EBV-associated primary CNS lymphoma occurring after immunosuppression is a distinct immunobiological entity.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV- HIV- PCNSL and 2 EBV- HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV- PCNSL. As with prior studies, EBV- HIV- PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV- PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV- HIV- PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.

Recent treatment advances in Hodgkin lymphoma: a concise review.

The majority of patients with Hodgkin lymphoma enjoy durable remissions following front-line treatment. This typically involves combination chemotherapy with or without radiotherapy. A significant minority of patients experience relapsed/refractory disease, of whom only approximately half can be 'salvaged' with conventional second-line treatments. Until recently, for those patients either failing or who are not fit for salvage, there have been few curative alternatives. Furthermore, there is a significant risk of delayed treatment complications to conventional therapies, including secondary malignancies and cardiac disease. However, novel targeted therapies are producing excellent results in clinical trials. They provide additional treatment options for those with relapsing/refractory disease; they may have potential in front-line therapy. The anti-CD30 antibody brentuximab vedotin (BV) has been tested as monotherapy and in combination in a variety of clinical settings, including in relapsed/refractory patients and as consolidative therapy following standard second-line therapy. Nivolumab and pembrolizumab, currently used in other malignancies that are known to utilise the programmed death pathway for survival, have shown outstanding results when used as single agents in heavily pre-treated (including BV refractory) patients. Individualising and adapting a patient's treatment course, whether augmenting or rationalising therapy, based on an interim positron emission tomography/computed tomography response is an important strategy currently under exploration to minimise toxicity while maximising response. Further work is needed to explore clinical and biological factors associated with improved outcomes. Knowledge of these factors combined with the movement of novel therapies into the front-line setting will enable individualised therapy to enhance clinical responses and minimise toxicities.

The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

A comprehensive analysis of the cellular and EBV-specific microRNAome in primary CNS PTLD identifies different patterns among EBV-associated tumors.

Primary central nervous system (pCNS) posttransplant lymphoproliferative disorder (PTLD) is a complication of solid organ transplantation characterized by poor outcome. In contrast to systemic PTLD, Epstein-Barr virus (EBV)-association of pCNS PTLD is almost universal, yet viral and cellular data are limited. To identify differences in the pattern of EBV-association of pCNS and systemic PTLD, we analyzed the expression of latent and lytic EBV transcripts and the viral and cellular microRNAome in nine pCNS (eight EBV-associated) and in 16 systemic PTLD samples (eight EBV-associated). Notably although 15/16 EBV-associated samples exhibited a viral type III latency pattern, lytic transcripts were also strongly expressed. Members of the ebv-miR-BHRF1 and ebv-miR-BART clusters were expressed in virtually all EBV-associated PTLD samples. There were 28 cellular microRNAs differentially expressed between systemic and pCNS PTLD. pCNS PTLD expressed lower hsa-miR-199a-5p/3p and hsa-miR-143/145 (implicated in nuclear factor kappa beta and c-myc signaling) as compared to systemic PTLD. Unsupervised nonhierarchical clustering of the viral and cellular microRNAome distinguished non-EBV-associated from EBV-associated samples and identified a separate group of EBV-associated pCNS PTLD that displayed reduced levels of B cell lymphoma associated oncomiRs such as hsa-miR-155, -21, -221 and the hsa-miR-17-92 cluster. EBV has a major impact on viral and cellular microRNA expression in EBV-associated pCNS PTLD.

The presence of KIR2DS5 confers protection against adult immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder of unknown aetiology, characterised by an isolated low platelet count in the absence of other identifiable causes. Genes influencing activation of the immune system have been identified as influencing predisposition. Killer cell immunoglobulin-like receptors (KIR) control T-cell and natural killer (NK) cell function via inhibitory and activating signalling pathways. The inhibitory KIR2DL3, KIR3DL2 and KIR3DL1 are up-regulated in the T-cells of patients with ITP in remission relative to those with active disease, and an association of KIR2DS2 and KIR2DL2 with ITP has also been reported. No comprehensive KIR analysis in ITP has been reported. We performed genotyping of all currently known KIR genes using sequence specific primer polymerase chain reaction (SSP-PCR) on a cohort of 83 adult patients with ITP (chronic/persistent or relapsed primary ITP identified by defined criteria) and 106 age matched healthy white volunteers. Non-white patients were not included in the analysis. There was an over-representation of KIR2DS3 (known to be in linkage disequilibrium with KIR2DS2 and 2DL2) and under-representation of KIR2DS5 (also protective against other immune mediated disorders) in adult ITP [odds ratio (OR) = 0.16, confidence interval (CI) 0.08-0.32, P < 0.001]. By multivariable binary logistic regression to adjust for age, sex and the effects of other KIR genes, the presence of KIR2DS2/2DL2 with KIR2DS5 abrogated the risk of KIR2DS2/2DL2 and the protective benefit of KIR2DS5. Further studies are required to establish the mechanistic basis for these observations and their potential impact on ITP therapy.

