Epstein-Barr virus-encoded miR-BART20-5p inhibits T-bet translation with secondary suppression of p53 in invasive nasal NK/T-cell lymphoma.

Nasal NK/T-cell lymphoma (NNL) is an Epstein-Barr virus (EBV)-associated lymphoma derived from cytotoxic NK or T cells of the nasal mucosa. NNLs are noninvasive in the earliest stage, and become invasive with disease progression. The EBV encodes at least 44 miRNAs, whose functions in the pathogenesis of NNL are mostly unknown. We evaluated the levels of 39 EBV-encoded miRNAs with quantitative real-time RT-PCR in a series of 20 noninvasive NNLs and 20 invasive NNLs. miR-BART20-5p was associated most strongly with invasion (P ≤ 0.001), and lack of T-bet, the master transcription factor for cytotoxic NK cells. However, we identified T-bet (official symbol, TBX21) transcripts in T-bet-negative NNLs, implying a block in the translation of T-bet by miR-BART20-5p. In co-transfection experiments, miR-BART20-5p inhibited T-bet translation in both non-Hodgkin and Hodgkin lymphoma cell lines. Endogenous mir-BART20-5p also inhibited translation of T-bet in EBV-infected YT lymphoma cells of NK-cell origin. Induction of T-bet in YT cells up-regulated p53, leading to increased sensitivity in response to doxorubicin. Finally, YT cells transplanted into severe combined immunodeficiency mice had an invasive behavior. Taken together, we conclude that EBV-encoded miR-BART20-5p inhibits T-bet translation with secondary suppression of p53 in invasive nasal NK/T-cell lymphoma. An antagomir to miR-BART20-5p might be an effective therapeutic agent through induction of T-bet and p53.

Chromosome instability modulated by BMI1-AURKA signaling drives progression in head and neck cancer.

Chromosomal instability (CIN) is widely considered a hallmark of cancer, but its precise roles in cancer stem cells (CSC) and malignant progression remain uncertain. BMI1 is a member of the Polycomb group of chromatin-modifier proteins that is essential for stem cell self-renewal. In human cancers, BMI1 overexpression drives stem-like properties associated with induction of epithelial-mesenchymal transition (EMT) that promotes invasion, metastasis, and poor prognosis. Here, we report that BMI1 mediates its diverse effects through upregulation of the mitotic kinase Aurora A, which is encoded by the AURKA gene. Two mechanisms were found to be responsible for BMI1-induced AURKA expression. First, BMI1 activated the Akt pathway, thereby upregulating AURKA expression through activation of the ß-catenin/TCF4 transcription factor complex. Second, BMI1 repressed miRNA let-7i through a Polycomb complex-dependent mechanism, thereby relieving AURKA expression from let-7i suppression. AURKA upregulation by BMI1 exerts several effects, including centrosomal amplification and aneuploidy, antiapoptosis, and cell-cycle progression through p53 degradation and EMT through stabilization of Snail. Inhibiting Aurora A kinase activity attenuated BMI1-induced tumor growth in vivo. In clinical specimens of head and neck cancer, we found that coamplification of BMI1 and AURKA correlated with poorer prognosis. Together, our results link CSCs, EMT, and CIN through the BMI1-AURKA axis and suggest therapeutic use from inhibiting Aurora A in head and neck cancers, which overexpress BMI1.

Expression of CD19 and lack of miR-223 distinguish extramedullary plasmacytoma from multiple myeloma.

AIMS: Extramedullary plasmacytoma (EMP) and multiple myeloma (MM) are both plasma cell (PC) tumours that are usually distinguished by clinical manifestations, but not by histopathological examination alone. However, EMP may express B-cell markers, such as CD79a and CD20, and MM may express germinal centre B-cell (GCBC)-associated microRNAs, such as miR-93 and miR-181b. Down-regulation of miR-30a or up-regulation of miR-223 is associated with the transition from GCBCs into PCs or memory B-cells, respectively. We studied B-cell markers and microRNAs to establish criteria that could distinguish EMP from MM. METHODS AND RESULTS: Immunostains for the B-cell markers CD19, CD20, CD79a and PAX5 were performed. Expression levels of microRNAs 30a, 93, 181b and 223 were measured by real-time reverse transcription polymerase chain reactions. 73% of EMPs expressed CD19 whereas MM cases were negative. EMP and MM had similar levels of miR-30a, miR-93, and miR-181b, but EMP lacked expression of miR-223. CONCLUSIONS: The presence of CD19 and lack of miR-223 suggested aberrant B-cell differentiation in EMP. Although the underlying mechanism for this differential expression was unclear, a CD19(+) /miR-223(-) phenotype could be used to distinguish EMP from the CD19(-) /miR-223(+) phenotype of MM.

