EBV-associated Lymphoproliferative Disorders Research Articles

Abstract SY41-04: Immune suppression by T cell exclusion in pancreatic cancer

Abstract In 1960, Klein and colleagues found that when mice developed primary methylcholanthrene-induced sarcomas, they also developed an acquired immune resistance mediated by lymph node cells to a secondary challenge comprised of cancer cells derived from the primary tumor. The paradoxical and critical finding of the study was that this anti-cancer immune response did not control the growth of the primary tumor, despite its ability to prevent the establishment of a secondary tumor comprised of cancer cells derived from the primary tumor. The primary tumor evaded immune control by establishing an immune suppressive tumor microenvironment (TME). Unambiguous evidence for the inability in humans of a systemic immune response to eliminate immunogenic cancer cells was provided by Boon's studies thirty years later of the antigens that elicit specific CD8+ T cell responses in melanoma patients. Cloned CD8+ T cells from a melanoma patient were used to identify the antigen expressed by that patient's cancer: MAGE-A1. The explicit demonstration of the co-existence of a progressing melanoma with melanoma-specific T cells in this patient implicitly raised the question of why the T cells did not control the growth of the cancer. Moreover, Rosenberg and colleagues reported evidence of disease recurrence in melanoma patients despite very high levels of vaccine-induced circulating T cells and no evidence of antigen loss. The discovery of melanoma-specific T cells in patients led not only to vaccine strategies to increase the frequency of cancer-specific T cells in patients, but also to a more direct means for accomplishing this goal, that of adoptively transferring large numbers of in vitro expanded tumor-infiltrating lymphocytes. This approach has shown some efficacy, but has not had the dramatic success of adoptively transferring virus-specific CD8+ T cells to immunodeficient bone marrow transplant recipients with CMV infection or EBV-associated lymphoproliferative disorders. Differences in the microenvironments of virally infected tissues and cancers must account for these distinct outcomes. The more recent strategy of enhancing the function of effector T cells by targeting CTLA-4 and PD-1, immunoregulatory receptors on T cells, has been successful in subsets of patients with melanoma, NSCLC, bladder cancer, and renal cell cancer. Nevertheless, it has become apparent that even if these T cell checkpoint antagonists overcome some of the immune suppressive effects of the TME, there must be other, more fundamental inhibitory reactions in the TME to explain why most patients, especially those with colorectal cancer (CRC), ovarian cancer, prostate cancer, and pancreatic ductal adenocarcinoma (PDAC) do not exhibit objective responses to these therapies. A clue to the nature of this dominant immune suppression mediated by the TME comes from studies that have examined the spatial relationship of CD8+ effector T cells to cancer cells in three of the tumors that do not respond to anti-PD-1/anti-PD-L1: CRC, ovarian cancer and PDA. In each of these cancers, CD8+ T cells are excluded from cancer cell nests, and correlates with a poor long term clinical outcome. Thus, the TME can limit the capacity of T cells to accumulate amongst cancer cells, and one must conclude that until this problem is overcome, the full potential of other approaches to T cell-mediated tumor immunotherapy will not be realized. Recent studies have begun to explain how this form of immune suppression is mediated. Pre-clinical studies in mouse models of cancer now implicate the major stromal cell types of the TME, cancer-associated fibroblasts (CAFs) and myelomonocytic cells as being responsible for restricting the accumulation of T cells in the vicinity of cancer cells. As would be predicted, overcoming this restriction has revealed the anti-tumor effects of T cell checkpoint antagonists that had been ineffective when administered as monotherapy. I will discuss my group's recent studies of how the CAF mediates the exclusion of T cells in an autochthonous moue model of PDAC. The discovery the mechanism of exclusion has led to a potential therapeutic approach that revealed the existence of a previously unsuspected spontaneous anti-cancer immune response in these mice, and uncovered the efficacy of the anti-PD-L1 T cell checkpoint antagonist. Citation Format: Douglas T. Fearon. Immune suppression by T cell exclusion in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY41-04. doi:10.1158/1538-7445.AM2015-SY41-04

A patient with elderly-onset atypical hydroa vacciniforme with an indolent clinical course

