B-cell Lymphoproliferative Disorders Research Articles

An overview of prognostic markers in patients with CLL

Chronic lymphocytic leukemia (CLL) is a low-grade B-cell lymphoproliferative disorder. It is the most prevalent type of leukemia in the western countries, with a median age at diagnosis of 70 years. In 2023, it is estimated that there will be 18,740 new cases of CLL, and an estimated 4,490 people will die of this disease. It represents 1.0% of all new cancer cases in the U.S. The rate of new cases was 4.6 per 100,000 men and women per year based on 2016–2020 cases, age-adjusted. Death rates from CLL are higher among older adults, or those 75 and older. The death rate was 1.1 per 100,000 men and women per year based on 2016–2020 deaths, age-adjusted. A common question that patients with CLL ask during their first clinic visit is: “How long will it be before I would need treatment?” Although this might seem like a simple question, the answer is not straight forward. CLL is a heterogenous disease, with a variable clinical course. Some patients may present with an aggressive disease requiring early initiation of treatment, while others have an indolent course and some, having so called smoldering CLL, may never need treatment. The variability in disease course can make predicting disease prognosis a complicated process. This brings forth the importance of establishing prognostic models that can predict disease course, time to treatment, and survival outcomes in such a heterogenous disease. The Rai and Binet staging systems were developed in the late 1970s to early 1980s. They separated patients into different stages based on clinical characteristics and laboratory findings. These simple staging systems are still in use; however, several prognostic markers need to be added for an individualized assessment and, with the recent development of genomic techniques leading to better understanding of CLL at the molecular level, newer prognostic markers have emerged.

Outcome of Hairy Cell Leukemia: A Single-center Study

Abstract: BACKGROUND: Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder. Modern therapy with purine analogs and immunotherapy can provide long-term remission, but the risk of recurrence remains about 40%–50%. The aim of this study was to evaluate the outcome of patients with HCL who received treatment in Nanakali Hospital. PATIENTS AND METHODS: A retrospective cross-sectional study was carried out on 50 patients of HCL diagnosed from 2004 to 2022 in Nanakali Hospital in Erbil City, Kurdistan Region, Iraq. Demographics, clinical presentation, treatment data, complications, response, recurrence, and survival data were collected from medical records. The results were presented with descriptive statistics. Variables were compared by Chi-square analysis. RESULTS: The mean age was 52.64 ± 12.37 years, and 84% were male. The most common presenting symptoms were splenomegaly (18%) and fatigue (14%). The majority (69.6%) received cladribine; the response rate was 73.9%, with a complete remission (67.4%). 47.8% had recurrent disease. The most common adverse effects were febrile neutropenia (58.7%) and Grade III and IV hematologic toxicity (41.3%). The results were significantly associated with ANC pretreatment (P = 0.019), comorbidity (P = 0.001), and treatment response (P = 0.004). Cladribine–rituximab combination resulted in complete remission (100%). Ten-year overall survival was 70%. CONCLUSIONS: The results were broadly consistent with literature reports, demonstrating the efficacy and safety of cladribine with/without rituximab as first-line therapy for HCL but with a 30% mortality of concern. Further studies should identify modifiable factors that affect poor prognosis in subgroups to guide improvements in risk management of HCL.

EBV-RELATED LYMPHOPROLIFERATIVE DISEASES: A REVIEW IN LIGHT OF NEW CLASSIFICATIONS

Epstein-Barr virus (EBV) is a prevalent virus that can be detected in the vast majority of the population. Most people are asymptomatic and remain chronically infected throughout their lifetimes. However, in some populations, EBV has been linked to a variety of B-cell lymphoproliferative disorders (LPDs), such as Burkitt lymphoma, classic Hodgkin lymphoma, and other LPDs. T-cell LPDs have been linked to EBV in part of peripheral T-cell lymphomas, angioimmunoblastic T-cell lymphomas, extranodal nasal natural killer/T-cell lymphomas, and other uncommon histotypes. This article summarizes the current evidence for EBV-associated LPDs in the light of the upcoming World Health Organization classification and the 2022 ICC classification. Keywords: Epstein Barr Virus: EBV, Lymphoma; Lymphoproliferative Diseases.WHO Classification.