E2A-positive gastric MALT lymphoma has weaker plasmacytoid infiltrates and stronger expression of the memory B-cell-associated miR-223: possible correlation with stage and treatment response.

Extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue of the stomach (gastric MALT lymphoma) is derived from memory B cells of the marginal zone. Normal memory B cells do not express markers of germinal-center B cells, such as E2A (immunoglobulin enhancer-binding factor E12/E47), B-cell chronic lymphocytic leukemia/lymphoma 6 (BCL6), or activation-induced cytidine deaminase (AID). E2A is a transcription factor that induces somatic hypermutations and blocks plasma cell differentiation. In 50 stage-I(E)/II(E1) gastric MALT lymphomas, we confirmed that all cases were BCL6(-)/AID(-), but a subset (50%, 25/50) was E2A(+). As E2A(-) and E2A(+) gastric MALT lymphomas had similar numbers of somatic hypermutations without intraclonal variations, which implied an origin from memory B cells, the expression of E2A was best regarded as a marker of aberrant follicular differentiation. Although the status of somatic hypermutation was not affected by E2A, E2A(+) gastric MALT lymphoma showed less plasmacytoid infiltrates and higher expressions of miRNA-223, a microRNA associated with memory B cells. Clinically, E2A(+) gastric MALT lymphomas were more likely to spread to perigastric lymph nodes and were less responsive to Helicobacter eradication therapy than were E2A(-) gastric MALT lymphomas. Taken together, aberrant E2A expression is a diagnostic feature of a subtype of gastric MALT lymphoma with weaker plasmacytoid infiltrates and stronger miR-223 expression. A prospective study would be necessary to verify the association between E2A expression and a poor response to Helicobacter eradication therapy.

Early low-grade gastric MALToma rarely transforms into diffuse large cell lymphoma or progresses beyond the stomach and regional lymph nodes.

BACKGROUND/PURPOSE: Gastric mucosa-associated lymphoid tissue lymphoma (MALToma) usually presents at an early stage involving only the stomach and/or regional lymph nodes. Although a sequential transformation from low-grade gastric MALToma (GM) to high-grade GM to secondary diffuse large B-cell lymphoma (DLBCL) is commonly assumed, documented cases of transformation are rare. We aim to determine the frequency of transformation. METHODS: We identified 55 early low-grade GMs, 18 early high-grade GMs, and 13 advanced GMs at the National Taiwan University Hospital from 1995 to 2005. The median follow-up time was 59 months. RESULTS: We found that only one early low-grade GM and two early high-grade GMs transformed into secondary DLBCLs and progressed outside the stomach and regional lymph nodes. Significantly, we identified 13 low-grade GMs that were refractory to Helicobacter eradication therapy or relapsed after initial response. All 13 cases had been followed-up for at least 3 years without development of secondary DLBCLs. The frequency of transformation for early low-grade GM was less than 2% (1/55). Although two lymphoma-unrelated mortalities were identified, none of the 55 patients with early-low grade GMs died of the disease. CONCLUSION: Compared with chronic lymphocytic leukemia, which has a 16% transformation rate and a median transformation time of 24 months, we conclude that early low-grade GM rarely transforms into secondary DLBCL or progresses beyond the stomach. Without transformation or progression, patients with early low-grade GM rarely die of the disease and should be treated conservatively.

Characterization of Epstein-Barr virus BGLF4 kinase expression control at the transcriptional and translational levels.