Hydroa vacciniforme (HV) is a rare photodermatosis that mainly affects children and manifests as vesiculopapular eruptions in sun-exposed areas without systemic symptoms. HV-like lymphoma (HVLL) is one of the Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD) of childhood. Its diagnosis is based on monoclonal T-cell proliferation; however, its degree of malignancy is controversial owing to its variable prognosis. Elderly-onset cases of these diseases are extremely rare, and the clinical features remain unknown. It has been shown that late onset is closely associated with a severe phenotype in EBV-associated LPD including atypical HV. Here we describe a case of elderly-onset atypical HV accompanied by T-cell monoclonality, but with a very indolent clinical course. Our patient indicates a possible case with elderly-onset atypical HV manifesting a favourable course, and that T-cell monoclonality and age of onset cannot always predict the disease severity, and highlights the difficulty of prognosis prediction in elderly-onset atypical HV.

TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation

The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

Lymphomatoid granulomatosis--a single institute experience: pathologic findings and clinical correlations.

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disorder. It is hypothesized that these patients have dysregulated immune surveillance of EBV. We reviewed the biopsies of 55 patients with LYG who were referred for a prospective trial at the National Cancer Institute (1995 to 2010) and evaluated the histologic, immunohistochemical, in situ hybridization, and molecular findings of these biopsies in conjunction with clinical information. Grading of the lesions was based on morphologic features and the number of EBV-positive B cells. The median age was 46 years (M:F 2.2:1). Clinically, all patients had lung involvement (100%), with the next most common site being the central nervous system (38%). No patient had nodal or bone marrow disease. All patients had past EBV exposure by serology but with a low median EBV viral load. We reviewed 122 biopsies; the most common site was lung (73%), followed by skin/subcutaneous tissue (17%); other sites included kidney, nasal cavity, gastrointestinal tract, conjunctiva, liver, and adrenal gland. Histologically, the lesions showed angiocentricity, were rich in T cells, had large atypical B cells, and were positive for EBV. Grading was performed predominantly on the lung biopsy at diagnosis; they were distributed as follows: LYG grade 1 (30%), grade 2 (22%), and grade 3 (48%). Necrosis was seen in all grades, with a greater degree in high-grade lesions. Immunoglobulin gene rearrangement studies were performed, and a higher percentage of clonal rearrangements were seen in LYG grade 2 (50%) and grade 3 (69%) as compared with grade 1 (8%). LYG is a distinct entity that can usually be differentiated from other EBV-associated B-cell lymphoproliferative disorders on the basis of the combination of clinical presentation, histology, and EBV studies. Grading of these lesions is important because it dictates the treatment choice.

Drug free remission after steroid-dependent disappearance of lymphoproliferative disorder in rheumatoid arthritis patient treated with TNF-alpha blockade: case study.

IntroductionTNF-α inhibitors plus MTX appear to have benefit in the longer-term reduction of RA. Boolean long-term remission under drug-free conditions is rare. The therapeutic mechanism and the factor of predicting response have not been clarified yet.Case descriptionA 24-year-old female rheumatoid arthritis (RA) patient, who once attained complete remission (CR) with the combination therapy with tumor necrosis factor alpha (TNF-alpha) inhibitor adalimumab (ADA) and methotrexate (MTX), showed the occurrence of Epstain- Barr virus (EBV)-associated lymphoproliferative disorder (LPD). Pulse treatment with methylprednisolone after the termination of anti TNF-α therapy resulted in the remission of EBV-associated LPD. The administration of prednisolone (PSL) was tapered off after the improvement of clinical symptoms and laboratory data. The patients achieved drug-free 12 months after urgent hospitalization and delivered healthy baby 2 years after hospital discharge. She has been complete drug-free Boolean remission for 5 years.Discussion and evaluationThe purpose of this brief case is report that we experienced the remission of LPD after CR with combined therapy with ADA and MTX. We believe this case report will be one of the paths for unveiling the pathogenesis and improving the treatment for RA.ConclusionsWe believe this case report will be one of the paths for unveiling the pathogenesis and improving the treatment for RA.

Overexpression of Activation-Induced Cytidine Deaminase in MTX- and Age-Related Epstein-Barr Virus-Associated B-Cell Lymphoproliferative Disorders of the Head and Neck.

