Treatment Of Lymphoproliferative Disease Research Articles

ABCL-274: Outcomes in Patients with Lymphoproliferative Diseases and COVID-19: Results of a Subgroup Analysis of the CHRONOS19 Registry

BackgroundPatients (pts) with lymphoproliferative diseases (LPD) are at high risk of COVID-19 severe and lethal course.ObjectiveTo evaluate treatment outcomes and risk factors in pts with LPD and COVID-19 in a real-world setting.MethodsCHRONOS19 is an ongoing nationwide observational cohort study of adult (≥18 y) pts with hematologic disease and COVID-19. Data from 15 centers were collected on a web-based platform and managed in a de-identified manner. We performed a subgroup analysis of pts with LPD and COVID-19. Primary endpoint was 30-day all-cause mortality. Long-term follow-up was at 90 and 180 days.ResultsAs of data cutoff on April 14, 2021, 626 pts were included, 170 of them with LPD were eligible for primary endpoint assessment n(%): CLL – 64 (38%), Hodgkin lymphoma – 18 (10%), B-cell lymphomas aggressive/indolent – 49(29%) / 19(11%); M/F 96 (56%) / 74(44%), median age 56 [19–90] years, induction phase/R/R/remission 62(36%) / 68(40%) / 40(24%); comorbidities in 86 (51%) pts. Complications occurred in 126 (74%) pts; one-third had severe COVID-19, 25% were admitted to ICU, and 20% required mechanical ventilation. Comparative analysis of basic characteristics between pts with LPD and all other hematologic diseases did not reveal any significant clinical differences. All-cause mortality at 30 days was 14% in pts with LPD vs 15% in pts with other hematologic diseases (p=0.87); 86% of deaths were due to COVID-19 complications. Eight additional deaths due to COVID-19 at 90 days and 5 deaths due to LPD progression at 180 days occurred. In multivariate analysis, ICU + mechanical ventilation (HR, 71.99 [19.5–271.0]), and age ≥60 years (HR, 5.360 [1.08–11.8] were the most significant risk factors of death. COVID-19 affected treatment of LPD in 55% of pts, and 52% experienced treatment delay for a median of 4 [1–10] weeks. Early relapsed/refractory disease occurred in 5 of 170 (2.9%) pts; the association of relapses with impaired treatment is unknown. COVID-19 re-infection occurred in 1 patient. Data on antibodies after SARS-CoV-2 infection are available for 65 patients with LPD: anti-SARS-CoV-2 IgG was detected in 44 (66%) pts and only in 60% of pts with CD20-positive LPD vs 100% of pts with T-cell and Hodgkin lymphomas (p=0.004).ConclusionsThirty-day all-cause mortality in SARS-CoV-2-infected pts with LPD was comparable with other hematological diseases and higher than in the general population. Lower frequency of anti-SARS-CoV-2 IgG in CD20-positive patients is probably due to anti-CD20 therapy.

Alterations in microRNA Expression during Hematopoietic Stem Cell Mobilization.

Simple SummaryLymphoproliferative disorders comprise a heterogeneous group of hematological malignancies characterized by abnormal lymphocyte proliferation. Autologous hematopoietic stem cell transplantation plays a very important role in the treatment of lymphoproliferative diseases. The key element in this process is the effective mobilization of hematopoietic cells from the marrow niche to the peripheral blood. Mobilization of HSC is regulated by many factors, out of which miRNAs present in the hematopoietic niche via targeting cytokines, and signaling pathways may play an important regulatory role. This study investigated the expression of selected miRNAs in patients with multiple myeloma, Hodgkin’s lymphomas, and non-Hodgkin’s lymphomas undergoing mobilization procedures. The aim of the study was to evaluate the expression of hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-34a-5p, hsa-miR-126-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-223-3p during the mobilization procedure, and to assess their role in mobilization efficacy. The level of miRNAs was tested at two time points before the initiation of mobilization and on the day of the first apheresis. Our results suggest that the investigated miRNAs, especially hsa-miR-146a-5p, may influence the efficacy of HSC mobilization.microRNAs play an important role in the regulation of gene expression, cell fate, hematopoiesis, and may influence the efficacy of CD34+ cell mobilization. The present study examines the role of hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-34a-5p, hsa-miR-126-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-223-3p in the course of hematopoietic stem cell mobilization. The numbers of CD34+ cells collected in patients with hematological malignancies (39 multiple myelomas, 11 lymphomas) were determined during mobilization for an autologous hematopoietic stem cell transplantation. The miRNA level was evaluated by RT-PCR. Compared to baseline, a significant decline in hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-126-3p, hsa-miR-146a-5p, and hsa-miR-155-5p was observed on the day of the first apheresis (day A). An increase was observed only in the expression of hsa-miR-34a-5p. On day A, a negative correlation was found between hsa-miR-15a-5p and hsa-miR-146a-5p levels and the number of CD34+ cells in peripheral blood. A negative correlation was observed between hsa-miR-146a-5p and the number of collected CD34+ cells after the first apheresis. Good mobilizers, defined according to GITMO criteria, demonstrated a lower hsa-miR-146a-5p level on day A than poor mobilizers. Patients from the hsa-miR-146a-5p “low expressors” collected more CD34+ cells than “high expressors”. Our results suggest that the investigated miRNAs, especially hsa-miR-146a-5p, may influence the efficacy of HSC mobilization.

