B-lymphoproliferative Disorders Research Articles

Dermatomyositis Associated With High-Grade B-Lymphoproliferative Disorders: A Case Report and Literature Review.

Dermatomyositis (DM) and polymyositis are idiopathic inflammatory myopathies (IIMs), most associated with solid organ malignancies, and less commonly hematological malignancies. We discuss a case of DM associated with diffuse large B-cell lymphoma, followed by a review of literature on the pathogenesis, clinical course, treatment, and prognosis. Various challenges with the diagnosis and management of underlying lymphoproliferative disorders (LPDs) in patients with IIM are discussed. The case demonstrates the importance of being vigilant of the association between IIM and LPD. Cancer screening in patients with IIM is discussed, including the recently published International Guideline for IIM-Associated Cancer Screening. More research is required to address knowledge gaps in cancer screening in IIM.

Frequency and Outcomes of Obinutuzumab-Induced Thrombocytopenia

Introduction Obinutuzumab (Obi) is a third-generation anti-CD20 monoclonal antibody widely used in the treatment of B-lymphoproliferative disorders (B-LPD) including follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL). It is infrequently associated with severe thrombocytopenia which can be life-threatening. The pathophysiology is unclear, but platelet clearance can be extremely rapid (<24 hours). Methods After obtaining local ethics review, we collected the total number of patients who received Obi as part of multi-agent chemotherapy for the following hematological diseases from 01/01/2015 -01/01/2023: FL (Obi-bendamustine +/- Obi maintenance; Obi-cyclophosphamide/vincristine/prednisone +/-doxorubicin +/- Obi maintenance, Obi-Zanubrutinib (Rosewood trial) and CLL (Obi-venetoclax; Obi-chlorambucil). All cases of clinically suspected Obi-induced thrombocytopenia were reviewed and presented below. All suspected cases were confirmed by evidence of temporal relationship with Obi, spontaneous resolution off Obi, and recapitulation with Obi rechallenge. Results Four out of 147 patients receiving Obi-based multi-agent chemotherapy were found to have Obi-induced thrombocytopenia (frequency 2.7%). Two patients were females (50%), and the median age was 78 (range, 72-82) years. Figure 1 demonstrates the trend of platelet and neutrophils in a patient who received Obi and venetoclax (Ven). Ven was postulated as the cause of the thrombocytopenia, but Ven cessation had no effect on the thrombocytopenia. After suspending Obi, there was a steady increase in the platelet count to 104 x 10 9/L. Obi was re-commenced at cycle 7 (eighth dose) which was followed by a prompt relapse to 4 x 10 9/L 48 hours later. A steady recovery in the platelet count was again seen after ceasing Obi. The other 3 patients received Obi-bendamustine for FL. For the first patient, thrombocytopenia occurred on day 7 of first cycle (41 x 10 9/L) with a nadir of 14 x 10 9/L on day 11. She received intravenous immunoglobulin (IVIg) and oral corticosteroids due to associated bleeding. The second patient had thrombocytopenia on day 28 of the first cycle (71 x 10 9/L) which decreased to a nadir 13 x 10 9/L while receiving Obi-maintenance. There was no bleeding and Obi was ceased followed by a gradual improvement in thrombocytopenia. For the third patient, thrombocytopenia (41 x 10 9/L) occurred on day 18 of first cycle without bleeding. Obi was changed to rituximab. Discussion This is the first known study on the frequency of Obi-induced thrombocytopenia (2.7%). Obi-associated thromboctopenia is severe, sudden-onset, not exclusive to the first cycle of therapy, and the mechanism remains unknown. The diagnosis may be challenging as patients are often receiving other myelosuppressive therapies. Various strategies may help to elucidate the cause, including correlation with timing of exposure and single agent elimination. Suspension of Obi on suspicion is both an important diagnostic and therapeutic strategy. Obi rechallenge can be considered however thorough patient discussion is required with close monitoring and strategies to manage thrombocytopenia. In multi-agent chemotherapy studies containing Obi, severe thrombocytopenia of any cause occurred in up to 13% of cases including fatal hemorrhage. The optimal management is unclear. As our cases demonstrate, thrombocytopenia may resolve spontaneously after withdrawing Obi. Analogous to ITP, steroids, and intravenous immunoglobulin (IVIg) have been associated with response whereas platelet transfusions may be ineffective. Two recent cases with severe thrombocytopenia were treated with thrombopoietin receptor agonist (TPO-RA). Other important considerations are regular blood count monitoring and withholding medications which may increase bleeding risk (e.g., anticoagulants) especially during the first cycle. Summary Obi is prevalent in the treatment of B-LPD, being used in 6 different regimens in local guidelines. However, clinicians need to be aware of the risk of thrombocytopenia which may be severe. The frequency was 2.7% in this single-center retrospective audit. Correlation with timing of exposure is important. The optimal management is unclear. It may be self-limiting whereas some cases have been treated with steroids, IVIg, and TPO-RA. Further studies are required to delineate the incidence, pathophysiology, diagnosis, and management.

