Joanne R. Flavell

Target cells of Epstein-Barr-virus (EBV)-positive post-transplant lymphoproliferative disease: similarities to EBV-positive Hodgkin's lymphoma.

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) encompasses a histologically broad range of lesions, arising from the expanded pool of EBV-infected B cells in the immunocompromised host. Identification of the precise cellular origin of these tumours could clarify their pathogenesis. METHODS Of 13 cases of EBV-positive cases of PTLD characterised by histological analysis, pattern of EBV gene expression, and clinical course, 11 had monoclonal or biclonal lesions in which we determined the progenitor B cell by immunoglobulin heavy chain (IgH) genotyping. RESULTS Two tumours had a naive B cell genotype and two showed patterns of IgH somatic mutation typical of antigen-selected (post-germinal-centre) memory cells. All four arose early post-transplant and expressed the markers of EBV transformation--Epstein-Barr nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1. However, seven tumours, either of early or late onset and including some with downregulated EBNA 2 and LMP 1, arose from post-germinal cells with randomly mutated or sterile IgH genotypes usually incompatible with B-cell survival in vivo. INTERPRETATION PTLD can arise from a broad range of target B cells and not only from the pool of antigen-selected memory cells that EBV generally colonises in immunocompetent individuals. Tumour development seems frequently associated with the EBV-induced rescue and expansion of B cells that have failed the physiological process of germinal centre selection into memory. This finding shows an unexpected connection between pathogenesis of PTLD and that of EBV-positive Hodgkins lymphoma, another B-cell malignancy of atypical post-germinal-centre cell origin.

Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

In approximately 50% of patients with Hodgkins lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Epstein–Barr virus-encoded latent infection membrane protein 1 regulates the processing of p100 NF- κ B2 to p52 via an IKK γ /NEMO-independent signalling pathway

The oncogenic Epstein–Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) constitutively activates the ‘canonical’ NF-κB pathway that involves the phosphorylation and degradation of IκBα downstream of the IκB kinases (IKKs). In this study, we show that LMP1 also promotes the proteasome-mediated proteolysis of p100 NF-κB2 resulting in the generation of active p52, which translocates to the nucleus in complex with the p65 and RelB NF-κB subunits. LMP1-induced NF-κB transactivation is reduced in nf-kb2−/− mouse embryo fibroblasts, suggesting that p100 processing contributes to LMP1-mediated NF-κB transcriptional effects. This pathway is likely to operate in vivo, as the expression of LMP1 in primary EBV-positive Hodgkins lymphoma and nasopharyngeal carcinoma biopsies correlates with the nuclear accumulation of p52. Interestingly, while the ability of LMP1 to activate the canonical NF-κB pathway is impaired in cells lacking IKKγ/NEMO, the regulatory subunit of the IKK complex, p100 processing remains unaffected. As a result, nuclear translocation of p52, but not p65, occurs in the absence of IKKγ. These data point to the existence of a novel signalling pathway that regulates NF-κB in LMP1-expressing cells, and may thereby play a role in both oncogenic transformation and the establishment of persistent EBV infection.

Interleukin 6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of 'B' symptoms and failure to achieve complete remission in patients with advanced Hodgkin's disease.

Summary. Interleukin 6 (IL‐6) is a potent immunomodulatory cytokine that has pathogenic and prognostic significance in a number of disorders. Previous studies in Hodgkins disease (HD) have demonstrated the association between elevated serum levels of IL‐6 and unfavourable prognosis, including advanced stage and the presence of ‘B’ symptoms and with reduced survival. Although IL‐6 expression has been demonstrated in both the malignant Hodgkin/Reed–Sternberg (HRS) cells and in the various non‐malignant cells present in HD biopsies, a relationship between expression of IL‐6 by the tumour and outcome measures has not been established. The study group comprised of 97 patients with advanced HD who were recruited to two related clinical trials. IL‐6 expression was determined on paraffin‐wax sections of biopsy material by means of an immunohistochemical assay. Of the 97 patients, 27 (28%) showed staining for IL‐6 in HRS cells. IL‐6 expression by HRS cells was significantly correlated with a decreased likelihood of achieving a complete response to chemotherapy (P = 0·02) and with an increased prevalence of ‘B’ symptoms (P = 0·04). IL‐6 expression by HRS cells was not associated with Epstein–Barr virus status (P = 0·57). In summary, the results suggest that IL‐6 expression by HRS cells may contribute to the presence of ‘B’ symptoms and to a decreased likelihood to achieve a complete remission in HD patients.

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector

Viruses that replicate selectively in cancer cells constitute an exciting new class of anticancer agent. The conditionally replicating adenovirus (CRAd) dl1520, which lacks the E1B-55K gene, has elicited significant clinical responses in humans when used in combination with chemotherapy. A convergent development has been to use replication-defective viruses to express prodrug-activating enzymes in cancer cells. This can sensitize the cancer to prodrug, but depends upon achieving sufficient level, distribution and specificity of enzyme expression within the tumour. In this study, we have expressed the prodrug-activating enzyme nitroreductase (NTR) in the context of an E1B-55K-deleted adenovirus, CRAd-NTR(PS1217H6). We show that CRAd-NTR(PS1217H6) retains oncolytic growth properties, and expresses substantially more NTR than a comparable, replication-defective adenovirus. The combination of viral oncolysis and NTR expression results in significantly greater sensitization of SW480 and WiDr colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, CRAd-NTR(PS1217H6) was shown to replicate in subcutaneous SW480 tumour xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replication-defective virus. Combination therapy of CRAd-NTR(PS1217H6) with CB1954 reduced tumour growth from 13.5- to 2.8-fold over 5 weeks, and extended median survival from 42 to 81 days, compared with no treatment.