Lymphoproliferative blood diseases as a cause of liver disease

The article presents a review of the literature on liver pathology at lymphoproliferative diseases (LPD) of the blood. Liver dysfunction and/or hepatomegaly may be due to haematological pathology or may develop as a result of specific chemotherapy. Sometimes the isolated liver damage can be the main manifestation of hematological disease. Involvement of the liver worsens the LPD course and prognosis, it causes a reduction in the dose of basic therapy, and even leads to fulminant liver failure. Liver damage can be caused by direct infiltration of the tumor mass, paraneoplastic process, biliary obstruction, hemophagocytic syndrome, activation of chronic viral hepatitis. The following histological types of liver damage are defined: diffuse infiltration of the portal tract, nodular infiltration with the formation of regeneration nodes, sinusoidal infiltration. Each LPD type has its own histological type of liver damage. Paraneoplastic phenomena at LPD are stipulated by the active release of cytokines from the tumor mass and include the bile duct disappearance syndrome (BDDS), peliosis hepatitis and sinusoidal ectasia. The cause of biliary obstruction is most often lymphadenopathy with compression of the extrahepatic bile ducts, less often, primary or secondary lesions of the pancreas. Hemophagocytic syndrome is a potentially lethal condition that occurs with recurrence or late-stage lymphoma. Against the background of treatment with LPD, there is a risk of activation of chronic viral hepatitis B. Nucleoside analogues are recommended for the prevention or treatment of HBV infection. Blood LPD is often associated with HCV infection. Concomitant treatment of LPD and HCV infection with interferon-free regimens is safe and effective. Chemotherapy for the underlying disease usually restores the structure and function of the liver.

HIV-Related Lymphoproliferative Diseases in the Era of Combination Antiretroviral Therapy.

HIV-positive patients have a 60- to 200-fold increased incidence of Non-Hodgkin Lymphomas (NHL) because of their impaired cellular immunity. Some NHL are considered Acquired Immunodeficiency Syndrome (AIDS) defining conditions. Diffuse large B-cell Lymphoma (DLBC) and Burkitt Lymphoma (BL) are the most commonly observed, whereas Primary Effusion Lymphoma (PEL), Central Nervous System Lymphomas (PCNSL), Plasmablastic Lymphoma (PBL) and classic Hodgkin Lymphoma (HL) are far less frequent. Multicentric Castleman disease (MCD) is an aggressive lymphoproliferative disorder highly prevalent in HIV-positive patients and strongly associated with HHV-8 virus infection. In the pre-Combination Antiretroviral Therapy (CART) era, patients with HIV-associated lymphoma had poor outcomes with median survival of 5 to 6 months. By improving the immunological status, CART extended the therapeutic options for HIV positive patients with lymphomas, allowing them to tolerate standard chemotherapies regimen with similar outcomes to those of the general population. The combination of CART and chemotherapy/ immuno-chemotherapy treatment has resulted in a remarkable prolongation of survival among HIV-infected patients with lymphomas. In this short communication, we briefly review the problems linked with the treatment of lymphoproliferative diseases in HIV patients. Combination Antiretroviral Therapy (CART) not only reduces HIV replication and restores the immunological status improving immune function of the HIV-related lymphomas patients but allows patients to deal with standard doses of chemotherapies. The association of CART and chemotherapy allowed to obtain better results in terms of overall survival and complete responses. In the setting of HIV-associated lymphomas, many issues remain open and their treatment is complicated by the patient’s immunocompromised status and the need to treat HIV concurrently.