Recent research has shown that activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Such aberrant AID expression is seen in B-cell leukemia/lymphomas, including Burkitt lymphoma which is associated with c-myc translocation. Moreover, Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) increases genomic instability through early growth transcription response-1 (Egr-1) mediated upregulation of AID in B-cell lymphoma. However, few clinicopathological studies have focused on AID expression in lymphoproliferative disorders (LPDs). Therefore, we conducted an immunohistochemical study to investigate the relationship between AID and LMP-1 expression in LPDs (MTX-/Age-related EBV-associated), including diffuse large B-cell lymphomas (DLBCLs). More intense AID expression was detected in LPDs (89.5%) than in DLBCLs (20.0%), and the expression of LMP-1 and EBER was more intense in LPDs (68.4% and 94.7%) than in DLBCLs (10.0% and 20.0%). Furthermore, stronger Egr-1 expression was found in MTX/Age-EBV-LPDs (83.3%) than in DLBCLs (30.0%). AID expression was significantly constitutively overexpressed in LPDs as compared with DLBCLs. These results suggest that increased AID expression in LPDs may be one of the processes involved in lymphomagenesis, thereby further increasing the survival of genetically destabilized B-cells. AID expression may be a useful indicator for differentiation between LPDs and DLBCLs.

EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management.

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus, affecting >90% of the adult population. EBV targets B-lymphocytes and achieves latent infection in a circular episomal form. Different latency patterns are recognized based on latent gene expression pattern. Latent membrane protein-1 (LMP-1) mimics CD40 and, when self-aggregated, provides a proliferation signal via activating the nuclear factor-kappa B, Janus kinase/signal transducer and activator of transcription, phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase pathways to promote cellular proliferation. LMP-1 also induces BCL-2 to escape from apoptosis and gives a signal for cell cycle progression by enhancing cyclin-dependent kinase 2 and phosphorylation of retinoblastoma (Rb) protein and by inhibiting p16 and p27. LMP-2A blocks the surface immunoglobulin-mediated lytic cycle reactivation. It also activates the Ras/PI3K/Akt pathway and induces Bcl-xL expression to promote B-cell survival. Recent studies have shown that ebv-microRNAs can provide extra signals for cellular proliferation, cell cycle progression and anti-apoptosis. EBV is well known for association with various types of B-lymphocyte, T-lymphocyte, epithelial cell and mesenchymal cell neoplasms. B-cell lymphoproliferative disorders encompass a broad spectrum of diseases, from benign to malignant. Here we review our current understanding of EBV-induced lymphomagenesis and focus on biology, diagnosis and management of EBV-associated B-cell lymphoproliferative disorders.

Lymphoproliferative disorders in inflammatory bowel disease patients on immunosuppression: Lessons from other inflammatory disorders.

Immunosuppressive agents, such as thiopurines, methotrexate, and biologics, have revolutionized the treatment of inflammatory bowel disease (IBD). However, a number of case reports, case control studies and retrospective studies over the last decade have identified a concerning link between immunosuppression and lymphoproliferative disorders (LPDs), the oncological phenomenon whereby lymphocytes divide uncontrollably. These LPDs have been associated with Epstein-Barr virus (EBV) infection in which the virus provides the impetus for malignant transformation while immunosuppression hampers the immune system's ability to detect and clear these malignant cells. As such, the use of immunosuppressive agents may come at the cost of increased risk of developing LPD. While little is known about the LPD risk in IBD, more is known about immunosuppression in the post-transplantation setting and the development of EBV associated post-transplantation lymphoproliferative disorders (PTLD). In review of the PTLD literature, evidence is available to demonstrate that certain immune suppressants such as cyclosporine and T-lymphocyte modulators in particular are associated with an increased risk of PTLD development. As well, high doses of immunosuppressive agents and multiple immunosuppressive agent use are also linked to increased PTLD development. Here, we discuss these findings in context of IBD and what future studies can be taken to understand and reduce the risk of EBV-associated LPD development from immunosuppression use in IBD.

Hydroa Vacciniforme-Like Eruption Associated with Epstein-Barr Virus Infection in an Older Adult.