Utility of Measurable Residual Disease (MRD) Assessment in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) treatment advances have significantly improved disease-free remission, with greater focus in clinical trials being placed on measurable residual disease (MRD) as a marker of subclinical disease assessment. While this concept is used extensively in other haematological neoplasms, there is yet to be a consensus on the threshold for MRD in MCL that demonstrates prognostic and therapeutic significance, and in this context has yet to reach routine clinical practice.The historical long-term method for MCL MRD assessment has been real-time quantitative polymerase chain reaction (PCR), targeting the clonal immunoglobulin heavy locus (IGH) rearrangement or the IGH::CCND1 translocation rearrangement. A significant problem at present relates to identifying alternative assays for patients who do not have a suitable molecular target by this method.This article reviews existing techniques used in MRD assessment for MCL and describes novel methods which may overcome existing limitations, including next-generation sequencing modalities. The use of circulating tumour DNA is explored, with techniques such as CAPP-Seq and PhasED-Seq demonstrating promise in B-lymphoproliferative disorders, though application in MCL requires further study.The other aspect of practice using MRD is identifying therapeutic options which can address a subclinical molecular relapse. Developing suitable interventions that can alter the disease trajectory based on longitudinal MRD kinetics are needed to justify its incorporation into standard care.

Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein\u2013Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV\u2009+\u2009lymphoproliferative disorders

BackgroundB-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein–Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible.MethodsAs a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay.ResultsRank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir.ConclusionsrAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Early Immune Profile Assessment By Flow Cytometry Predicts Severe COVID-19 Pneumonia