Lymphadenopathy and severe infection caused by cytomegalovirus: algorithm for the diagnosis and treatment of lymphoproliferative disease (clinical case)

Lymphadenopathy and severe infection caused by cytomegalovirus: algorithm for the diagnosis and treatment of lymphoproliferative disease (clinical case)

Specific Myocardial Lesion in a Patient with Diffuse Large B-Cell Lymphoma (a Case Report)

Introduction. Myocardial lymphoma is one of the rarest lesion localizations among all the lymphoproliferative diseases. Regardless of the current lack of generally accepted specific recommendations for the treatment of primary and secondary myocardial lymphomas the chemotherapy is carried out in accordance with clinical guidelines for the treatment of lymphoproliferative diseases, which in some cases results in a successful outcome. Materials and methods . The article presents a clinical example of the diagnostic procedure and treatment of a 65-year-old female with the following diagnosis: non-Hodgkin’s lymphoma st. II BE gr. II with the involvement of myocardium, cervical and mediastinal lymph nodes, and the right palatine tonsil. The patient received six courses of R-CEOP chemotherapy. Results and discussion . An echo-positive structure 28mm by 20 mm in size attached to the interatrial septum was found inside the right atrium following the second course of chemotherapy. Ejection fraction: 61%. Effect: regression exceeding 50%. Four months following the completion of treatment the ejection fraction was 63%, regression – exceeding 80%, thus implying a partial response. Conclusion. The diagnosis of lymphoma with a lesion in the myocardium has been positively verified by histology and IHC. Currently less invasive techniques are normally preferred, transesophageal cardiac echo guided biopsy being one. Similar to any other lymphoproliferative disease, myocardial lymphoma is a chemosensitive tumour that responds well to treatment. In each individual case, the treatment strategy must be patient-specific and take into account the somatic status of the patient, the tumour size and the degree of its spread, the tumour morphological type, and whether it would be possible to arrange adequate specialized support needed.

Значение коморбидности для больных хроническим лимфолейкозом

Chronic lymphocytic leukemia (CLL) remains an uncurable disease, in which the age, number and severity of comorbidities primarily determine the choice of therapeutic tactics and objectives. To evaluate actual comorbidity and polymorbidity in patients with CLL and a possible relationship between the diseases and comorbidities that are considered concurrent and side effects of the administered treatment. The study consisted of two parts. In a retrospective study, we analyzed records of patients with CLL from the Registry for Diagnostics and Treatment of Lymphoproliferative Diseases. In addition, we thoroughly evaluated and prospectively followed up 124 patients in the course of their preparation to a new stage of CLL tratement. Examining data from the Russian Registry for Diagnostics and Treatment of Lymphoproliferative Diseases (n=1361) showed that in Russia, the age of patients with newly diagnosed CLL has increased in the recent decade with the increase in life span, which might change the comorbidity structure. Comparing retrospective and our own data (n=124) showed that diagnoses of concurrent diseases are often recorded formally (p3 suggested a poor prognosis for patients with CLL. Diagnosis and treatment of comorbidities in patients with CLL require participation of different medical specialists working in a close contact with oncohematologists.