Dear Editor: A 70-year-old woman presented with a 2-week history of facial eruption. She was treated with corticosteroid, on the basis of a diagnosis of allergic contact dermatitis, which improved her condition. However, it recurred after 1 year. She did not complain about any subjective photosensitivity or hypersensitivity to mosquito bites. Physical examination revealed multiple erythematous papulopustules with ulcers and crusts on the face, accompanied by edema on both cheeks and eyelids (Fig. 1). She was otherwise healthy, except for having had cryptogenic liver cirrhosis 7 years previously. Histopathological examination was performed on the forehead. Small to medium-sized atypical lymphocytes infiltrated around the perivascular and periadnexal spaces in the entire dermis (Fig. 2A, B). These atypical cells were positive for CD2, CD3, CD5, and T-cell intercellular antigen-1, and focally positive for CD8 (Fig. 2C, D). Some reactive cells were positive for CD4, and none of the tumor cells expressed CD56, CD20, or CD30. Additionally, the infiltrating cells were positive for Epstein-Barr virus (EBV)-encoded RNA in in situ hybridization (Fig. 2E). Laboratory tests revealed mild leukopenia (white blood cell count, 2.4×103/µl), mildly elevated liver enzyme levels (aspartate aminotransferase and alanine aminotransferase levels of 100 and 98 U/L, respectively), and elevated lactate dehydrogenase levels of 233 U/L (reference range, 100~200 U/L). The serum was positive for EBV-capsid antigen immunoglobulin (Ig) G and EBV early antigen IgG and IgM. Quantification of EBV by using real-time polymerase chain reaction (PCR) revealed 200.5 copies/µl. Ultraviolet A provocation test on the back did not provoke symptoms. PCR revealed no monoclonal T-cell receptor (TCR)-gamma gene rearrangement. Except for hepatosplenomegaly, no other evidence of systemic involvement was found. Finally, a diagnosis of hydroa vacciniforme (HV)-like eruption was concluded. At 1-year follow-up, new lesions continually arose with the application of topical corticosteroid. Fig. 1 Multiple erythematous crusted papulopustules are observed, with ulcers on both cheeks and the forehead. Arrows indicate the biopsy sites: one pustule and one papule on the forehead. Fig. 2 Small to medium-sized atypical lymphoid cells infiltrate the perivascular and periadenexal areas throughout the entire dermis (hematoxylin and eosin staining; A: ×40, B: ×200). The atypical cells are positive for (C) CD3, focally positive ... HV-like eruption, or atypical HV, occurs predominantly in children and adolescents in East Asia and Latin America. It is characterized by an edematous, crusted papulovesicular eruption similar to HV1,2,3,4. Unlike HV, however, it could also occur in non-sun-exposed areas, and is accompanied by fever, lymphadenopathy, or hepatosplenomegaly1,2,3,4. Because HV-like lymphoma could occur in non-sun-exposed areas and is accompanied by systemic symptoms, it can be confused with HV-like eruption. However, as recent studies revealed that most lesions contain monoclonal TCR rearrangement, the World Health Organization classification system newly categorized it as one of the EBV-associated lymphoproliferative disorders of childhood in 20085. These three disease categories were related to EBV infection and predominantly occur in children and adolescents. However, HV-like eruption is occasionally observed in adults aged older than 60 years: one case was that of a 65-year-old woman with orogenital ulcer, whose symptoms were well controlled with corticosteroid and famciclovir3, whereas the other was a case of a 74-year-old man whose condition deteriorated rapidly and who died within 3 months of diagnosis4. Thus, we report a case of HV-like eruption in an older adult manifesting chronic feature. Furthermore, the disease entity needs to be clarified and the disease properties need to be investigated in future studies.

Determination of Optimal Target Antigen and Immune Modulatory Approaches to Improve and Standardize Epstein-Barr Virus-Specific Adoptive T Cell Therapy