Primary CNS posttransplant lymphoproliferative disease (PTLD): an international report of 84 cases in the modern era.

We performed a multicenter, International analysis of solid organ transplant (SOT)-related primary central nervous system (PCNS) posttransplant lymphoproliferative disease (PTLD). Among 84 PCNS PTLD patients, median time of SOT-to-PTLD was 54 months, 79% had kidney SOT, histology was monomorphic in 83% and tumor was EBV+ in 94%. Further, 33% had deep brain involvement, 10% had CSF involvement, while none had ocular disease. Immunosuppression was reduced in 93%; additional first-line therapy included high-dose methotrexate (48%), high-dose cytarabine (33%), brain radiation (24%) and/or rituximab (44%). The overall response rate was 60%, while treatment-related mortality was 13%. With 42-month median follow-up, three-year progression-free survival (PFS) and overall survival (OS) were 32% and 43%, respectively. There was a trend on univariable analysis for improved PFS for patients who received rituximab and/or high-dose cytarabine. On multivariable Cox regression, poor performance status predicted inferior PFS (HR 2.61, 95% CI 1.32-5.17, p = 0.006), while increased LDH portended inferior OS (HR 4.16, 95% CI 1.29-13.46, p = 0.02). Moreover, lack of response to first-line therapy was the most dominant prognostic factor on multivariable analysis (HR 8.70, 95% CI 2.56-29.57, p = 0.0005). Altogether, PCNS PTLD appears to represent a distinct clinicopathologic entity within the PTLD spectrum that is associated with renal SOT, occurs late, is monomorphic and retains EBV positivity.

Homozygous FCGR3A-158V alleles predispose to late onset neutropenia after CHOP-R for diffuse large B-cell lymphoma.

BACKGROUND: Recent reports suggest genetic polymorphisms influence susceptibility to rituximab-induced late-onset neutropenia (LON), which in turn may be a predictor of good outcome in B-cell lymphoma. AIMS: We report the largest study to date assessing FCGR3A-V158F polymorphisms in diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide/hydroxydaunorubicin/Oncovin (vincristine)/prednisone/rituximab (CHOP-R). The influence of C1qA-A276G polymorphisms in DLBCL, and the impact of both polymorphisms on susceptibility to LON and outcome were also examined. METHODS: 115 DLBCL patients treated with CHOP-R were compared with 105 healthy White people controls with regards to FCGR3A-V158F and C1qA-A276G polymorphisms. LON incidence and event-free and overall survival (EFS and OS) were analysed for linkage to either polymorphism. RESULTS: The FCGR3A-V158F but not the C1qA-A276G polymorphism influenced the risk of developing LON. 50% of FCGR3A-158V/V patients experienced LON. In contrast, only 7% V/F and 2% F/F experienced LON. The FCGR3A-158V/V genotype was associated with LON compared with V/F (P = 0.028) and F/F genotypes (P = 0.005). Although no patients with either LON or FCGR3A-158V homozygosity relapsed compared with 33% FCGR3A-158F/F and 21% non-LON, this did not translate into improved EFS or OS. CONCLUSIONS: Polymorphic analysis may be a predictive tool to identify those at high risk of LON. Prospective studies are required to establish definitively if LON or FCGR3A-158V/V genotype influences outcome.

A novel immunodeficiency disorder characterized by genetic amplification of interleukin 25.

Many primary immunodeficiency disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of disease. Here, we have used a whole-genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper T cell bias in the immunodeficiency case provides a mechanistic explanation for recurrence of infections by pathogens met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses.

Epstein-Barr Virus-related post-transplant lymphoproliferative disorders: pathogenetic insights for targeted therapy.

Post-transplant lymphoproliferative disorder (PTLD) is a spectrum of major, life-threatening lymphoproliferative diseases occurring in the post-transplant setting. The majority of PTLD is of B-cell origin and is associated with several risk factors, the most significant being Epstein-Barr virus (EBV) infection. EBV's in vitro transforming abilities, distinctive latency, clonality within the malignant cells and response to targeted therapies implicate a critical role in the biology of PTLD. This minireview focuses on EBV-related PTLD pathogenesis, in particular the interplay between aspects of the EBV life cycle and latency with nonviral factors resulting in the wide spectrum of histology and clinical presentations encountered in PTLD. With the increased prevalence of transplantation a rise in the incidence of PTLD may be expected. Therefore the importance of laboratory and animal models in the understanding of PTLD and the development of novel therapeutic approaches is discussed.