Abstract 2686: Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia

Abstract Introduction: The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is clinically active in lymphoproliferative diseases driven by B-cell receptor (BCR) and Toll-like receptor (TLR) signaling, including chronic lymphocytic leukemia (CLL) and Waldenstrom macroglobulinemia (WM), respectively. However, deep remissions are uncommon and resistance to single agent has been described. The hallmark of WM is an activating mutation in MYD88 in the TLR signaling pathway. While mutations in MYD88 are uncommon in CLL, our previous study identified gene signatures indicative of active BCR and TLR signaling in CLL cells residing in lymphoid tissues (Herishanu, Blood 2011). Further, TLR9 activating CpG oligonucleotides induce proliferation and extend CLL cell survival in vitro. These observations suggest that BCR and TLR signaling may cooperate to activate CLL cells in the tissue microenvironment. Here, we tested the hypothesis that targeting both BCR and TLR signaling could improve therapy for CLL. Methods: CLL PBMCs were treated with ibrutinib and/or an IRAK1/4 inhibitor (Calbiochem) for 1h and then stimulated with soluble αIgM, CpG, or both. We quantified changes in phosphorylation of BTK, PLCγ2 and ERK (BCR pathway) and STAT3 and STAT1, as well as total IRAK1 (TLR pathway). Results: As expected, ibrutinib inhibited phosphorylation of BTK, PLCγ2 and ERK (P<.05) and decreased the survival of CLL cells stimulated with αIgM (P=.001). CpG stimulated TLR signaling degrades IRAK1 and the stimulates cytokine secretion that can, in autocrine fashion, activate STAT phopsphorylation. The IRAK1/4 inhibitor effectively inhibited TLR signaling resulting in stabilization of IRAK1 (P=.002), decreased phosphorylation of STAT1/3 (P=.04) and decreased viability compared to CpG stimulated but not IRAK inhibitor treated cells. Ibrutinib had no effect on CpG-induced IRAK1 degradation, but was comparable to IRAK1/4 inhibition in reducing STAT phosphorylation, suggesting that inhibition of BTK can antagonize downstream effects of TLR activation but not upstream IRAK dependent steps. In contrast, IRAK1/4 inhibition had no effect on αIgM-induced BCR activation. Next we evaluated the effect of dual BCR and TLR activation, modelling co-operative activation of both pathways in the tumor microenvironment. Under these in vitro conditions, ibrutinib prevented BCR activation and partial TLR activation, while IRAK1/4 affected only the TLR pathway. When ibrutinib and the IRAK1/4 inhibitor were combined, activation of both BCR and TLR signaling was prevented resulting in a significant reduction in CLL cell viability (P=.01) compared to co-activation of both pathways. Conclusion: While ibrutinib partially inhibited TLR signaling, an IRAK1/4 inhibitor was required for full inhibition of the pathway. The combination of BTK and IRAK1/4 inhibition for the treatment of lymphoproliferative diseases warrants further investigation. Citation Format: Eman L. Dadashian, Sarah Herman, Adrian Wiestner. Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2686. doi:10.1158/1538-7445.AM2017-2686

Therapy-related acute myeloid leukemia following treatment of lymphoid malignancies.

Therapy-related acute myeloid leukemia (t-AML) is a heterogeneous entity most frequently related to breast cancer or lymphoproliferative diseases (LD). Population-based studies have reported an increased risk of t-AML after treatment of lymphomas. The aim of this study was to describe the characteristics and outcome of 80 consecutive cases of t-AML following treatment of LD. t-AML accounted for 2.3% of all AML cases, occurred 60 months after LD diagnosis, and were characterized by a high frequency of FAB M6 AML and poor-risk cytogenetic abnormalities. Time to t-AML diagnosis was influenced by patient age, type of LD, and treatment. Among the 48 t-AML patients treated with intensive chemotherapy, median overall survival (OS) was 7.7 months compared to 26.1 months in de novo, 4.2 months in post-myeloproliferative neoplasm, 9.4 months in post-myelodysplastic syndrome, 8.6 months in post-chronic myelomonocytic leukemia AML, 13.4 months in t-AML secondary to the treatment of solid cancer, and 14.7 months in breast cancer only. OS of post-LD t-AML patients was significantly influenced by age, performance status, myelodysplastic syndrome prior to LD/t-AML, and treatment regimen for LD. Thus, t-AML following lymphoid malignancies treatment should be considered as very high-risk secondary AML. New treatment strategies in patients with LD/t-AML are needed urgently.

Association of Chronic HBV Infection with Chronic Lymphoproliferative Disorders: A Review and Case Report

This article presents a clinical report on the associated course of chronic hepatitis B virus (HBV) infection with Castleman’s disease (CD). We noticed the reactivation of previously latent chronic hepatitis B (CHB) with high replicative activity of HBV DNA during the treatment of lymphoproliferative disease. This clinical case dictates the need for pre-emptive therapy of HBV infection with nucleoside analogues in patients who are receiving chemotherapy. (Int J Biomed. 2016;6(2):128-132.).