Epstein-Barr Virus (EBV) is a ubiquitous gamma herpesvirus that infects lymphocytes and epithelial cells, establishing lifelong latency. Immunosuppression is associated with a transition from a quiescent, latent EBV infection to either a lytic or active latent life cycle, which involves transformation of memory B cells to rapidly growing lymphoblastoid cell lines (LCLs). Due to the immunogenicity of EBV viral proteins, cells expressing most latent or lytic peptides are eradicated through immune surveillance mechanisms. EBV-associated lymphoproliferative disorders (LPDs) arise in immunocompromised individuals and are facilitated by the expression of the same viral proteins that are expressed in the immune tolerant environment. Furthermore, EBV-LPDs acquire various mechanisms to suppress immune function, such as expression of the programmed death ligand (PD-L1), CTLA-4, amino acid depletion, and propagation of suppressive immune cell subsets, including tumor-associated macrophages (TAMs) that attenuate T cell receptor signaling and cytokine production. These immunosuppressive mechanisms increase the risk of acquiring EBV viremia and can facilitate the development of LPDs. In other work reported by our group (Patton et al, ASH abstract 2014), a TAM-like population has recently been discovered to spontaneously expand in peripheral blood mononuclear cell (PBMC)/LCL co-cultures. These TAM-like macrophages are capable of potent cytotoxicity against EBV-specific T cells, thus novel strategies to counteract this negative regulatory network are needed.One effective strategy developed to reestablish EBV-specific immunity is adoptive T cell therapy. However, the optimal combination of target antigens required to protect against EBV-LPD is not well characterized. It remains plausible that expansion of cytotoxic and/or helper T cell populations specific for multiple EBV antigens will improve the efficiency of the T cell response. The suppression of negative immune regulators may further enhance the effectiveness of the adoptive therapies to provide optimal control of viremia and effectively treat EBV-driven malignancies.Here, we generated EBV-specific T cell preparations by culturing PBMCs supplemented with interleukins 4 and 7 (IL4, IL7) in the presence of latent (EBNA1, EBNA3, LMP1, LMP2) or lytic (BZLF1, BRLF, BMLF1, BMRF1) PepMixes (complete protein-spanning pools of overlapping peptides). Cells were separated into two groups, a test and expansion group, to determine optimal ex vivo expansion. Activity of T cells was measured by direct flow-based cytotoxicity against LCL targets and interferon gamma (IFNγ) production via Enzyme-Linked Immuno Spot (ELISpot).Preliminary ELISpot assays from two donors in the test group showed that BZLF1-specific T cells produced as much as 34-fold more IFNγ in comparison to other EBV antigens (standardized to a negative control). T cells produced in culture with a peptivator PepMix, a collection of 13 latent and lytic peptide pools, on average exhibited the second highest levels of IFNγ release, expressing as much as 1.4-fold more IFNγ than the next highest antigen-specific response, depending on the donor. Additionally, LMP1 produced as much as 24-fold higher levels of IFNγ. Therefore, given the heterogeneous EBV viral gene expression profiles and variation of immune dominance across human leukocyte antigen (HLA) types, use of polyclonal immunogen-specific T cells may be more effective in improving survival and long-term protection from EBV-driven complications via improved memory T cell surveillance. Additional ex vivo work is currently underway examining combinations of lytic and latent antigens based on immunodominance analysis across HLA types. Finally, use of immune modulatory mechanisms to address newly discovered TAM-like populations contributing to reduced immune effector function against EBV-LPD are currently being evaluated. We are testing strategies to promote PD-L1 and CTLA-4 blockade, inhibition of amino acid depletion, and down regulation of regulatory T cells, to allow for enhanced expansion of antigen-specific T cell populations. Confirmation of these hypotheses will be directed toward developing streamlined methods for efficient, rapid, and personalized T cell preparation that can be standardized for translation into clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Epstein-Barr virus-associated posttransplant lymphoproliferative disorder after allogeneic stem cell transplantation.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder is an increasingly life-threatening complication after allogeneic stem cell transplantation with the use of more complex transplant procedures. Reduced intensity conditioning regimens in combination with in-vivo or ex-vivo T-cell depletion are particularly important risk factors. Prospective monitoring of EBV viremia by real-time quantitative polymerase-chain reaction (PCR) should be performed after high-risk allogeneic stem cell transplantation. However, lack of standardization and concerns about sensitivity and low positive-predictive value challenge the interpretation of PCR monitoring. Preemptive treatment is feasible and can reduce EBV-related mortality but may lead to overtreatment in some patients. Readily available rituximab and methods of adoptive transfer of T-cells are valuable tools. Rituximab is probably the most attractive agent showing the most robust data in this setting. Rituximab seems to offer a good balance between efficacy and toxicity for the treatment of established EBV-associated posttransplant lymphoproliferative disorder. But most often there is a need to combine with adoptive immunotherapy with T-cells to maintain long-term disease control, with either simple unmanipulated donor lymphocyte infusion or more specific and complex adoptive EBV-specific cytotoxic T-cells. EBV-associated posttransplant lymphoproliferative disorder can often be prevented or treated, especially in earlier stages. The specific role and timing of the different treatment strategies need to be defined.