▪Background and ObjectiveThe rapid spread of the COVID -19 pandemic and the high variability of the course of the disease make it essential to search for early predictors of outcome. The objective of our study is to predict severe SARS COV2 pneumonia using early cytometric profilesMaterial and MethodsProspective and observational study of adults with confirmed COVID-19 infection admitted on Emergency Department (ED). We collected epidemiological, clinical and laboratory data of every patient until they were discharged or died. Multiparametric flow cytometry (FC) analysis of T-lymphocytes (CD4, CD8, CD4 activated, CD8 activated, naïve (Tn), central-memory (Tcm), effector-memory (Tem), effector (Te) and Th17 subsets), B-lymphocytes (naïve, memory, transitional subsets, and assessment of clonality), NK cells, plasmablasts, p-DCs (plasmacytoid dendritic cells), m-DCs (myeloid dendritic cells), basophils, and monocytes (MO1, MO2, MO3, slan+ MO3) was performed on whole peripheral blood collected on EDTA, before immunosuppressive therapy was started. We designed a 7-tube 8-color experimental panel. Cell surface staining of 2 × 106 cells was performed and at least 500 000 total events were acquired for the assessment of plasmablasts, p-DCs, m-DCs, basophils, and the monocyte subsets; for the study of B, T and NK-lymphocyte populations we acquired at least 100 000 total events (FACS Canto II; BD Biosciences). Severity was assessed on the basis of World Health Organization´s (WHO) international 10 level ordinal scale (WHOs) and also according to 4 respiratory status, based on SpO2(peripheral blood oxygen saturation)/FIO2 (fraction of inspired oxygen) ratio (SpFi). SpFi group 1 >452, SpFi2 2: 315-452; SpFi 3 236-315, SpFi 4: <236 (respiratory distress)Results53 patients were included: epidemiological and clinical data available on table 1. 23 patients (43.39%) arrived to SpFi4 status. WHOs >6 (WHO 6:oxygen by non invasive ventilation or high flow) was achieved by 20 patients (37.7%)Good prognosis (meaning SpFi1 as the worse respiratory status in the follow -up) was associated to cytometric profiles: there was a significant increase in CD3, p-DCs, m-DCs, basophils, monocytes, Tcm, % of lymphocytes and CD3/CD19 ratio whereas there was a significant reduction in CD19, % of neutrophils and % Neutrophils/% lymphocyte ratio. In the SpFi4 group, there was a significant reduction of CD3, p-DCs, m-DCs, plasmablasts and CD3/NK ratio. In patients starting in SpFi group 1-2 in the ED but progressing to SpFi 3-4 during the follow up (27 patients), there was a statistically significant relation with Tn, Te and Tn/Te ratio (Tn/Te ratio <0.717: OR 13.5 (p 0.002, [95% CI 2.552-71.403). Initial SpFi1 patients that evolved to SpFi 3-4 during follow up (10 patients), presented a Tn/Te ratio < 0.717 with an OR 11.556 (p =0.005, [95% CI 2.059-64.853]).Plasmablasts < 0.075 and CD3/NK <5.71, were identified as independent risk factors for SpFI4 during follow up. After multivariable analysis, both variables kept their significance: CD3/NK (OR 11,247, p=0.005) and plasmablasts (OR 12,524 , p=0.004). About prediction of WHOs >6, multivariable analysis showed CD3/NK <5.71 (OR 22,240 [95%CI 2,340-211,342] p= 0.007) and plasmablasts<0.075 (OR 28.635 [95% CI 3,187-257,301] p=0.003).A score (0,1,2) comprising both risk factors, was significantly predictive of SpFI4, regardless of the initial respiratory status, age or days from symptoms onset. In our cohort, only 1/15 (6.7%) patients with 0 points (neither plasmablasts nor CD3/NK score), arrived to SpFi4. However, 10/11 (90.9%) patients with 2 points, reached to SpFi4 respiratory status (C-index = 0.837) Same score was applied to predict WHOs > 6: 90.9% with 2 points progressed to WHO>6 and 0/15 patients with 0 points reached the same goal (C-index = 0.872)An incidental finding of 4 indolent B-lymphoproliferative disorders (2CLL-like MBLs and 2non -CLL-like MBL), was found, and they were associated with older age and progression to death.ConclusionsFlow cytometry on whole peripheral blood samples of SARS-COV2 pneumonia patients, collected before corticosteroid or immunosuppressive therapy, could identify cytometric patterns associated to prognosis. Plasmablasts, mDCs and pDCs levels as well as CD3/NK ratio, are associated to a worse respiratory status, while Tn/Te ratio could detect non-severe patients who will require high-flow oxygen devices during follow up. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute gout flare of bilateral first metatarsophalangeal joints due to ibrutinib use in chronic lymphocytic leukemia

Bruton tyrosine kinase inhibitors represent important tools in the therapeutic armamentarium against chronic lymphocytic leukemia (CLL) and other B-lymphoproliferative disorders. We describe herein a unique 65-year-old patient who presented with bilateral foot pain four months after starting treatment with ibrutinib for CLL. Of note, the patient had previously been diagnosed with gout, and was taking allopurinol prophylactically at the time of the event. Compliance with allopurinol was in excess of 99%. Yet, he was diagnosed with acute gout flare of bilateral first metatarsophalangeal (MTP) joints.Management & Outcome: Ibrutinib dose was reduced by one third, and the patient's gout flare up was treated with ibuprofen as needed. After symptoms abated, ibrutinib was continued at 2/3rds of the dose, with an excellent CLL control. The patient tolerated this dose without any further adverse effects.Discussion/Conclusions: We have reported a unique side effect of acute bilateral first MTP joint gout flare likely triggered by ibrutinib use for CLL while the patient was taking a xanthine oxidase inhibitor. The mechanism by which ibrutinib caused this phenomenon remains to be elucidated.