The Role of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration in the Diagnosis and the Subtyping of Lymphoma in the Mediastinum

Endobronchial ultrasound-guided transbronchial needle aspiration is a commonly performed procedure in patients with mediastinal lymphadenopathy. Its role has been widely established in the diagnosis and the staging of lung cancer and other benign disease, but there is still debatable evidence regarding its role in the diagnosis, the subtyping, and the treatment of lymphoproliferative diseases. Consequently, there are no guidelines or statements to guide the choice of diagnostic modality in such cases. We review the available literature to evaluate the myth that endobronchial ultrasound-guided transbronchial needle aspiration does not provide sufficient diagnostic material for the accurate diagnosis and subtyping of mediastinal lymphoma.

Role of rituximab in the treatment of postpartum acquired haemophilia A: a systematic review of the literature.

Acquired haemophilia A (AHA) is a rare, but often severe, haemorrhagic disorder characterised by the development of autoantibodies directed against coagulation factor VIII (FVIII:C), with an estimated incidence of approximately one case per million persons per year1–4. The age at onset of AHA is distributed in a biphasic pattern, with a small peak in young individuals, primarily postpartum women, and a major peak in those aged 68–80 years of either sex5–7. This bleeding disorder may be associated with pregnancy, malignancies, autoimmune diseases and drug exposure, but in a consistent proportion of cases, about 50%, no underlying cause can be identified at diagnosis8–11. At variance with congenital haemophilia, in which haemarthroses are the most common bleeding symptoms, haemorrhages in AHA are most frequently reported to involve soft tissues (muscle, skin). The diagnosis of AHA is based on the laboratory detection of a prolonged activated partial thromboplastin time, not corrected after mixing and incubating equal volumes of normal plasma and plasma from the patient for 2 hours at 37 °C, and a reduction of FVIII:C plasma levels with a FVIII-inhibiting activity in patients with a negative personal or family history of bleeding12,13. Prompt recognition and treatment of AHA are mandatory, as inadequate management and complications of the disease are associated with high mortality rates (up to 22%)14. The aims of the therapeutic approach are the control of bleeding, eradication of the inhibitor and, when possible, treatment and elimination of the associated disease15. While the bypassing agents activated prothrombin complex concentrates (APCC) and recombinant activated factor VII (rFVIIa) are effective therapies for the management of acute bleeding in AHA patients16–19, immunosuppression with steroids in association with cyclophosphamide is able to eradicate the inhibitor in about 70% of cases5,20. In recent years, various reports have suggested a significant role of rituximab in the treatment of patients with acquired FVIII inhibitors refractory to standard immunosuppressive regimens21,22. Rituximab is a chimeric murine/human monoclonal antibody directed against CD20 transmembrane protein expressed on the surface of premature and mature B lymphocytes. It depletes B cells from the blood, lymph nodes and bone marrow and has demonstrated efficacy in the treatment of CD20-positive lymphoproliferative diseases as well as in a variety of autoimmune disorders, such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura and AHA23. With regards to this last indication, rituximab (at a dose of 375 mg/m2 once a week for 4 weeks), alone or in association with corticosteroids or other immunosuppressive drugs, has been used both as first-line therapy and as salvage treatment in cases refractory to conventional immunosuppressive agents with very high response rates. In particular, two systematic reviews conducted by Franchini and colleagues and Garvey found complete responses in 88% and 79% of cases, respectively22,24. However, only few cases were patients with postpartum AHA. Thus, in order to elucidate the role of rituximab in the treatment of pregnancy-associated FVIII autoantibodies, we have performed a systematic review.

Epidemiology Of Invasive Aspergillosis (IA) During Induction Therapy In Adults With Acute Lymphoblastic Leukemia (ALL): A Graall-2005 Study