Improvement in Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation upon Administration of Mesenchymal Stem Cells from Third-Party Donors: A Pilot Prospective Study

Poor graft function (PGF) is a refractory complication that occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the present study, we prospectively evaluated the efficacy and safety of mesenchymal stem cells (MSCs) expanded from the bone marrow of a third-party donor to patients with PGF after allo-HSCT. Twenty patients with PGF (7 with primary and 13 with secondary PGF) received MSCs (1 × 10(6)/kg) one to three times at 28-day intervals. Seventeen patients were responsive to MSCs, whereas three were not. Within the first 100 days after MSC treatment, 13 patients developed 20 episodes of infection. Moreover, five patients experienced cytomegalovirus-DNA viremia, and seven experienced Epstein-Barr virus (EBV)-DNA viremia within the first 100 days after MSC treatment; three of the latter developed EBV-associated posttransplant lymphoproliferative disorders (PTLD) within the follow-up period. Grade II acute graft-versus-host disease (GVHD) occurred in one patient, and local chronic GVHD occurred in two patients after receiving MSC treatment, including one acute GVHD and one chronic GVHD, respectively, after accepting donor lymphocyte infusions due to PTLD. After a follow-up period of an average of 508 days (range 166-904 days) posttransplantation, 11 patients died. No short-term toxic side effects were observed after MSC treatment. Two patients experienced leukemic relapse. With the exception of three patients with PTLD, no secondary tumors occurred. These results indicate that MSCs derived from the bone marrow of a third-party donor are beneficial in the treatment of both primary and secondary PGF that develops after allo-HSCT. However, additional studies will be needed to determine whether such treatment might increase the risk of EBV infection and reactivation or the development of EBV-associated PTLD.

Combination of SAHA and bortezomib up-regulates CDKN2A and CDKN1A and induces apoptosis of Epstein-Barr virus-positive Wp-restricted Burkitt lymphoma and lymphoblastoid cell lines.

Epstein-Barr virus (EBV) latent proteins exert anti-apoptotic effects on EBV-transformed lymphoid cells by down-regulating BCL2L11 (BIM), CDKN2A (p16(INK4A) ) and CDKN1A (p21(WAF1) ). However, the potential therapeutic effects of targeting these anti-apoptotic mechanisms remain unexplored. Here, we tested both in vitro and in vivo effects of the combination of histone deacetylase (HDAC) and proteasome inhibitors on the apoptosis of six endemic Burkitt lymphoma (BL) lines of different latency patterns (types I and III and Wp-restricted) and three lymphoblastoid cell lines (LCLs). We found that the combination of HDAC and proteasome inhibitors (e.g. SAHA/bortezomib) synergistically induced the killing of Wp-restricted and latency III BL and LCLs but not latency I BL cells. The synergistic killing was due to apoptosis, as evidenced by the high percentage of annexin V positivity and strong cleavage of PARP1 (PARP) and CASP3 (caspase-3). Concomitantly, SAHA/bortezomib up-regulated the expression of CDKN2A and CDKN1A but did not affect the level of BCL2L11 or BHRF1 (viral homologue of BCL2). The apoptotic effects were dependent on reactive oxygen species generation. Furthermore, SAHA/bortezomib suppressed the growth of Wp-restricted BL xenografts in nude mice. This study provides the rationale to test the novel application of SAHA/bortezomib on the treatment of EBV-associated Wp-restricted BL and post-transplant lymphoproliferative disorder.

CD8+ T Cell Transcriptional Profiles of Pediatric Heart Transplant Recipients That Carry EBV Loads in Peripheral Blood.