VILLOUS LYMPHOCYTES IN BLOOD AND BONE MARROW IN SOME FORMS OF B-CELL LYMPHOID MALIGNANCIES

The cytological and immunocytochemical features of the lymphocytes with villous morphology in peripheral blood and bone marrow in some B-lymphoproliferative disorders were studied. The diagnosis of hairy cell leukemia, a hairy cell leukemia variant, splenic marginal zone lymphoma and splenic diffuse red pulp small B-cell lymphoma was ascertained in accordance with the new revision of the WHO classification (2016). The neoplastic cells of hairy cell leukemia were determined by the presence of high tartrate resistant acid phosphatase (TRAP) activity. Cell surface expression of CD19, CD20 and CD21 antigens was detected. Also, the expression of CD25, CD103 and CD200, and in some cases cyclin D1, was found out. CD5, CD10 and CD23 were not detected. The immunophenotype of cells in splenic marginal zone lymphoma with villous processes also corresponded to the mature B cells. The expression of CD19, CD20 and CD21 was observed in all cases, CD11c – in 50% of patients, CD25 or CD5 – in 10% of patients. In 80% of patients, the pathologic cells did not show TRAP activity. In the bone marrow and peripheral blood cells of patients with diffuse red pulp lymphoma, TRAP activity was not detected. An immunophenotype in the hairy cell leukemia variant was different from those of classic HCL (CD19+CD20+CD22+CD103+CD11c+CD5–CD10–CD23–). Characterized immunophenotypical markers, which have differential diagnostic values in several forms of lymphoid tumors of B cell origin, will be important for the choice of treatment methods and prognosis

Monitoring anti-B cell immunotherapies in autoimmune diseases: Go with the flow. A Position Paper of the Italian Society for Clinical Cell Analysis (ISCCA)

During the past decades autoimmune diseases have been usually treated with immunosuppressive drugs mostly active on T-Cell mediated responses. Only in recent years, with our extended knowledge of the pathogenic mechanisms of autoreactive disorders and the tremendous development of new therapeutic monoclonal antibodies, anti-B-Cell therapies have emerged as a new option for treating autoimmune diseases. The rationale for this changeover from T-Cell to B-Cell targeted therapies resides in the recently accumulated evidence of the role of B-Cells in the pathogenesis of autoimmune diseases and in the generation of tissue damage. Targeting memory and effector BCells may then disrupt the production of pathogenic antibodies, counteract the role of B-Cells in sustaining antigen presentation to T-Cells and block the synthesis of B-Cell activation cytokines. The anti-CD20 monoclonal antibody Rituximab was first introduced more than 20 years ago for the treatment of CD20+ chronic B-lymphoproliferative disorders, and was then successfully experimented in the treatment of an ever-increasing spectrum of autoimmune diseases. Newer anti-CD20 monoclonal antibodies have been introduced more recently, which vary in their biological effects. The need for laboratory indicators that may help the rational usage and follow-up of anti-CD20 treatments has now emerged, due to the high variability of individual response, to the markedly different outcomes in the various diseases and to the controversial role of pathogenic autoantibodies as indicators of disease activity. Flow cytometric (FCM) analyses to identify and enumerate the B-cell functional subsets in the peripheral blood have been developed in recent years. They can be used to assess the degree and the persistence of memory B-Cell depletion, the quality and the timing of B-Cell reconstitution, along with the highly sensitive FCM counting technique needed for the detection of extremely low cell levels. The long-term aim of this innovative approach is to provide clinicians with a tool for a safer and more rational usage of anti-CD20 agents.