The BGLF4 protein of Epstein-Barr virus (EBV) is a serine/threonine protein kinase that phosphorylates several viral and cellular substrates at cellular cyclin-dependent kinase target sites. BGLF4 is required for efficient viral DNA replication and release of mature virions. It also stimulates the transactivation activity of the immediate-early transactivator Zta (BZLF1) and suppresses the transactivation activities of BMRF1 and EBNA-2. This study aimed to characterize further the regulation of BGLF4 expression at the transcriptional and translational levels. It was shown that BGLF4 was expressed with early kinetics and reached maximal levels after DNA replication. The promoter activity of BGLF4 was upregulated mainly by the immediate-early transactivator Rta, rather than Zta, as revealed by Zta-specific short hairpin RNA in EBV-positive cells and by luciferase reporter assays. By rapid amplification of 5' cDNA ends, two major transcriptional start sites were identified at 201 and 255 nt upstream of the first in-frame ATG of BGLF4 in P3HR1 cells. An additional transcript initiated from -468 was detected in Akata cells. The translation initiation site of BGLF4 was confirmed by mutagenesis, in vitro translation and transient transfection. The translation regulatory effect mediated by the long 5'-untranslated region (5'UTR) of BGLF4 was demonstrated by dual reporter assays in 293T and EBV-positive NA cells. These results suggested that different promoter usage and 5'UTR-mediated translation enhancement may ensure the proper expression of BGLF4 at various stages of virus replication.

EBV-positive Hodgkin lymphoma is associated with suppression of p21cip1/waf1 and a worse prognosis.

BACKGROUND: About 30-50% of Hodgkin lymphomas (HLs) harbor the Epstein-Barr virus (EBV), but the impact of EBV infection on clinical outcomes has been unclear. EBV-encoded small RNAs (EBERs) are presented in all EBV-infected cells, but their functions are still less understood. RESULTS: EBER1 was transfected into two HL cell lines, KMH2 and L428, and microarrays were used to screen for EBER1-induced changes. We found that EBER1 suppressed p21cip1/waf1 transcription in HL cell lines. In addition, positive regulators of p21cip1/waf1 transcription, such as p53, EGR1, and STAT1, were decreased. Suppression of p21cip1/waf1 in the EBER1+ HL cell lines was associated with increased resistance to histone deacetylase inhibitors or proteasome inhibitors, drugs known to cause apoptosis by increasing p21cip1/waf1 levels. On biopsy specimens, EBV+ HLs had weaker expression of both p21cip1/waf1 and active caspase 3. Clinically, suppression of p21cip1/waf1 in EBV+ HLs was associated with a worse 2-year disease-free survival rate (45% for EBV+ HLs vs. 77% for EBV- HLs, p = 0.002). CONCLUSION: Although the underlying mechanisms are still relatively unclear, EBER1 inhibits p21cip1/waf1 transcription and prevents apoptosis through down-regulation of p53, EGR1, and STAT1. The anti-apoptotic activity of EBER1 may be important in the rescue of Reed-Sternberg cells from drug-induced apoptosis and in the clinical behaviors of EBV+ HLs.

Loss of CD7, independent of galectin-3 expression, implies a worse prognosis in adult T-cell leukaemia/lymphoma.

AIMS: Loss of CD7 is characteristic of adult T-cell lymphoma/leukaemia (ATLL). Galectin-3 (Gal-3) is strongly induced in cultured human T lymphotropic virus-1-infected T lymphocytes, and may cause apoptosis through interaction with CD7. The aim was to investigate the clinical relevance of the Gal-3-CD7 pathway in ATLL. METHODS AND RESULTS: Immunohistochemistry for Gal-3 and CD7 was performed on 22 cases of ATLL in the leukaemic phase. We found that the lymphoma cells were not necessarily Gal-3+, but Gal-3+ stromal cells could always be found. Independent of the status of Gal-3, there was an association of loss of CD7 with a worse prognosis. CONCLUSIONS: These data suggest that, by down-regulating CD7, ATLL cells could have escaped Gal-3-induced apoptosis to run a more aggressive clinical course.

CD94 1A transcripts characterize lymphoblastic lymphoma/leukemia of immature natural killer cell origin with distinct clinical features.