Pediatric heart transplant (HTx) recipients display impaired CD8+ T cellular immune surveillance due to chronic immunosuppression and thus are at increased risk of EBV-associated post-transplant lymphoproliferative disorders (PTLD). Approximately 70% of asymptomatic pediatric HTx patients who seroconvert after HTx carry EBV loads in peripheral blood. One third of these patients display high chronic EBV loads (> 16,000 genomic copies/ml), and have up to 45% rate of progression to late onset PTLD. Here we used microarray analyses to investigate the hypothesis that CD8+ T cell transcriptional signatures are different between asymptomatic pediatric HTx patients that carry EBV loads vs. asymptomatic patients that do not carry EBV loads in peripheral blood. Six EBV seropositive asymptomatic, gender-, age-, and immunosuppression-matched pediatric HTx recipients were recruited to the study and consisted of: (i) undetectable viral load (UVL, n=2) carriers, resembling “normal” EBV latency; (ii) low viral load (LVL n=2) carriers; (iii) high viral load (HVL n=2) carriers. RNA was extracted from highly purified CD8+ T cells, amplified and hybridized to the Affymetrix H133 2.0 Plus Array. Perfect match data were normalized using the RMA method as implemented in BRB Array Tools. Differentially expressed genes were found for each comparison using the J5 test at threshold |J5|>12. Impacted pathways were studied using Pathway Express. Transcriptional signatures (transcripts, pathways, and networks) for pro-inflammatory cytokines and chemokines were significantly increased in CD8+ T cells from LVL and HVL carriers as compared to UVL carriers. In addition, CD8+ T cell from HVL carriers uniquely revealed up-regulation of transcripts relevant for apoptosis and for cross-talk to B lymphocytes, as well as macrophage-specific transcripts critical for cell migration and inflammation. These results indicate significant differences in the transcriptional profiles of CD8+ T cells from patients that carry EBV loads suggesting that these transcriptional signatures could be used as early surrogate biomarkers to monitor and assess patients at risk of complications that may benefit for prompt clinical intervention.

Synchronous tuberculosis, Epstein-Barr virus-associated lymphoproliferative disorder and cytomegalovirus infection in an allogeneic transplant recipient: a case report.

BackgroundAllogeneic stem cell transplant recipients are prone to infections by various organisms. Tuberculosis (TB) represents a rare infectious complication, especially in countries non-endemic for TB.Case reportHere, we report the case of a German patient with exposure to TB decades before he was diagnosed with disseminated TB as well as synchronous Epstein-Barr virus associated lymphoproliferative disorder and cytomegalovirus infection after allogeneic stem cell transplantation for refractory acute myeloid leukemia. Tuberculostatic and virostatic therapy was administered and the patient could be discharged with no apparent signs of infection two weeks after initiation of therapy.ConclusionThis case illustrates the need for awareness of mycobacterial infections in patients from non-endemic regions undergoing stem cell transplantation even if other reasons for fever are present.

CNS Lymphoma

The concept and understanding of central nervous system (CNS) lymphoma have greatly evolved in the past few years. Better characterization of a number of lymphoproliferative neoplasms through clinical, immunophenotyping, and molecular studies is reflected in a much more complex WHO Classification of Tumours of Hematopoietic and Lymphoid Tissue. The term "primary CNS lymphoma" is now restricted to primary diffuse large B-cell lymphoma confined to the CNS (and/or to the eye) that occurs in immunocompetent patients. Many other lymphoma subtypes, some of which are primary or exclusive to the CNS, such as lymphomas of the dura and immunodeficiency-associated lymphomas, are excluded from this definition. We describe the clinical and morphologic features of a diverse group of lymphomas occurring in the CNS, including primary CNS lymphoma, primary vitreoretinal lymphoma, lymphomatosis cerebri, Epstein-Barr virus-associated lymphoproliferative disorders, low-grade B-cell lymphoma, T-cell lymphoma, anaplastic large cell lymphoma, intravascular large B-cell lymphoma, and Hodgkin lymphoma. The purpose of this review is to provide a practical approach to the diagnosis of an often-challenging entity, focusing on how to maximize the use of small tissue biopsies and prevent diagnostic traps, which we have encountered with similar cases. Clinical, radiologic, and histologic examples are presented.