Activation of NF-κB in B cell receptor signaling through Bruton's tyrosine kinase-dependent phosphorylation of IκB-α.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

EBV-Positive B-Cell Proliferations of Varied Malignant Potential: 2015 SH/EAHP Workshop Report-Part 1.

The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review B-cell proliferations of varied malignant potential associated with immunodeficiency. The Workshop Panel reviewed all cases of B-cell hyperplasias, polymorphic B-lymphoproliferative disorders, Epstein-Barr virus (EBV)-positive mucocutaneous ulcer, and large B-cell proliferations associated with chronic inflammation and rendered consensus diagnoses. Disease definitions, boundaries with more aggressive B-cell proliferations, and association with EBV were explored. B-cell proliferations of varied malignant potential occurred in all immunodeficiency backgrounds. Presentation early in the course of immunodeficiency and in younger age groups and regression with reduction of immunosuppression were characteristic features. EBV positivity was essential for diagnosis in some hyperplasias where other specific defining features were absent. This spectrum of B-cell proliferations show similarities across immunodeficiency backgrounds. Localized forms of immunodeficiency disorders arise in immunocompetent patients most likely due to chronic immune stimulation and, despite aggressive histologic features, often show indolent clinical behavior.

PAX5 interacts with RIP2 to promote NF-κB activation and drug-resistance in B-lymphoproliferative disorders.

Paired box protein 5 (PAX5) plays a lineage determination role in B-cell development. However, high expression of PAX5 has been also found in various malignant diseases, including B-lymphoproliferative disorders (B-LPDs), but its functions and mechanisms in these diseases are still unclear. Here, we show that PAX5 induces drug resistance through association and activation of receptor-interacting serine/threonine-protein kinase 2 (RIP2; also known as RIPK2), and subsequent activation of NF-κB signaling and anti-apoptosis gene expression in B-lymphoproliferative cells. Furthermore, PAX5 is able to interact with RIP1 and RIP3, modulating both RIP1-mediated TNFR and RIP2-mediated NOD1 and NOD2 pathways. Our findings describe a new function of PAX5 in regulating RIP1 and RIP2 activation, which is at least involved in chemotherapeutic drug resistance in B-LPDs.

Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice.

ABSTRACTHistone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). Here, we report that the Eμ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eμ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.

Obinutuzumab: a new class of anti-CD20 monoclonal antibody.

Obinutuzumab is a new anti-CD20 monoclonal antibody which demonstrated clinical superiority compared with rituximab in a recent phase III study. There is a need to better understand how this antibody differs from rituximab and why it could modify the landscape of the treatment of CD20 malignancies in the near future. Antibody-dependent cellular cytotoxicity plays a critical role in clinical activity of rituximab. To increase antibody-dependent cellular cytotoxicity, a strategy improving the affinity between the Fc portion of the antibody and FcγRIIIa expressed by effector cells has been recently developed. This strategy modifies the carbohydrate located between the two Fc arms. Thus, the lack of fucose on IgG oligosaccharide improves binding to FcγRIII and antibody-dependent cellular cytotoxicity. Obinutuzumab recognized a CD20 epitope different from that bound by rituximab. This property confers different features to obinutuzumab mechanisms of action with a noncaspase-dependent direct-cell death and the lack of complement-dependent cytotoxicity. Obinutuzumab demonstrated significant activity in animal models, and phase I or II studies showed clinical activity in different subtypes of CD20 diseases. Obinutuzumab, a type II glycoengineered monoclonal antibody, is characterized by an increased antibody-dependent cellular cytotoxicity and direct-cell death but no complement-dependent cytotoxicity. Recent clinical data demonstrated a superiority of obinutuzumab compared with rituximab, suggesting that this antibody should be, in the future, the backbone of the treatment of B-lymphoproliferative disorders.

Hepatitis C among Egyptian Patients Referred for Bone Marrow Examination: Seroprevalence and Analysis of Hematological Findings.