Sodium valproate in combination with ganciclovir induces lysis of EBV-infected lymphoma cells without impairing EBV-specific T-cell immunity.

Histone deacytelase inhibitiors (HDACi) represent a new class of anti-lymphoma therapeutics. Data in the clinical setting regarding on- and off-target effects of these agents are limited. Epstein-Barr virus (EBV)-positive lymphomas represent a highly defined system in which to make these observations. We present a case of a patient with multiple relapsed EBV-positive Diffuse Large B-cell Lymphoma that was chemo-refractory to anthracylcines, alkylating agents and rituximab. Treatment was commenced with the HDACi sodium valproate (VPA) in combination with the anti-viral nucleoside analogue ganciclovir (GCV). Therapy resulted in detectable cell-free unencapsulated circulating EBV-DNA providing supportive evidence for the first-time that lysis of virus infected lymphoma cells is induced using this therapeutic combination. EBV-specific CD8+ effector T-cell immunity was not impaired by VPA/GCV. Although GCV/VPA was insufficient to induce clinical remission, our data furthers the rationale that more potent HDAC inhibitors such as butyrate or gemcitabine together with GCV, perhaps in combination with chemotherapy, should be further investigated as therapy in relapsed/refractory EBV-positive lymphomas.

Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders.

Adoptive immunotherapy using autologous Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes (auto-CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto-CTL remains constrained. Generation is time-consuming, and auto-CTL cannot be established in patients treated with the B-cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo-CTL) offers immediate accessibility. Allo-CTL has previously shown efficacy in "early" polyclonal- PTLD. We treated three patients with aggressive, advanced monoclonal-PTLD following solid-organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide-specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor-derived tumor targeted EBV-specific CTL; the other died of non-PTLD related pathology. In the third patient, autopsy demonstrated homing of allo-CTL at the tumor site. Larger prospective studies of EBV-specific allo-CTL in PTLD are warranted.

Correlation of T-cell immune response with spontaneous resolution and subsequent relapse of Hodgkin's lymphoma.

To our knowledge, there has been no report of spontaneous regression in a non-immunocompromised adult with classical Hodgkin's lymphoma (HL) in the absence of chemotherapy. We describe spontaneous regression and subsequent relapse of Epstein - Barr virus (EBV)-positive HL in an otherwise healthy male adult. The clinical course was associated with an increase in regulatory T-cell markers within the peripheral blood and diseased lymph node at the time of relapse and with a concomitant reduction in cellular immunity against relevant EBV latent membrane protein tumor-associated antigens. Our findings are in keeping with previous observations that implicate impaired cellular immunity in the immunopathogenesis of EBV-positive HL.

Antibody responses to vaccinations given within the first two years after transplant are similar between autologous peripheral blood stem cell and bone marrow transplant recipients.

As a consequence of the significantly larger inoculum of lymphoid cells present in peripheral blood stem cell (PBSC) harvests compared to bone marrow (BM), it is possible that autoPBSCT recipients may have an earlier and*or enhanced response to vaccines. Until data to confirm this become available, the European Blood and Marrow Transplantation Association (EBMT) recommend that all transplant recipients be immunized in the same way regardless of stem cell source. We performed a prospective study comparing serological responses to influenza, pneumococcal polysaccharide and tetanus toxoid vaccines between autoPBSCT with autoBMT recipients. Antibody responses in sibling HLA-matched allogeneic BMT (alloBMT) survivors were also evaluated. All vaccines were administered within the first 2 years after stem cell transplantation. Fifty patients were enrolled. The time of vaccination after transplant was similar between autoPBSCT (mean 11 months for each vaccine) and autoBMT recipients (mean 12 months except 13 months for tetanus toxoid) (P = NS). Serological responses were poor and no significant difference in response to any of the vaccines used was seen between the three transplant cohorts. We provide no evidence that current EBMT guidelines be modified. Large prospective vaccine studies are needed to address the issue more fully.

A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations.

S-Methylation by thiopurine methyltransferase (TPMT) is an important route of metabolism for the thiopurine drugs. About one in 300 individuals are homozygous for a TPMT mutation associated with very low enzyme activity and severe myelosuppression if treated with standard doses of drug. To validate the use of molecular genetic techniques for the detection of TPMT deficiency, we have determined red blood cell TPMT activity in 240 adult blood donors and 55 normal children. Genotype was determined by restriction fragment length analysis of polymerase chain reaction products in a cohort of 79 of the blood donors and five cases of azathioprine-induced myelosupression, and this confirmed a close relationship between genotype and phenotype. In 17 of the 24 cases in which mutations were found, DNA was also available from remission bone marrow. In one of these cases, DNA from the remission marrow sample indicated the presence of a non-mutated allele that had not been seen in the blast DNA sample obtained at presentation. These results indicate that polymerase chain reaction-based assays give reliable and robust results for the detection of TPMT deficiency, but that caution should be exercised in relying exclusively on DNA obtained from lymphoblasts in childhood leukaemia.

