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    • br Material and Methods br Results br

    2018-10-23


    Material and Methods
    Results
    Discussion We document the expression of EBV viral miRNAs by three independent methods, small RNA sequencing, qRT-PCR and miRNA-ISH in malignant B cells, plasma and bone marrow biopsies of patients with CLL. It is noteworthy that more than 90% of adults older than 40years are EBV positive (Rickinson, 2001). We also found that only one healthy donor and 4% of patients with CLL had negative EBNA-1 IgG titers. Furthermore, we observed that BHRF1-1 expression levels in plasma from untreated CLL patients are significantly higher than in plasma of healthy donors. This result is in apparent contradiction with the absence of similar findings in leukemic B-cells. However, EBV infects not only B lymphocytes, but also epithelial cells, smooth muscle cells, T- and NK- cells which can eventually release it into the plasma via microvesicles (Jochum et al., 2012); this phenomenon is likely to explain the discrepancy between plasma and B-cell expression levels in patients with CLL. An alternative explanation is that malignant order GSK2126458 are actively secreting microvesicles and/or exosomes that contain viral miRNAs (Ghosh et al., 2010). Interestingly, none of the seronegative (EBNA-1 IgG) patients had an EBV-DNA load above the threshold of positivity, suggesting that the presence of EBV-miRNAs could not be explained by the onset of active infection and/or the absence of EBV-specific immunoglobulin due to CLL-related defects of antibody response. Another interesting finding of our study was that patients who apparently did not experienced EBV infection express BHRF1-1 and BART4 miRNAs. This finding was confirmed in two different groups of patients with CLL (Figs. 3D and S5). However, none of the patients showed a clear positivity for LMP-1 and EBER staining on their respective bone marrow specimens. LMP-1 mRNA has been shown to be expressed in a significant proportion of patients with CLL and to correlate with the degree of bone marrow involvement (Tarrand et al., 2010). However, unlike the study reported by Tarrand et al. where serum levels or mRNA were measured, we evaluated LMP-1 expression by IHC directly on bone marrow specimens. In addition, expression of BHRF1-1 and miR-155 by miRNA-ISH was found diffusely positive in bone marrow specimens from patients. Based on our experience, the measurement of miRNAs (by RT-PCR and/or miRNA-ISH) may represent a more sensitive method to detect viral components compared with assays detecting viral proteins only (ELISA and IHC). In order to exclude that the positive results could be attributed to the presence of human miRNAs with a homologous sequence, we performed a search within the NCBI database using the BLAST algorithm and did not find a human miRNA with an identical sequence for BHRF1-1. Next, we found a positive correlation in the studied patients between the plasma levels of miR-155 and those of BHRF1-1 miRNA. We have previously reported that miR-155 is a marker of the risk of progression from monoclonal B cell lymphocytosis (MBL) to CLL and a predictor of response to treatment (Ferrajoli et al., 2013). Although DNA sequences were previously identified in the plasma of patients with various cancers, this is the first study to identify an EBV-associated miRNA in the plasma of patients with CLL (Ghosh et al., 2010). These findings support the possibility that EBV miRNA(s) released by infected cells could be taken up by CLL cells or other cells and affect the gene expression levels in the new “host” cells. It is known that patients with CLL can produce microvesicles (Jochum et al., 2012) as well as exosomes (N Kay, personal communication) and have increased levels of microvesicles in their plasma. Our findings support the possibility that leukemic B-cells can have effective cross talk and exchange of cellular contents with other neighboring cells. Furthermore, this potential interaction expands our understanding of the way CLL may be initiated: “latent” EBV-infected cells communicate with the surrounding cells and the microenvironment by releasing pathogenetic viral miRNAs and induction of the expression of miR-155 as well as other oncogenic cellular miRNAs that affect cell survival and apoptosis. Furthermore, cellular protein coding genes such as TP53 are targets of EBV viral miRNAs. The fact that TP53 level is reduced after BFRF1-1 infection in primary culture of malignant B-cells from the majority of cases but not in all, suggest that other cancer genes involved in the BHRF1-1 signaling pathway have to be identified. However several factors, other than miRNAs, could explain the role of EBV in the pathogenesis of CLL. For example, in the elderly a significantly greater frequency of EBV-specific CD8+ T-cells has been noted along with a lower frequency of EBV antigen-specific interferon-gamma-producing T-cells (Ouyang et al., 2003). Thus, it is possible that an abnormal response to EBV stimuli favors a robust burst of B clones that are EBV-driven, especially in an older individual.