Variations in ATM Protein Expression During Normal Lymphoid Differentiation and Among B-Cell-Derived Neoplasias

The ataxia telangiectasia mutated (ATM) protein plays a central role in the cellular response to DNA double-strand breaks (DSBs). Developmentally programmed DSBs are restricted to cellular subsets within lymphoid tissues and we asked whether ATM expression is differentially regulated during lymphoid differentiation. We showed that immature B cells in bone marrow and immature T cells of the thymic cortex were negative or weakly ATM-positive. T cells of thymic medulla and peripheral tissues strongly expressed ATM. High levels of ATM were present in the B lymphocytes of the mantle zone and in plasma cells, while the majority of germinal center B cells were negative or weakly labeled. Therefore, ATM expression appears to be down-regulated at those stages of lymphoid development where physiological DNA DSBs occur. In B-chronic lymphocytic leukemia and mantle cell lymphoma we observed two categories: ATM-negative tumors, most likely reflecting the presence of ATM mutation, and tumors with abundant ATM expression. Most follicular center-cell lymphomas and diffuse large B-cell lymphomas, which rarely show inactivation of the ATM gene, were negative or weakly ATM-positive. Tumor cells from most cases of Hodgkins disease were ATM-negative. Therefore, unless ATM inactivation occurs, ATM expression in lymphoid tumors is likely to reflect their cellular origin. As a result, immunostaining to identify lymphoid neoplasias with ATM inactivation might only be feasible for tumors derived from the stages where ATM is constitutively highly expressed.

Expression of the tumour necrosis factor receptor‐associated factors 1 and 2 in Hodgkin's disease

The tumour necrosis factor receptor‐associated factors (TRAFs) 1 and 2 participate in the signal transduction of various members of the tumour necrosis factor receptor (TNFR) family, including TNFR1, TNFR2, CD40, CD30, and the Epstein–Barr virus (EBV)‐encoded latent membrane protein‐1 (LMP1). Previous in situ hybridization studies have demonstrated TRAF1 transcripts in the malignant cells of the majority of Hodgkins disease (HD) tumours, where the expression of TRAF1 was higher in EBV‐associated tumours than in their EBV‐negative counterparts. In order to determine whether TRAF1 and also TRAF2 were expressed at the protein level in HD and whether there was any relationship to EBV status, immunohistochemistry has been used to detect these proteins in a series of HD specimens. TRAF1 protein was detected more frequently in Hodgkin/Reed–Sternberg (HRS) cells from EBV‐positive tumours than in their EBV‐negative counterparts. This difference was statistically significant (p=0.01). In contrast, TRAF2 expression by HRS cells appeared to be independent of EBV status. Using a sequential labelling approach, co‐localization of LMP1 with either TRAF1 or TRAF2 was also demonstrated in HRS cells from EBV‐positive tumours. Copyright

Apoptosis of malignant cells in Hodgkin's disease is related to expression of the cdk inhibitor p27KIP1.

Previous results from B‐cell chronic lymphocytic leukaemia suggest that expression of p27KIP1 might be important in protection from apoptosis. Given the relevance of apoptosis to the pathogenesis of Hodgkins disease (HD), it was decided to examine the expression of p27KIP1 in relation to apoptosis in these lesions. Paraffin‐wax sections from a total of 65 histologically confirmed HD tumours were used to derive apoptotic index (AI) and DNA fragmentation index (DFI) scores, which were compared with the expression of various cell‐cycle‐regulating proteins, including p27KIP1 (p27), p21WAF1/CIP1 (p21) and cyclin D1, and with Epstein–Barr virus (EBV) status. The DFI was measured by TdT‐mediated dUTP‐FITC nick end‐labelling (TUNEL), and the AI by conventional morphology. Cells showing the typical morphology of apoptosis, together with those resembling so‐called ‘mummified’ Hodgkin/Reed–Sternberg (HRS) cells, were included in AI measurements. Increasing numbers of p27‐positive HRS cells were associated with lower levels of apoptosis in these cells, as indicated by significantly lower AI and DFI scores. There was a trend towards poorer survival in those patients with the highest numbers of p27‐positive HRS cells and with lower AI and DFI scores, but these differences were not statistically significant. p21‐positive HRS cells were significantly more frequent in those cases with lower AI scores. A similar trend was observed for p21 and DFI, although this relationship was not statistically significant. There was also a trend towards higher levels of cyclin D1 protein in HD cases with high AI and DFI values. A tendency for increasing numbers of p27‐positive and p21‐positive HRS cells in EBV‐positive cases was noted, but this relationship was not statistically significant. EBV status did not correlate with either AI or DFI scores. The results of this study suggest that p27, and possibly also p21, may be involved in protection from apoptosis in HD. Copyright

Expression of the matrix metalloproteinase 9 in Hodgkin's disease is independent of EBV status

Background—In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix. Aim—To test whether the expression of MMP-9 in Hodgkins disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkins disease cell line, L428. Methods—MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkins disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkins disease cell line, L428. Results—MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkins disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkins disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkins disease cell line. Induction of LMP-1 expression in the Hodgkins disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography. Conclusions—These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkins disease tumours and by the Hodgkins disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.

Detection of Epstein-Barr virus in archival Hodgkin's disease specimens

The Epstein-Barr virus (EBV) is associated with several malignancies, including endemic Burkitts lymphoma, undifferentiated nasopharyngeal carcinoma, post-transplant lymphoproliferative disease, and Hodgkins disease. The “gold standard” for the detection of EBV infection in clinical tissues is RNA in situ hybridisation that targets the abundantly produced Epstein-Barr virus early RNAs (EBERs).1 This approach is effective in the detection of latent EBV infection in routinely processed, paraffin wax embedded histological material, and has been widely used to analyse the association of EBV with …