Most lymphoblastic lymphomas (LBLs) are regarded as neoplasms of immature T cells because they express cytoplasmic CD3 and frequently carry T-cell receptor (TCR) gene rearrangements. Immature natural killer (NK) and T cells, however, have a common bipotent T/NK-cell precursor in the thymus, and NK cells also express cytoplasmic CD3. Thus, some LBLs could arise from immature NK cells. Mature NK cells express 2 CD94 transcripts: 1A, induced by interleukin 15 (IL-15), and 1B constitutively. Because immature NK cells require IL-15 for development, CD94 1A transcripts could be a marker of NK-LBL. To test this hypothesis, we used laser capture microdissection to isolate IL-15 receptor alpha(+) lymphoid cells from the thymus and showed that these cells contained CD94 1A transcripts. We then assessed for CD94 transcripts in 21 cases of LBL that were cytoplasmic CD3(+), nuclear terminal deoxynucleotidyl transferase positive (TdT(+)), and CD56(-), consistent with either the T-cell or NK-cell lineage. We found that 7 LBLs expressed CD94 1A transcripts without TCR gene rearrangements, suggesting NK-cell lineage. Patients with NK-LBL were younger than patients with T-LBL (15 years versus 33 years; P = .11) and had a better 2-year survival (100% versus 27%; P < .01). These results improve the current classification of LBL and contribute to our understanding of NK-cell differentiation.

Oligoclonal T cells in histiocytic necrotizing lymphadenopathy are associated with TLR9+ plasmacytoid dendritic cells.

Histiocytic necrotizing lymphadenopathy (HNL), a disease of unknown cause, is characterized pathologically by the presence of plasmacytoid dendritic cells (pDCs), which are frequently mixed with oligoclonal T cells (OTCs) and myeloid cells. Toll-like receptors (TLRs 1-10) are a family of pattern recognition receptors of DCs. To investigate the interactions between pDCs and T cells, and to look for an etiology of HNL, we studied 24 HNLs for the profile of TLRs. Transcripts of TLR7, a receptor on pDCs for single-stranded RNA, were found in every case, confirming the universal presence of pDCs. Transcripts of TLR9, another receptor on pDCs for microbial unmethylated CpG-rich DNA, were correlated with OTCs, implying T-cell expansion stimulated by TLR9+ pDCs in response to a microbe. Because PCRs for bacterial 16S rDNAs were negative in the lymph nodes, a bacterial origin seems unlikely, but a virus remains a possible candidate. The pDCs lacked the maturation marker CD83, which suggested ineffective stimulation of T cells and might account for the usually benign course of HNL. Taken together, these data illustrate a novel approach, based upon TLR transcript analysis, for the integration of pathology, immunology, and clinical findings of HNL.

CD94 transcripts imply a better prognosis in nasal-type extranodal NK/T-cell lymphoma.

Transcription of natural killer (NK) cell antigen receptors (NKRs), such as CD94, NKG2, and killer immunoglobulin-like receptors (KIRs), is developmentally regulated and clonally distributed. We have shown a restricted KIR repertoire (rKIR-R) without monoclonal T-cell receptor rearrangement (mTCR-R) supports a NK lineage in nasal-type extranodal NK/T-cell lymphoma (NTENL) but does not correlate with clinical outcomes. Developing NK cells express first CD94, then NKG2A, NKG2E, and finally NKG2C. This sequence suggests an immature CD94- and a mature CD94+ subtype of NTENL. Using a rKIR-R without a mTCR-R as a criterion in 25 cases of NTENL, we confirmed a true NK lineage in 19 cases, including 10 CD94+ and 9 CD94- patients by reverse transcriptase-polymerase chain reaction (RT-PCR). Eight of the 10 CD94+ patients but only 2 of the 9 CD94- patients survived beyond 1 year (median survival, 60 months versus 10 months by Meier-Kaplan survival analysis, P =.026 by Cox F test). The remaining 6 patients had a rKIR-R plus a mTCR-R, suggesting mixed NK/T differentiation. They were CD94- by RT-PCR, found predominantly in young women, and had a median survival of 35 months. Thus, on the basis of the transcripts of NKRs, a division of NTENLs into CD94+, CD94-, and mixed NK/T types reflects a true biologic divergence with different clinical behaviors.