Alan Kwok Shing Chiang

CD56 ˛ NK lymphomas: clinicopathological features and prognosis

The surface molecule CD56 marks a category of malignant lymphoma of putative natural killer (NK) cell origin. We conducted a retrospective analysis of 24 cases of CD56+ NK lymphoma/leukaemia to define the clinicopathologic and prognostic features of this specific group of lymphomas. 56 cases of nasal lymphomas and 204 cases with an initial diagnosis of peripheral T‐cell lymphoma were retrospectively analysed. To specifically examine lymphomas of putative NK origin, only those that were negative for surface expression of CD3 but positive for CD56 were analysed. 24 cases were identified. The initial predominant sites of involvement were nasal (n =18), palate (n = 1), nodal (n = 1) and multi‐organ (n =4). Clinically, in patients with disease localized to one anatomical site (n = 20), most had symptoms confined to the nose, with a high percentage in early stage (I: 91%; IV: 9%). The marrow was not involved in any of these cases. However, patients with multi‐organ involvement at presentation (n = 4) behaved differently. All presented acutely with pancytopenia, hepatosplenomegaly, and marrow infiltration with haemophagocytosis. A leukaemic phase was observed in one case. Anthracycline containing combination chemotherapy resulted in complete remission in 75% of patients with localized disease, but only in 25% with multi‐organ involvement. The median survival of patients with localized disease was 12 months, compared with 2 months in the multi‐organ group (P =0.06); the disease‐free survival was significantly better in the former (P <0.01). The overall median survival of all patients was still poor at 11 months.

Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease

Nasal T/NK‐cell lymphomas can be further separated into those of natural killer (NK) cell lineage or of T‐cell lineage, with differences in cellular phenotype, T‐cell receptor (TcR) gene rearrangement and TcR transcript expression. Both NK‐ and T‐cell subtypes are closely associated with Epstein‐Barr virus (EBV). In this study, EBV gene expression was determined in 23 cases of nasal lymphoma (NL) by in situ hybridisation (ISH), reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunohistochemistry (IH). Of the 23 cases, 19 were classified as NK‐cell and 4 as T‐cell tumours. ISH for EBV‐encoded small non‐polyadenylated RNAs showed that all cases, whether NK or T, harboured EBV in virtually all tumour cells. RT‐PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern, the latent membrane proteins, LMPI and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs, compatible with the latency type II pattern. In addition, analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single‐cell level consisting of both LMPI+ and LMPI‐ tumour cells, suggesting a mixture of latency I and II. Although 2 early lytic transcripts, BZLFI and BHRFI, were also detected in 13 and 10 cases, respectively, the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle. The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkins disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types. Down‐regulation of immunogenic proteins (EBNA2‐EBNA6) in nasal lymphoma may enable tumour cells to evade host cytotoxic T‐cell surveillance.

Nasal T/natural killer (NK)-cell lymphomas are derived from Epstein-Barr virus–infected cytotoxic lymphocytes of both NK- and T-cell lineage

The cellular nature of nasal T/natural killer (NK)‐cell lymphomas (NLs) remains controversial. It is still debatable whether these represent T‐cell lymphomas with extensive loss of surface antigens or are, in fact, true NK‐cell lymphomas. They are associated closely with Epstein‐Barr virus (EBV), to the extent that EBV‐encoded small non‐polyadenylated RNAs (EBER) expression can be used as a marker for the neoplastic cells. The cell lineage of this group of lymphomas was examined further by correlating immunophenotype, genotype and EBV status with the expression of cytotoxic granule‐associated proteins, perforin and T‐cell intracellular antigen‐1 (TIA‐1) in 13 cases of NL. Combined immunophenotypic and gene rearrangement analyses demonstrated that NLs can be identified clearly as either NK‐cell or T‐cell tumours. Nasal NK‐cell lymphomas lacked clonal rearrangement of both T‐cell receptor (TCR) γ and immunogloulin heavy chain (IgH) genes and were either CD3(Leu4)−CD56+ (8 cases) or CD3(Leu4)+CD56+ (2 cases), whereas nasal T‐cell lymphomas had rearranged TCRγ and germ‐line IgH genes and were either CD3(Leu4)+CD56+ (2 cases) or CD3(Leu4)+CD56− (1 case). Immunohistochemical (IH) studies showed that both perforin and TIA‐1 were expressed universally in NL, irrespective of NK‐ or T‐cell lineage. Dual labelling of TIA‐1 by IH and EBER by in situ hybridisation demonstrated that the granule proteins were expressed predominantly by the EBER+ tumour cells. Our results indicate that NLs are derived from EBV‐infected cytotoxic lymphocytes of both NK‐ and T‐cell lineage. We postulate that cytotoxic lymphocytes generated during the cellular immune response to EBV infection or re‐activation at the nasal region themselves may become targets for EBV infection and subsequent transformation. Int. J. Cancer 73:332–338, 1997.