BackgroundIA is a major cause of morbidity and mortality for patients with hematological malignancies and has been mainly reported in patients with acute myeloblastic leukemia (AML) and stem cell transplantation. Much less is known about the role of IA during the treatment of lymphoproliferative diseases like ALL. Here, we retrospectively evaluated the characteristics of patients and the incidence of IA occurring during the induction course in adult ALL patients enrolled in the GRAALL-2005 trial. MethodsWe collected the data of 36 patients with IA during induction chemotherapy. All were included between May 2006 and October 2012 in the multicentric GRAALL-2005 phase III trial. According to ALL characteristics, these patients were treated in 3 different substudies: GRAALL, GRAALL-Rituximab (GRAALL-R) for CD20+ ALL, GRAAPH for Philadelphia (Ph) chromosome-positive ALL. IA was defined retrospectively using the EORTC modified criteria (De Pauw, CID 2008). ResultsAmong the 969 patients enrolled, 36 (3.7%) developed IA during induction therapy. The median age was 30 years for all patients receiving induction and 47 years (range, 18 to 59) for patients with IA. We observed 18 IA (3%) by the GRAALL protocol (593 patients enrolled), 16 (8.3%) by the GRAALL-R protocol (191 patients enrolled), and 2 (0.7%) by the GRAAPH protocol (270 patients enrolled). In the GRAALL-R protocol, IA was diagnosed in 6 patients randomly assigned to receive rituximab and in 10 patients treated in the control group. The median time between first day of induction therapy and IA diagnosis was 20 days (range, -2 to 71). The median time between the first day of neutropenia and IA diagnosis was 18 days (range, 0 to 76). At the time of IA diagnosis, 13 patients were hospitalized in laminar airflow rooms, 10 patients in rooms with overpressure, 1 patient in a room with high-efficiency particulate filter, 5 patients in conventional rooms, 1 patient received home care and the type of hospitalization was unknown for the 6 remaining patients. Four patients with IA (11%) had received antifungal drug for invasive fungal infection (IFI) prophylaxis: one patient received fluconazole, one received caspofungin, and two received liposomal amphotericin B. All patients with IA presented pulmonary symptoms associated with a sinusal or cutaneous localization in 1 and 2 patients respectively. The diagnosis of IA was classified as possible in 7 episodes (19 %), probable in 22 episodes (61%), proven in 4 episodes (11%), and indeterminate in the remaining 3 episodes. Detection of Aspergillus antigen in serum by latex agglutination was positive in 24 cases (67%). Chest CT scans were taken for 33 patients (92%): nodules were found in 19 patients, halo sign in 20 patients, and “air crescent sign” in 1 patient. Bronchoalveolar lavage was performed in 11 patients (31%): culture was positive in 7 of them and Aspergillus antigen was positive in 2 of them. Biopsies where positive in 4 cases: in pulmonary biopsy in 3 patients and in cutaneous biopsy in 1 patient. Of all the Aspergillus isolates identified to the species level, Aspergillus fumigatus was isolated from 4 patients and Aspergillus flavus from 1 patient. Overall and IA-attributable 12-week mortality was 8 (0.8%) and 6 (16.7%) patients respectively. ConclusionIA is less frequently observed during induction therapy in adult ALL as compared to adult AML patients. However, the attributable mortality appears to be high in these patients. They are as at risk patients for IA and should be included in prophylactic and empirical antifungal clinical trials. Disclosures:No relevant conflicts of interest to declare.

Clinical Study of Post-Transplantation Lymphoproliferative Diseases in Children with Acquired Aplastic Anemia After Allogeneic Hematopoietic Stem Cell Transplantation

Objective To explore the epidemiology,clinical characteristics,diagnosis,treatment and prognosis of post-transplantation lymphoproliferative diseases (PTLD) in children with acquired aplastic anemia (AAA) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods From July 2002 to May 2012,a total of 71 children who underwent allo-HSCT with AAA at the Institute of Shanghai Children's Medical Center were included in this study.All of these children's clinical data were analyzed.The conditioning regimen included fludarabine,cyclophosphamide and rabbit-antithymocyte globulin with or without total body radiotherapy,and graft-versus-host disease prophylaxis mainly consisted of cyclosporine and methotrexate.For these patients who were monitored by Epstein-Barr virus (EBV)-DNA copies in peripheral blood mononuclear cells after allo-HSCT,a preemptive treatment with anti-CD20 monoclonal antibody rituximab was given when EBV-DNA copies rose persistently.Once PTLD was diagnosed,reduction or withdrawal of immunosuppression was administered.Results The incidence of PTLD for children with AAA after allo-HSCT was 4.2％ (3/71).All the PTLD patients were presented with persistent fever with no reaction to any antibiotics,progressive antiadoncus and lymphadenectasis.Two cases of the PTLD patients were monitored by EBV-DNA copies after allo-HSCT,and both had persistent high level of EBV-DNA copies before the diagnosis of PTLD,also after the first course of rituximab.After therapies,two of the three PTLD patients were cured and alive till now; however,another one had no effect to the therapy and died 34 days after the diagnosis of PTLD.Conclusions PTLD is a rare and potentially fatal complication for children with AAA after allo-HSCT.Dynamic monitoring of EBV-DNA copies could have certain guiding on the early diagnosis and treatment for PTLD.The preemptive treatment with rituximab could reduce the incidence and mortality of PTLD. Key words: post-transplantation lymphoproliferative diseases; acquired aplastic anemia; allogeneic hematopoietic stem cell transplantation; children; Epstein-Barr virus; rituximab