The minimum CD34 threshold depends on prior chemotherapy in autologous peripheral blood stem cell recipients.

We analysed 57 patients with non-myeloid malignancies who received a non-purged autologous PBSCT. All had similar mobilisation and conditioning regimens. A high prior chemotherapy score and the number of chemotherapy lines used (P = 0.015 and P = 0.01, respectively) were adverse predictors of CD34 cell yields. Lower CD34 values (P = 0.002) were seen in patients treated with potent stem cell toxins (BCNU, melphalan, CCNU and mustine), designated toxicity factor 4 agents (TF4). All patients infused with grafts containing CD34 cell doses between 1.0 and 2.0 x 10(6)/kg (range 1.25-1.90) engrafted by day 51. The only variable associated with slow platelet recovery was exposure to TF4 (P = 0.007). The majority of patients with CD34 >1.0 x 10(6)/kg achieved rapid and sustained engraftment and the only predictive factor of delayed recovery is prior exposure to stem cell toxins. Potential PBSCT candidates should if possible avoid first line and salvage chemotherapy containing TF4 drugs. We therefore advocate a minimum CD34 threshold of >1.0 x 10(6)/kg in patients without extensive prior chemoradiotherapy, and > or = 2.0 x 10(6)/kg in all other patients.

A trend towards an increased incidence of chronic graft-versus-host disease following allogeneic peripheral blood progenitor cell transplantation: a case controlled study.

Allogeneic peripheral blood progenitor cell transplantation (alloPBPCT) is increasingly used as an alternative to bone marrow transplantation (alloBMT). Early data suggest that the incidence and severity of acute graft-versus-host disease (GVHD) following alloPBPCT is no higher than that seen with alloBMT, despite the increased number of cytotoxic T cells infused with mobilised blood. We compared 12 patients undergoing alloPBPCT with 12 well-matched alloBMT controls. All patients received identical GVHD prophylaxis. No T cell depletion or CD34 purification was performed. Median engraftment times for neutrophils >0.5 x 10(9)/l and platelets >20 x 10(9)/l were 14 and 12 (alloPBPCT) and 21 and 23 days (alloBMT), respectively (P = 0.0035 and P = 0.002). There was no difference in antibiotic requirements (P = 0.83), platelet support (P = 0.59) or days in hospital (P = 0.51). After alloPBPCT, five patients developed > or =grade II acute GVHD vs five patients after alloBMT (P = 0.99). There was one death (alloBMT) at 100 days and three at 1 year (all due to relapse). There was one death at 100 days with alloPBPCT, and 11 patients remain alive (range 9-21 months) to date. Chronic GVHD occurred in five patients in the PBPC arm and one patient in the BM arm (P = 0.14). This case-controlled analysis indicates that alloPBPCT results in more rapid engraftment kinetics but in no significant difference in transplant-related morbidity or mortality. There is no difference in the incidence of acute GVHD. However, there is a trend towards increased incidence of chronic GVHD in patients allografted with PBPC. Prospective randomised trials are required to determine further the role of alloPBPCT.

An unusual case of hypersomnolence syndrome.

Hypersomnolence syndrome has been well described in patients with acute lymphoblastic leukaemia (ALL) following cranial irradiation. It is most frequently seen in children. In adults it is clinically very mild. We describe a case of a particularly severe and prolonged post radiation hypersomnolence syndrome in an adult with ALL. The hypersomnolence syndrome is discussed in the context of side effects of central nervous system directed therapy in elderly patients with ALL.

A case of transfusion-acquired hepatitis C.

The Blood Transfusion Service introduced screening for Hepatitis C antibody (HCV) in September 1991. This is done by second generation enzyme linked immunosorbent assay (ELISA) tests. We present a case of post-transfusion hepatitis C hepatitis in a patient with myeloma. Infection was acquired before screening was introduced. Both the patient and the infected blood donor were diagnosed using ELISA assays and the polymerase chain reaction (PCR). In this way we prevented the blood donor from spreading the virus via subsequent blood donations. There were some interesting discrepancies in the HCV assays. Blood samples, when tested by different methods, gave both positive and negative results. The results also varied according to when the blood samples to be tested were taken. The case illustrates the importance of confirming positive results and that no single laboratory test is entirely satisfactory in diagnosing HCV infection.