Phenotypic and cytotoxic characteristics of peripheral T‐cell and NK‐cell lymphomas in relation to Epstein–Barr virus association

We investigated the phenotypic and cytotoxic characteristics of different types of peripheral T‐cell and NK‐cell lymphomas and correlated the findings of cytotoxic phenotype with Epstein–Barr virus (EBV) association.

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitts lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8+ T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.

Differences in T-cell-receptor gene rearrangement and transcription in nasal lymphomas of natural killer and T-cell types : Implications on cellular origin

Although nasal lymphomas showing midfacial destructive lesions had been classified as T-cell lymphomas, their exact cellular origin is still unclear. Although they usually express a restricted number of T-cell-related antigens, namely, CD2, CD43, and CD45RO, other pan-T or subset-T-lineage antigens, such as CD3 (membrane), CD5, CD4, CD8, and CD7, are frequently absent. Conversely, they often express a natural killer (NK) cell-associated antigen, CD56, but lack other mature NK markets. To study their cellular origin further, the authors analyzed T-cell receptor (TCR) gene transcription in three cases of nasal lymphomas and correlated the findings with the phenotype and gene rearrangement data. Two cases of nasal lymphomas with CD2+CD3(Leu4)-CD19-CD56+ phenotype were shown to express truncated 1.0-kb Tbeta and multiple unrearranged Tdelta transcripts with germline TCR beta, gamma, delta, and immunoglobulin heavy-chain joining region (JH) genes, consistent with NK cell lineage. In contrast, one case of nasal lymphoma with CD2+CD3(Leu4)+CD8+CD19-CD56+ phenotype expressed full-length Talpha, Tbeta, and Tgamma transcripts rearranged TCR beta, gamma, and deleted TCR delta genes, indicating T-lineage, These results support the view that nasal lymphomas can separated into NK-cell and T-cell neoplasms, based on differences genotypic characteristics. The possibility of these tumors being derived from a putative common precursor cell merits further investigation.

Suberoylanilide hydroxamic acid induces viral lytic cycle in Epstein-Barr virus-positive epithelial malignancies and mediates enhanced cell death†

In Epstein‐Barr virus (EBV)‐associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV‐positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8‐fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV‐positive AGS/BX1 cells when compared with that of EBV‐negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus‐targeted drug for treatment of EBV‐associated epithelial malignancies.

Bortezomib and SAHA Synergistically Induce ROS-Driven Caspase-Dependent Apoptosis of Nasopharyngeal Carcinoma and Block Replication of Epstein–Barr Virus

A novel drug combination of a proteasome inhibitor, bortezomib, and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was tested in nasopharyngeal carcinoma (NPC), both in vitro and in vivo. Dose-response of different concentrations of bortezomib and SAHA on inhibition of cell proliferation of NPC was determined. Mechanisms of apoptosis and effects on lytic cycle activation of Epstein–Barr virus (EBV) were investigated. Combination of bortezomib and SAHA (bortezomib/SAHA) synergistically induced killing of a panel of NPC cell lines. Pronounced increase in sub-G1, Annexin V–positive, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)–positive cell populations were detected after treatment with bortezomib/SAHA when compared with either drug alone. Concomitantly, markedly augmented proteolytic cleavage of PARP, caspase-3, -7, -8, and -9, reactive oxygen species (ROS) generation, and caspase-8–dependent histone acetylation were observed. ROS scavenger, N-acetyl cysteine, diminished the apoptotic effects of bortezomib/SAHA, whereas caspase inhibitor Z-VAD-FMK significantly suppressed the apoptosis without decreasing the generation of ROS. Bortezomib inhibited SAHAs induction of EBV replication and abrogated production of infectious viral particles in NPC cells. Furthermore, bortezomib/SAHA potently induced apoptosis and suppressed the growth of NPC xenografts in nude mice. In conclusion, the novel drug combination of bortezomib and SAHA is highly synergistic in the killing of NPC cells in vitro and in vivo. The major mechanism of cell death is ROS-driven caspase-dependent apoptosis. Bortezomib antagonizes SAHAs activation of EBV lytic cycle in NPC cells. This study provides a strong basis for clinical testing of the combination drug regimen in patients with NPC. Mol Cancer Ther; 12(5); 747–58. ©2013 AACR.

Cellular Gene Expression That Correlates with EBER Expression in Epstein-Barr Virus-Infected Lymphoblastoid Cell Lines

ABSTRACT Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.

Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein‐Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV‐positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV‐infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV‐positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP‐ribose) polymerase and caspase‐3, ‐7 and ‐9 in EBV‐positive versus EBV‐negative NPC cells were observed. More than 85% of NPC cells expressing immediate‐early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase‐3. Tracking of expression of EBV lytic proteins and cleaved caspase‐3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHAs induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus‐targeted therapy against EBV‐positive cancer.