An unfavorable and a successful pregnancy outcome during and after treatment of gamma heavy chain disease.

Gamma heavy chain disease (gHCD) is a rare B-cell lymphoproliferative disorder that mostly occurs after childbearing age. Here we report the first case of gHCD in a pregnant patient that was diagnosed in the second trimester, and another pregnancy in the same patient after initial treatment for gHCD. The former pregnancy ended in intrauterine fetal death, believed to be caused by insufficient maternal blood flow due to multiple placental infarcts. The latter pregnancy course was uneventful. Although we cannot rule out the possibility that the poor outcome of the former pregnancy was due to an unfortunate complication independent of gHCD, the courses of these pregnancies suggest that non-lymphomatous gamma heavy chain may have a significant impact on pregnancy and that its removal by treatment may improve outcomes.

BET bromodomain inhibitors PFI-1 and CPI-203 suppress the development of follicular lymphoma via regulating Wnt/β-catenin signaling

ObjectiveFollicular lymphoma (FL) is an indolent B-cell lymphoproliferative disorder, characterized by a lymphoid follicular pattern of growth. PFI-1 or CPI-203 has been known to effectively promote the inhibition of primary effusion lymphoma progression. This study aimed at investigating the anti-tumor properties of PFI-1 and CPI-203 on FL cells and uncover the underlying mechanism of action. MethodsFL cells were treated with PFI-1 and CPI-203, and the treated cells were evaluated for their cell viability, cell cycle and apoptosis using CCK8, flow cytometry, and Western blot assays. A xenograft mouse model was used for assessing the in vivo effects of CPI-203 on tumorigenesis. ResultsPFI-1 or CPI-203 showed potential inhibitory effects on the cell viability of DOHH2 and RL cells in a dose-response-dependent manner. Furthermore, PFI-1 and CPI-203 inhibited cell growth, induced apoptosis of FL cells in vitro, and facilitated the translocation of β-catenin into cytoplasm both in vitro and in vivo. After engrafted with FL cells, CPI-203-treated mice got a longer duration of survival and a smaller tumor size than control mice. Mechanistically, PFI-1 and CPI-203 impede the activity of β-catenin and its downstream molecules by regulating the DVL2/GSK3β axis. ConclusionIn conclusion, PFI-1 and CPI-203 may serve as potential anti-tumor inhibitors for the therapy of FL.

Features and Factors Associated With Myeloid Neoplasms After Chimeric Antigen Receptor T-Cell Therapy

This case-control study examines the incidence and risks of myeloid neoplasms in adults treated for B-cell lymphoproliferative disorders or multiple myeloma.

Submucosal Epstein-Barr Virus Positive Polymorphic B-cell Lymphoproliferative Disorder of the Larynx: A Case Report.

Epstein-Barr virus (EBV) belongs to the group of human herpes virus and can cause clinical and subclinical infections. Although EBV-related disease presentations are similar, they can lead to oncogenic transformation with various clinical manifestations. A thorough workup with morphology, immunohistochemistry, and molecular studies is crucial for the diagnosis of EBV-positive polymorphic B-cell lymphoproliferative disorder, not otherwise specified (NOS), which is a new entity introduced by International Consensus Classification in 2022. We describe an interesting presentation of EBV-positive polymorphic B-cell lymphoproliferative disorder with laryngeal involvement to bring awareness to this entity and we would like to address the need for more accessible treatment options.

HIV-associated CD8+ T-cell Skin Infiltrative Disease and EBV-associated Polymorphic B-cell Lymphoproliferative Disorder in an AIDS Patient Who Improved Dramatically with Antiretroviral Therapy Alone.

Human immunodeficiency virus (HIV)-associated CD8+ T-cell skin infiltrative disease with severe erythroderma has rarely been reported. While HIV-positive patients are prone to develop lymphoma, which is often associated with Epstein-Barr virus, polymorphic lymphoproliferative disorder is rare, accounting for <5% of cases. We herein report a 41-year-old HIV-positive man who presented with a fever, erythroderma, and lymphadenopathy and was diagnosed with the coexistence of both diseases. His condition improved significantly with continued antiretroviral therapy. This case suggests that HIV-induced immunodeficiency is central to the pathogenesis of both entities and that improvement of the immunodeficient state is an effective treatment.

