II molecules compared to EBV-negative cHL patients. HLA expression (in relation to EBV) in Asian cHL patients has not been previously investigated.Methodology/principal findingsWe randomly selected 145 cHL patients with formalin-fixed, paraffin embedded tissue blocks available from 5 hospitals from the Northern part of China. Hematoxylin & Eosin-stained slides were used to re-classify the histological subtypes according to the WHO classification. EBV status was determined by visualization of EBERs in tumor cells using in situ hybridization. Membranous expression of HLA molecules was detected by immunohistochemistry using antibodies HC-10 (class I heavy chain) and anti-beta2-microglobulin for HLA class I, and CR3/43 for HLA class II. EBV+ tumor cells were observed in 40% (58/145) of the cHL patients. As expected, the percentage of EBV+ cases was much higher in the mixed cellularity subtype (71%) than in the nodular sclerosis subtype (16%) (pConclusionsThe results in the Northern China population were similar to those in the Caucasian population for HLA class I, but not for HLA class II.