A Case of Sinonasal Extranodal NK/T-Cell Lymphoma with Indolent Behaviour and Low-Grade Morphology

Extranodal NK/T-cell lymphoma, nasal type (ENKL), is an uncommon subtype of EBV associated lymphoma usually characterized by aggressive clinical course. We report an atypical sinonasal ENKL case with long-lasting indolent behaviour, developed in the setting of a polymorphic EBV-associated lymphoproliferative disorder (LPD). A 52-year-old woman had suffered from chronic sinusitis and nasal obstruction since 2000, moderately worsened during the last years (marked enlargement of the sino-nasal mucosa at MRI in 2011) with elevated anti-VCA IgG and IgM titers. Three subsequent biopsies revealed slightly increasing morphophenotypic atypia, ranging from a polymorphic B- and T-cell EBV positive proliferation (diagnosed in 2011, but not fulfilling CAEBV diagnostic criteria) to an overt monomorphic mildly atypical T LPD without necrosis and angiocentricty diagnosed as ENKL in 2013 upon immunophenotype and TCR-γ gene clonal rearrangement. Clinically indolent ENKL with low-grade morphology is extremely rare and diagnostically challenging; while the few reports in the literature describe long-survival in ENKL treated patients comparing histologically neoplastic lesions at onset and recurrences, no reports are published on the slow progression from a polymorphic EBV-related T/NK proliferation to a histologically overt clinically indolent ENKL in an untreated patient who only received occasional steroid administration.

EBV-associated post-transplant lymphoproliferative disorder after umbilical cord blood transplantation in adults with hematological diseases

We analyzed the incidence, clinicopathological features, risk factors and prognosis of patients with EBV-associated post-transplant lymphoproliferative disorder (EBV-PTLD) in 288 adults undergoing umbilical cord blood transplantation (UCBT) at a single institution. Twelve patients developed proven EBV-PTLD at a median time of 73 days (range, 36-812). Three-year cumulative incidence (CI) of EBV-PTLD was 4.3% (95% CI: 1.9-6.7). All patients presented with extranodal involvement. Most frequently affected sites were the liver, spleen, central nervous system (CNS), Waldeyer's ring and BM in 7, 6, 4, 3 and 3 patients, respectively. One patient had polymorphic and 11 had monomorphic EBV-PTLD (7 diffuse large B-cell lymphomas not otherwise specified, 4 plasmablastic lymphomas). We confirmed donor origin and EBV infection in all histological samples. EBV-PTLD was the cause of death in 11 patients at a median time of 23 days (range, 1-84). The 3-year CI of EBV-PTLD was 12.9% (95% CI: 3.2-22.5) and 2.6% (95% CI: 0.5-4.7) for patients receiving reduced-intensity conditioning (RIC) and myeloablative conditioning, respectively (P<0.0001). In conclusion, adults with EBV-PTLD after UCBT showed frequent visceral and CNS involvement. The prognosis was poor despite routine viral monitoring and early intervention. An increased risk of EBV-PTLD was noted among recipients of RIC regimens.

Two rare cases of Epstein–Barr virus‐associated lymphoproliferative disorders in inflammatory bowel disease patients on thiopurines and other immunosuppressive medications

The setting of chronic immunosuppression in inflammatory bowel disease (IBD) may promote the proliferation of Epstein-Barr virus-positive neoplastic clones. We report two rare cases of Epstein-Barr virus-associated lymphoproliferative disorder in IBD patients: one resembled lymphomatoid granulomatosis, and the other was a lymphoma resembling Hodgkin lymphoma. There are currently no guidelines for the prevention of lymphoproliferative disorder in IBD patients on immunosuppressive therapy.

Heterogeneity in clinical course of EBV-associated lymphoproliferative disorder after allogeneic stem cell transplantation

Objective and ImportancePost-transplant lymphoproliferative disorder (PTLD) is a severe complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) associated with Epstein–Barr virus (EBV).Clinical presentationsAmong 263 individuals treated with allo-HSCT for severe aplastic anemia, pure white cell aplasia, T-prolymphocytic leukemia, and relapsed Hodgkin lymphoma, we diagnosed EBV-PTLD in 5 patients. Median age was 29 years (range 19–70 years) and four of five patients were EBV-seropositive prior to HSCT. All five had unrelated EBV-positive donors. In all cases, PTLD occurred within the first year post-transplant (median 4 months).InterventionThere were two rapidly fatal courses with extensive organ involvement. Both patients showed lymphopenia and thrombocytopenia. In contrast, the three surviving patients had higher lymphocytes and normal platelet counts, while PTLD was restricted to one site and resolved after 2–4 cycles of rituximab.ConclusionIn this case series courses of PTLD showed substantial diversity.