Epstein-Barr virus-positive mucocutaneous ulcer in a patient with ulcerative colitis: a recently described entity to take into account.

Epstein-Barr virus-positive mucocutaneous ulcer (EBVMCU) is a new entity recently included in the classification of B-cell lymphoproliferative disorders associated with Epstein-Barr virus (EBV). It is related to immunosuppression and it usually appears in the oropharynx or the skin, being the colon an uncommon location. We present the case of a 31-year-old man with ulcerative proctitis being treated with azathioprine (AZA) and adalimumab (ADA), who was admitted to the hospital due to suspicion of a moderate-severe flare of ulcerative proctitis. Microbiological stool analyses were negative, with a positive fecal calprotectin test (2700 mg/kg). Rectoscopy showed severe endoscopic activity, taking multiple biopsies. Intravenous steroids were started at a dose of 60 mg/day. He presented a favorable clinical and analytical evolution, being discharged from the hospital. The histological results were received at gastroenterology consultation, being compatible with EBVMCU. AZA and ADA were withdrawn, whereas descending steroid regimen and oral and topical mesalazine were continued, being the clinical response adequate.

Deep Characterization of Immune Dysfunction in Patients with Multiple Myeloma (MM) and Identification of Cellular Biomarkers for Tailored Vaccination Strategies

Background: Infection is the leading cause of death in MM. However, the extent of immune dysfunction compared to other B-cell lymphoproliferative disorders (B-CLPD) and healthy adults remains poorly studied. Greater knowledge is needed for tailored vaccination strategies and to improve outcomes upon new pathogens or breakthrough infections. Aim: Generate an atlas of the basal immune status and its response to vaccination in MM vs B-CLPD patients and age-matched health care practitioners (HCP), using the COVID-19 mRNA vaccines as a case study. Methods: A total of 1,099 blood and serum samples were collected from 177 individuals: 28 MM, 53 B-CLPD and 96 HCP older than 50. They were studied before COVID-19 mRNA vaccination, at days 7 and 14 after the first dose, at days 7 and 62 after the second dose, as well as before and at day 17 after the booster. Immune profiling was performed using multidimensional and computational flow cytometry that systematically analyzed 56 immune cell types per sample and time point: 17 B, 30 T, 6 antigen-presenting cell (APC) and 3 granulocytic subsets. Serum levels of IgM, IgG and IgA against the receptor-binding domain (RBD) of the spike (S) glycoprotein, S glycoprotein, nucleocapsid (N) and main protease were quantified using a multiplex-microsphere-based flow cytometry assay. SARS-CoV-2-specific CD8 T cells were quantified using a dextramer panel of S, N, membrane, and ORF3 proteins. Results: When compared to HCP and/or B-CLPD, MM patients showed abnormal distribution of 17/17 B, 22/30 T, 4/6 APC and 1/3 granulocytic cell subsets prior to vaccination. The most deviated cell types in MM were naïve CD21+ B-cells, naïve CD4 T-cells, PD1- and PD1+CD127low effector memory (EM) CD8 T cells, classical monocytes and neutrophils. The B cell compartment of MM patients showed impaired response to COVID-19 mRNA vaccination. While B-CLPD patients and HCP displayed significant expansions of naïve CD21-, IgM+IgD+CD27+CD21-, IgG+CD27-CD21-, and IgG+CD27+CD21- B-cell subsets, as well as of IgA+ circulating plasma cells, such expansions were not observed in MM patients. Accordingly, anti-RBD IgM, IgA and IgG titers were significantly reduced in MM compared to B-CLPD and HCP after the second dose ( P ≤.002). Importantly, the booster increased anti-RBD IgG levels in HCP (20,184 to 186,629 IU/mL, P &amp;lt;.001) but not in MM (1,991 to 5,998 IU/mL, P =.14) and B-CLPD (33,548 to 41,288 IU/mL, P =.19) patients. The immune response of the T-cell compartment was also altered in MM patients. The significant expansion of CD127lowPD1+ and CD127+CD25+ EM CD4, naïve CD4 and CD127lowPD1+ EM CD8 subsets observed in HCP was unnoted in MM and B-CLPD patients. Accordingly, the percentage of virus-specific CD8 T cells after the second dose did not increase in MM and B-CLPD patients. The booster increased virus-specific CD8 T cells in HCP (0.08 to 0.14%, P =.02), but not in MM (0.08 to 0.09%, P =.94) and B-CLPD (0.23 to 0.16%, P =.85). Furthermore, the booster induced virus-specific CD8 T cell differentiation into an EM phenotype in HCP but not in MM and B-CLPD patients. In addition to the dysfunctional B and T cell response to vaccination, MM patients showed abnormal kinetics of APC such as intermediateclassical and non-classical SLAN- and SLAN+ monocytes, as well as of granulocytic subsets such as basophils and neutrophils. Based on differences in immune-cell distribution using HCP as a reference, we calculated an immune dysregulation longitudinal cumulative score in each individual that included 48,496 immune parameters. Compared with HCP, up to 34% MM and 17% B-CLPD patients showed high immune dysregulation scores. Among them, 75% and 33%, respectively, had low seroconversion after the second dose. We found 7/30 T and 14/17 B-cell subsets associated with poor vaccine response in MM patients, namely CD127lowPD1+CXCR5+ EM CD8 T cells, as well as CD21- and CD21+ naïve, transitional, IgM+IgD+CD27+CD21+, IgM+CD27-CD21+, and IgG+CD27-CD21+ B cells. Conclusion: We provide an atlas of the immune dysfunction in MM patients and how it affects the efficacy of vaccination strategies such as for COVID-19. The schedule of vaccine doses may thus benefit from individualization according to patients' immune status, which could act as a surrogate of host, tumor and treatment-related immune dysfunction. Accordingly, we have identified key cell types for readily monitoring of patients' immune status using routinely available flow cytometry.