NF-κB Is Constitutively Activated in EBV-Infected T or NK Cells and Can Be a Molecular Target for EBV-Positive T/NK-Cell Lymphoproliferative Disease Treatment

AbstractAbstract 1998Epstein-Barr virus (EBV) can infect not only B cells but also rarely T or NK cells and causes EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), such as extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV). However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies have not been elucidated to date. Furthermore, optimal chemotherapy for these, especially for CAEBV, has not been established and the prognosis of EBV-T/NK-LPD remains very poor. NF-kB is a transcription factor that mediates anti-apoptotic molecular signaling and promotes proliferation of cancer cells. NF-kB is known to be constitutively activated in some hematological malignancies; the proteasome inhibitor bortezomib suppresses its activity and is used clinically as an anticancer reagent. To clarify the molecular mechanism underlying the development of EBV-T/NK-LPD, we focused on NF-kB in four EBV-positive T- and NK-cell lines, SNT8, SNT15, SNT16, and SNK6, which had been established from primary lesions of ENKL patients (SNT8 and SNK6) and the peripheral blood (PB) of a CAEBV patient (SNT15, 16), respectively. In these cells, it was demonstrated that p50, p52, RelA, and RelB—the components of NF-kB—existed constitutively in the nucleus. On the other hand, the EBV-negative T-cell lines, Jurkat and Molt4, and an NK-cell line, KHYG1, were negative for nuclear localization of these molecules. Elevated nuclear NF- kB–DNA binding activity was demonstrated by electrophoretic mobility shift assay (EMSA), using oligonucleotides encoding an NF-kB-binding sequence as a probe. Supershift EMSA revealed that NF- kB–DNA binding complexes in these cells involved p50, p52, and RelA. In addition, reporter assay using NF-kB-dependent luciferase reporter gene consisting of an NF-kB-binding site as a promoter demonstrated constitutive activation of NF-kB in SNT8 cells, whereas not in Jurkat cells. From these results, we concluded that NF-kB was constitutively activated through the canonical and noncanonical pathways in these EBV-infected T- or NK-cell (EBV-T/NK cell) lines. Next, we investigated NF-kB activation in EBV-T/NK cells derived from CAEBV patients. CAEBV was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in PB, and detection of EBV-infected T or NK cells with clonal proliferation. To detect infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. The EBV DNA of each fraction was quantified using a real-time quantitative polymerase chain reaction assay. Ten patients (aged 8–72 years; 4 male, 6 female; infected cell types CD4:3, CD8:3, γδ:1, and CD56:3) were diagnosed as CAEBV. Then, we examined NF-kB activation in these cells. Similar to the cell lines, immunoblotting and EMSA demonstrated evidence of constitutive activation of NF-kB such as nuclear localization and DNA binding of p50, p52, and RelA in EBV-T/NK cells from CAEBV patients. To investigate the molecular mechanism of NF-kB activation in EBV-T/NK cells, we examined the influence of viral proteins expressed in EBV-T/NK cells, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We performed luciferase reporter assay using expression vectors for these proteins in Jurkat cells. LMP1, most significantly, and LMP2A, to a lesser extent, upregulated NF-kB-dependent reporter gene expression in Jurkat cells, whereas the other viral proteins did not, suggesting that LMP1 and LMP2A mediated NF-kB activation in T/NK cells. Finally we investigated the contribution of NF-kB to the development of EBV-T/NK-LPD. Bortezomib, in a dose-dependent manner, inhibited translocation of p50, p52, and RelA to the nucleus and reduced the expression of Bcl-XL, which was a transcriptional target of NF-kB in SNT8, SNK6, and CAEBV patients' cells. In addition, it was also confirmed that bortezomib (0.5nM -5nM) suppressed the survival of and induced apoptosis of EBV-T/NK cells derived from 5 CAEBV patients. These results suggest that constitutive activation of NF-kB contributes to the development of EBV-T/NK-LPD and that the NF-kB signaling pathway can be an attractive molecular target for treatment. Disclosures:No relevant conflicts of interest to declare.

Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients

AbstractT-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)–stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV+ lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV+ LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility. The current trial was registered at www.clinicaltrials.gov as #NCT00058812.

Current status of older and new purine nucleoside analogues in the treatment of lymphoproliferative diseases.

For the past few years more and more new cytotoxic agents active in the treatment of hematological malignancies have been synthesized and become available for either in vitro studies or clinical trials. Among them the class of antineoplastic drugs belonging to the purine nucleoside analogues group (PNAs) plays an important role. Three of them: pentostatin (DCF), cladribine (2-CdA) and fludarabine (FA) were approved by Food and Drug Administration (FDA) for the treatment of hematological malignancies. Recently three novel PNAs: clofarabine (CAFdA), nelarabine (ara-G) and forodesine (immucillin H, BCX-1777) have been synthesized and introduced into preclinical studies and clinical trials. These agents seem to be useful mainly for the treatment of human T-cell proliferative disorders and they are currently undergoing clinical trials in lymphoid malignancies. However, there are also several studies suggesting the role of these drugs in B-cell malignancies. This review will summarize current knowledge concerning the mechanism of action, pharmacologic properties, clinical activity and toxicity of PNAs accepted for use in clinical practice, as well as new agents available for clinical trials.

Application of anti-CD20 monoclonal antibodies in the treatment of lymphoproliferative diseases

Out of numerous studied monoclonal antibodies, only a few reached the stage of clinical application. The CD20 molecule, non-glycolysed phospholipoprotein (usually termed B1), belonging to the tetraspan (TM4SF) family, 35-37 kD, is characteristic for all mature B lymphocytes, including CLL cells. The CD20 receptors, characteristic for 'B' lymphoproliferative diseases, have been demonstrated to be a good target for therapeutic effects to be achieved. Rituximab is a chimeric anti-CD20 IgG1 monoclonal antibody, with the sequences of the human constant region and sequences of the murine variable region. It is specifically bound to the B-lymphocyte CD20 antigen. The mechanism of all rituximab antitumor activity has not been established, but ADCC and CDC are believed to be the principal, with possible complementary effects. Therapeutic use of anti-CD20 monoclonal antibodies has demonstrated a significant benefit in the patients with 'B' CD20 positive lymphoproliferative diseases. Rituximab is today a golden standard for the comparation with other treatment modalities, increasingly in combination with chemotherapy.

Essential Requirement for Sphingosine Kinase 2 in a Sphingolipid Apoptosis Pathway Activated by FTY720 Analogues

The clinical immunosuppressant FTY720 is a sphingosine analogue that, once phosphorylated by sphingosine kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for sphingosine 1-phosphate. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. Sphingosine itself is pro-apoptotic, and apoptosis induced with FTY720 or sphingosine is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including sphingosine. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered 1 mg/kg AAL(R), correlating with levels of AAL(R)-phosphate (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base phosphates and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.

Rituximab for lymphoproliferative disease prior to haematopoietic stem cell transplantation for X‐linked severe combined immunodeficiency

Lymphoproliferative disease (LPD) is a complication of congenital and acquired immunodeficiency states. There are a number of treatment options for LPD arising after haematopoietic stem cell or solid organ transplantation including reduction of immunosuppression, targeted therapies, such as the anti-CD20 monoclonal antibody, rituximab, and EBV specific cytotoxic lymphocytes. Treatment of LPD in children with congenital immunodeficiency syndromes remains unsatisfactory and is associated with a high mortality rate. We recently managed an infant found to have polymorphic LPD concurrent with X-linked severe combined immunodeficiency (SCID). Haematopoietic stem cell transplantation (HSCT) had to be deferred because of progressive LPD. Treatment with rituximab resulted in regression of the LPD following which the patient received a 5/6 HLA matched umbilical cord blood (UCB) transplant. The patient remains well 20 months following transplantation. Rituximab treatment may have a useful role in the control of LPD associated with congenital immunodeficiency prior to HSCT.