Hepatitis C is a significant public health problem in Egypt where the highest prevalence (14.7%) of hepatitis C virus (HCV) exists. HCV prevalence is even higher among clinical populations and groups at risk of exposure to infection. Chronic HCV infection is associated with several hematological complications that may necessitate bone marrow (BM) examination. The aim of this study is to estimate HCV prevalence among patients referred for BM examination and to explore hematological and BM findings among HCV positive patients. One hundred adult patients referred for BM examination were included in the study and screened for HCV antibodies. Patients' clinical, hematological, and BM findings were recorded. The seroprevalence of HCV among patients referred for BM examination was 42%. The most common indication for BM examination among HCV positive patients was peripheral cytopenias (88.1%). The most common cytopenia detected was thrombocytopenia (85.7%). The most common diagnosis among HCV positive patients was hypersplenism (52.4%) followed by B-lymphoproliferative disorders (19%) and then immune thrombocytopenic purpura (11.9%). In conclusion, HCV prevalence among patients referred for BM examination is higher than that estimated in the general population. Patients with unexplained peripheral cytopenias should be tested for HCV.

The Utility of the BIOMED-2 Primers in the Detection of 2 Clonal, B-Lymphoproliferative Disorders Simultaneously Involving the Same Site

Molecular tests for clonality performed on atypical lymphoid lesions may yield abnormal results because of the coexistence of monoclonal B lymphocytosis or monoclonal gammopathy of undetermined significance in the sample investigated. To investigate the ability of the BIOMED-2 sets of primers to identify 2 clonal populations in the same formalin-fixed, paraffin-embedded tissue sample. Design.-Ten cases with 2 B-lymphoproliferative disorders at the same site were analyzed using 5 BIOMED-2 primer sets (IGH FR1, FR2, FR3, IGK VJ, and IGK VKde). All 10 cases (100%) showed at least 1 clone; 8 of 10 cases (80%) had 2 clones with at least 1 primer set, and the 2 clones were shown by 4 or 5 primer sets in none of the cases (0%), by 3 sets in 1 of 10 cases (10%), by 2 sets in 4 of 10 cases (40%), and by 1 set in 3 of 10 cases (30%). The most effective set was IGH FR2, detecting 4 of 10 biclonal cases (40%). The IGK VJ and IGK VKde each showed 2 clones in 3 of 10 cases (30% each). The least effective sets were IGH FR1 and FR3, with 2 of 10 cases (20%) each, with IGH FR1 being the least useful. The BIOMED-2 primers are effective in the detection of 2 clonal populations in the same sample.

Treatment of Epstein Barr virus‐induced haemophagocytic lymphohistiocytosis with rituximab‐containing chemo‐immunotherapeutic regimens

Haemophagocytic lymphohistiocytosis (HLH) is a life threatening complication of Epstein-Barr virus (EBV) infection. The anti-CD20 antibody rituximab depletes B cells, leading to improved outcomes for patients with EBV-associated B-lymphoproliferative disorders. To gather data on the use of rituximab in EBV-HLH, we performed a retrospective investigation involving 42 EBV-HLH patients who had received treatment with rituximab-containing regimens. On average, patients received 3 rituximab infusions (range 1-10) at a median dose of 375 mg/m(2) . In all patients, rituximab was administered with other HLH-directed medications, including steroids, etoposide and/or ciclosporin. Rituximab-containing regimens appeared well tolerated and improved clinical status in 43% of patients. Examination of laboratory data obtained prior to and within 2-4 weeks after the first rituximab dose revealed significant reductions in EBV load (median load pre-rituximab: 114,200 copies/ml, median post-rituximab: 225 copies/ml, P = 0.0001) and serum ferritin levels (median ferritin pre-rituximab: 4260 μg/l, median post-rituximab: 1149 μg/l, P = 0.001). Thus, when combined with conventional HLH-directed therapies, rituximab improves symptoms, reduces viral load and diminishes inflammation. These data support the incorporation of rituximab into future prospective clinical trials for patients with EBV-HLH.

Waldenström’s macroglobulinemia harbors a unique proteome where Ku70 is severely underexpressed as compared with other B-lymphoproliferative disorders

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70—a component of the non homologous end-joining pathway—might be relevant in disease pathophysiology.

Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53

Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266). The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73. p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel. Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.

Prospective Study to Evaluate the Efficacy and Safety of Gemcitabine Combined with GMCSF and Rituximab in Heavily Pre-Treated Relapsed / Refractory B-Lymphoproliferative Disorders

Abstract 1616 Background:Aggressive non-Hodgkin lymphomas (NHLs) are commonly treated with anthracycline-containing combination chemotherapy regimens. Although a proportion of patients can be cured, 40 –50% of patients relapse or are refractory to frontline therapy. High dose chemotherapy (HDT) with autologous stem cell transplantation (ASCT) is the current standard of care of relapsed aggressive lymphomas in eligible patients. Many patients cannot proceed to HDT as a result of reasons like advanced age, significant co morbidities and resistance to salvage chemotherapy. Moreover, the optimal salvage regimen is not known. Hence there is need to develop novel regimens that are likely to benefit these patients. Gemcitabine and rituximab are active agents in relapsed or refractory lymphoma and have demonstrated synergistic effects. GMCSF has been shown to potentiate the efficacy of Rituximab. In this study we evaluated the clinical activity, toxicity and tolerability of a salvage regimen that incorporated sequential combination of gemcitabine, rituximab and GMCSF (Sargramostim, Leukine). Methods:This trial was designed as a prospective, open and uncontrolled phase II study. 13 patients with relapsed or refractory B cell lymphoma were enrolled from Nov 2001 to Mar 2006 at Rush University Medical Center, Chicago, IL. All patients were scheduled to receive up to 6 cycles of Gemcitabine 800mg/m2 IV infusion over 120 minutes on days 1 and 8, GMCSF 250mcg/m2 subcutaneously once daily on day 9 through 15 (7doses) and Rituximab 375mg/m2 IV infusion on day 16. Cycles were repeated every 21 days to a total of 6 cycles. Responses were evaluated by CT scans and bone marrow exam (if involved) after the third course and at the end of the study. Primary endpoint was response rate. Secondary endpoints were toxicity evaluation, progression free survival (PFS) and overall survival (OS). Patients were followed for toxicity, response, PFS and OS. Results:Of the 13 patients in the study, 9 patients completed 6 cycles of therapy and were evaluable for response. The median age was 43 years (range 24 –75 years) with 6 males (66.6%) and 3 females (33.3%). 92% of patients had very advanced disease with stage III or IV disease (Stage IV-61.5% and stage III-30.8%), and 30.8% were poor risk (IPI 3–5). Major histological subtypes were diffuse large B-cell lymphoma (33.3%), Hodgkin lymphoma (33.3%) and follicular lymphoma (11.1%). Eight patients (88%) demonstrated extra nodal disease at one or more sites at presentation indicating a more aggressive lymphoma. Most of the patients were heavily pretreated with multiple combination chemotherapy or chemo immunotherapy with a median of 3 prior chemotherapy regimens (range 2–6). The majority of patients were complete responders after first line induction therapy.In the intent to treat analysis of responses, the overall response rate was 55.5% with 4 complete responses (44.5%) and 1 partial response (11.1%). Disease progression occurred in 4 patients (44%). Progression free survival ranged from 2 to 71 with a median of 6 months. Overall survival measured from 2 to 102 months with a median of 10 months. At last follow-up 22% of (2 of 9) were alive. Toxicity profile of this regimen was similar to other salvage therapies used in this patient population with an increased incidence of grade 3/4 anemia (15.3%) and thrombocytopenia (7.6%).No grade 3 and 4 non-hematologic toxicities were observed. Conclusions:Based on the result of our study, we conclude that gemcitabine combined with GMCSF and rituximab is an effective, feasible and tolerable regimen in heavily pre-treated aggressive relapsed lymphomas. Long-term remissions are possible in patients with heavily pre-treated relapsed/refractory lymphoma with complete remissions lasting for 78–102 months even in patients who relapsed after autologous peripheral blood stem cell transplantation. Disclosures:Shammo:Celgene Corporation: Honoraria, Research Funding, Speakers Bureau.