Targeting EBV Encoded Viral Kinases with GCV, AZT, Rituximab and Dexamethasone (GARD) Results in Durable Responses in Patients with CNS Lymphoproliferative Disease

Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. EBV+ CNS-LPD often occurs in the setting of immune suppression and comorbidities (solid organ transplant, autoimmunity). Treatment with high-dose methotrexate has increased survival but is not a viable option for all patients, highlighting the need for alternative treatments. Previous research has reported EBV+ CNS-LPD exhibits unique genetic and immunobiological signatures, however, few studies have reported on the epigenetic landscape. We have previously reported use of ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone (GARD) regimen induced complete and durable responses in a cohort of 13 patients with primary CNS post-transplant lymphoproliferative disease (PCNS-PTLD). Here we present 24 patients (10 from prior cohort and 14 new) with EBV+ CNS-LPD treated with GARD. We extend follow-up time for the previous cohort and add molecular context to clinical observations. We performed DNA methylation and expression analysis of EBV viral kinases in available tumor samples and revealed unique viral epigenetic states leading to expression of antiviral drug targets in an array of EBV+ CNS-LPD. Methods: We conducted an IRB-approved, retrospective review of patients with EBV+ CNS-LPD treated with GARD at The Ohio State University Wexner Medical Center between 1998-2022. Patient information was extracted from the electronic medical record. Treatment consisted of two-week induction of twice daily IV GCV/AZT (5mg/kg and 1,500 mg) IV dexamethasone (10-40mg), and weekly rituximab (375mg/m 2). After induction, dexamethasone was tapered, and GCV/AZT was switched to maintenance oral dosing of 450mg valganciclovir twice daily and 300mg of AZT twice daily. Rituximab was administered on days 1, 8, 15, and 22. Two-year overall survival (OS), five-year OS, and overall response rate were assessed. Available CNS-LPD biopsy samples were obtained, and expression of EBV lytic genes BZLF1, BXLF1 (thymidine kinase), and BGLF4 (protein kinase) were measured via qRT-PCR. Mass spectrometry based EpiTYPER assay was used to report the percentage of DNA CpG methylation in the gene promoters for viral BZLF1, BXLF1, BGLF4 and LMP1 genes . Biopsies from 17 patients with systemic PTLD with no CNS involvement were used for comparison. Results: 24 patients with EBV+ CNS-LPD were identified. 21 (87.5%) patients were diagnosed with a primary CNS-LPD (PCNS-LPD). Of the PCNS-LPD, 14 patients were PCNS-PTLD, 4 were receiving immunosuppression for autoimmune conditions, 1 patient had type 2 diabetes and two had no known history of immunosuppression. The remaining three patients had systemic PTLD with secondary CNS involvement. Diffuse large B-cell lymphoma was the most common histology (11; 45.8%) followed by grade 3 lymphomatoid granulomatosis (5; 20.8%), B-cell LPD (5;20.8%), polymorphic PTLD (2;8.3%) and one large cell lymphoma of ambiguous lineage. All patients were HIV negative. EBER was positive in all biopsies and EBV PCR of cerebrospinal fluid was positive in patients without biopsy. Sixteen patients (66.7%) achieved a completed response, with an overall response rate of 83%. Two-year OS was 62.5% (95% CI, 45.8-85.2%) and five-year OS was 52.8% (95% CI, 35.7%-77.9%). Five CNS-LPD brain biopsies were available for analysis. qRT-PCR of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BGLF4 was significantly higher (p=1.35e-5) in CNS-LPD when compared to systemic EBV+ PTLD. EpiTYPER data showed significantly decreased promoter methylation for viral kinases BXLF1 and BGLF4 (p=0.0281 p=0.0089). Conclusions: We have shown that GARD regimen shows efficacy in the treatment of EBV+ CNS-LPD, with 62.5% two-year OS and 52.8% five-year OS. Our results provide the molecular basis for expression of lytic viral kinases BXLF1 and BGLF4 in CNS-LPD by showing that both genes exhibit decreased promotor methylation. This expression of viral kinases in CNS-LPD supports the mechanistic rationale of this antiviral approach and could be further investigated as a predictive biomarker. Overall, this research highlights unique epigenetic features of EBV in CNS-LPD that support the use of antiviral, specifically the GARD regimen, in EBV+ CNS-LPD.

