Abstract: Rare single nucleotide variants play an important role in genetic diversity and heterogeneity of specific human disease. For example, an individual clinical sample can harbor rare mutations at minor frequencies. Genetic diversity within an individual clinical sample is oftentimes reflected in rare mutations. Therefore, detecting rare variants prior to treatment may prove to be a useful predictor for therapeutic response. Current rare variant detection algorithms using next generation DNA sequencing are limited by inherent sequencing error rate and platform availability.
Abstract: Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies towards actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved by using immunoglobulin somatic mutations and BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic events during lymphomagenesis. We provide evidence that BCL2 translocations and CREBBP mutations are early events, while MLL2 and TNFRSF14 mutations likely represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.
Abstract: Epstein-Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas BARF1 expression is restrained to the viral lytic replication cycle. In this study the transcriptional regulation of BARF1 during lytic replication is unraveled.Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter Luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins, BZLF1 (Z) and BRLF1 (R). R was found to activate the BARF1 promoter up to 250 fold, independent of Z and unaffected by BARF1 promoter methylation. Chromatin Immune Precipitation (ChIP), Electrophoretic Mobility Shift Assay (EMSA) and specific mutagenesis of the R responsive elements (RREs) demonstrated direct binding of R to RREs between -554 and -327 nucleotides relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 hours in context of the viral genome.In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region, and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.
Abstract: The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in ∼90% of the world population. Constitutive activation of NF-κB by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-κB overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-κB activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) cells.
Abstract: We have performed the first extensive profiling of Epstein-Barr virus (EBV) miRNAs on in vivo derived normal and neoplastic infected tissues. We describe a unique pattern of viral miRNA expression by normal infected cells in vivo expressing restricted viral latency programs (germinal center: Latency II and memory B: Latency I/0). This includes the complete absence of 15 of the 34 miRNAs profiled. These consist of 12 BART miRNAs (including approximately half of Cluster 2) and 3 of the 4 BHRF1 miRNAs. All but 2 of these absent miRNAs become expressed during EBV driven growth (Latency III). Furthermore, EBV driven growth is accompanied by a 5-10 fold down regulation in the level of the BART miRNAs expressed in germinal center and memory B cells. Therefore, Latency III also expresses a unique pattern of viral miRNAs. We refer to the miRNAs that are specifically expressed in EBV driven growth as the Latency III associated miRNAs. In EBV associated tumors that employ Latency I or II (Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric carcinoma), the Latency III associated BART but not BHRF1 miRNAs are up regulated. Thus BART miRNA expression is deregulated in the EBV associated tumors. This is the first demonstration that Latency III specific genes (the Latency III associated BARTs) can be expressed in these tumors. The EBV associated tumors demonstrate very similar patterns of miRNA expression yet were readily distinguished when the expression data were analyzed either by heat-map/clustering or principal component analysis. Systematic analysis revealed that the information distinguishing the tumor types was redundant and distributed across all the miRNAs. This resembles "secret sharing" algorithms where information can be distributed among a large number of recipients in such a way that any combination of a small number of recipients is able to understand the message. Biologically, this may be a consequence of functional redundancy between the miRNAs.
Abstract: Exosomes are specialized membranous nano-sized vesicles derived from endocytic compartments that are released by many cell types. Microvesicles are distinctive from exosomes in that they are produced by shedding of the plasmamembrane and usually larger in size (>1 µm). Exosome biogenesis involves the tightly controlled process of inward budding from the limiting membrane of multivesicular bodies (MVBs). This results in numerous intraluminal vesicles in the lumen of MVBs that contain distinct protein repertoires. It has been suggested that microvesicles shed by certain tumor cells hold functional messenger RNA (mRNA) that may promote tumor progression. We discovered that purified exosomes contain functional microRNAs (miRNAs) and small RNA, but detected little mRNA. Although a clear and decisive distinction between microvesicles and exosomes cannot be made and different subsets of exosomes exist, we speculate that exosomes are specialized in carrying small RNA including the class 22-25 nucleotide regulatory miRNAs. To demonstrate this we developed a co-culture system and found that exosomes are continuously secreted and transferred from Epstein Barr virus (EBV)-infected cells to uninfected neighboring cells. Throughout exosome transfer, the exogenous EBV-encoded miRNAs were delivered to subcellular sites of miRNA-mediated gene repression. Additionally, we found evidence that mature miRNAs are transferred between circulating cells in humans, since we detected EBV-miRNAs in non-infected cells in the peripheral blood of patients that include monocytes and T cells. In this addendum we discuss these findings in the context of recently published papers that advanced our current knowledge of exosome physiology, (mi)RNA function and intercellular RNA transfer. Based on this information we propose that an intercellular (miRNA-based) mode of signal transmission may be well suited in controlling space-confined processes such as the initiation of immune responses in the secondary (peripheral) lymphoid tissues or in a tumor microenvironment. Deciphering the molecular mechanism(s) that control small RNA loading into exosomes and transfer to recipient cells in vitro will provide new evidence for the physiological relevance of vesicle-mediated intercellular communication in vivo.
Abstract: Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human gamma-herpesvirus EBV.
Abstract: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon.
Abstract: Although most gastric cancers occur in elderly patients, a substantial number of cases of this common disease occur in young patients. Gastric cancer is a heterogeneous disease at the genomic level and different patterns of DNA copy number alterations are associated with different clinical behaviour. The aim of the present study was to explore differences in DNA copy number alterations in relation to age of onset of gastric cancer. DNA isolated from 46 paraffin-embedded gastric cancer tissue samples from 17 patients less than 50 years of age [median 43 (21-49) years] and 29 patients greater than or equal to 70 years of age [median 75 (70-83) years] was analysed by genome-wide microarray comparative genomic hybridization (array CGH) using an array of 5000 BAC clones. Patterns of DNA copy number aberrations were analysed by hierarchical cluster analysis of the mode-normalized and smoothed log(2) ratios of tumour to normal reference fluorescence signal intensities using TMEV software, after which cluster membership was correlated with age group. In addition, supervised analysis was performed using CGH Multi-array. Hierarchical cluster analysis of the array CGH data revealed three clusters with different genomic profiles that correlated significantly with age (p = 0.006). Cluster 1 mainly contained young patients, while elderly patients were divided over clusters 2 and 3. Chromosome regions 11q23.3 and 19p13.3 contributed most to age-related differences in tumour profiles. Gastric cancers of young and old patients belong to groups with different genomic profiles, which likely reflect different pathogenic mechanisms of the disease.
Abstract: Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.