CD5-Negative Monoclonal B-Cell Lymphocytosis (MBL) Is Detectable In 9% of Adults Aged Over 40 but Phenotypes Associated with Clinically Common Non-Hodgkin Lymphomas Are Infrequent (

AbstractAbstract 2002 Introduction:Monoclonal B-cells with a Chronic Lymphocytic Leukemia (CLL) phenotype are detectable in more than 10% of adults in the general population using high sensitivity flow cytometry assays designed to detect minimal residual disease after treatment. However, the prevalence of MBL with a phenotype corresponding to other B-lymphoproliferative disorders (B-LPD) is less than 2% of the general population even using the most sensitive assays. No studies have reported CD10+ MBL whereas several studies have demonstrated that the t(14;18) is frequently detectable in the general population at a level which should be detectable by flow cytometry. The lack of CD10+ MBL may indicate that the t(14;18) alone rarely results in the expansion of a clonal B-cell population in the blood, or that currently available assays are inadequate for detecting circulating follicular lymphoma. We have previously investigated 66 markers to determine the best candidates for diagnosis and monitoring B-LPD, which were then tested in over 1500 cases. We have developed a single-tube assay to screen for residual disease that can detect lymphoma cells when they represent as few as 1 in 10,000 leucocytes. The aim of this study was to asses the frequency with which lymphoma-phenotype monoclonal B-cells are detected in the general population using a high sensitivity assay. Methods:Cells from 679 individuals (342 male, 337 female, median age 64, range 40–99) with a normal blood count and no current or prior history of cancer were incubated with antibodies to Kappa, Lambda, CD19, CD20, CD5, CD10, LAIR1, CXCR5 and 0.5 million cells were acquired using a BD FACSCanto II cytometer. In cases with detectable MBL further phenotyping was performed and B-cells were selected and stored for FISH and molecular clonality studies. Results:MBL was detected 129/679 cases (19.0%): CLL-type MBL in 86/679 cases (12.7%), non-CLL MBL in 60/679 cases (8.8%) with both CLL-type and non-CLL MBL were present in 17/679 cases (2.5%). Within the non-CLL MBL group, in 21/60 cases the monoclonal B cells had no additional features to confirm a neoplastic population and it was not possible to ascertain whether these were neoplastic cells or a reactive population with a highly skewed kappa/lambda ratio. Of the remaining 39 specimens: none showed evidence of germinal centre differentiation; 12 (1.7% of total) showed a phenotype most consistent with marginal zone lymphoma/lymphoplasmacytic lymphoma; and 27 cases expressed strong levels of LAIR1 coupled with strong CD19 and CD20 and an extended phenotype that is relatively rare in clinical B-LPD, restricted to hairy cell leukemia and a small proportion (<15%) of marginal zone lymphomas. Conclusions:Using an assay designed for detection of residual disease in lymphoma it was possible to detect non-CLL phenotype MBL in 8.8% of individuals over 60 years of age with normal blood counts and no history of cancer. Only a small number of these cases had a phenotype comparable to follicular lymphoma or marginal zone lymphoma, which constitute the majority of clinically significant indolent B-LPD. The complete absence of cases with a germinal centre phenotype contrasts with the high frequency of detection of BCL2-IGH rearrangement by PCR in the peripheral blood of healthy individuals. The flow cytometry assay used in this study readily detects circulating cells with appropriate phenotype in patients known to have follicular lymphoma, diffuse large B-cell lymphoma or marginal zone lymphoma/lymphoplasmacytic lymphoma and the data indicate a high specificity for peripheral blood based diagnosis and monitoring in these conditions. The most clinically prevalent lymphomas are rarely detectable in peripheral blood in individuals without lymphadenopathy but there is a surprisingly frequent development in the general population of both CLL and a restricted subset of marginal zone lymphomas which may indicate a common developmental pathway. Disclosures:No relevant conflicts of interest to declare.