Establishment of the Bcwm.2 Cell Line As a BTK-Inhibitor Resistant, BCL2 Inhibitor Sensitive in Vitro and In Vivo Study Model for Waldenström's Macroglobulinemia

Background: Waldenström's macroglobulinemia (WM) is an indolent B-cell lymphoproliferative disorder characterized by bone marrow (BM) infiltration with lymphoplasmacytic lymphoma and monoclonal immunoglobulin M (IgM) production. Mutations in MYD88 are present in 95-97% of WM patients. Deletions involving chromosome 6q (del6q) are found in up to 50% of WM patients, and include genes that regulate MYD88, BCL2 and apoptotic signaling. Cell lines can play a pivotal role as disease models, contributing to a comprehensive understanding of WM biology and advancing therapeutic strategies. The availability of cell lines for the study of WM remains limited, and none demonstrate del6q. Patient and Methods: We developed and characterized a novel cell line (BCWM.2) from long-term cultures of CD19 + selected BM lymphoplasmacytic cells from a symptomatic, treatment naive WM patient who then received ibrutinib monotherapy on a clinical trial. The patient attained a major response to treatment, and after 2 years progressed. The patient underwent serial bone marrow biopsies, and extensive genomic and transcriptome analysis of his tumor during the study, and the corresponding cell line established at baseline. An in vivo xenograft mouse model of BCWM.2 was also established in NOD SCID mice, and the origin of the xenografted tumor was confirmed by genomic sequencing and immunohistochemistry. Results: BCWM.2 cells exhibited morphologic and immunophenotypic characteristics resembling lymphoplasmacytic cells and demonstrated robust propagation when co-cultured with HS-5 stromal cells in IMDM medium supplemented with 20% FBS. Flow cytometric analysis revealed that BCWM.2 exhibited an immunophenotype consistent with the source WM patient. Whole genome sequencing demonstrated that both BCWM.2 cells and original patient WM cells carried somatic activating mutation in MYD88 (S243N) and shared trisomy in chromosomes 3 and 12, heterozygous deletion of 6q, and amplification of 6p. Notably, a novel mutation in the SRC family member LYN (I297N; T&amp;gt;A), a component of BCR, was identified in BCWM.2 but not the original patient tumor. The I297N mutation found in the regulatory hinge region is predicted to cause activation of LYN. At time of progression on ibrutinib, the dominant clone that emerged in the patient's tumor also carried the I297N mutation. Additionally, mutated genes in BCWM.2 included histone deacetylase HDAC5, tumor suppressor RUNX3, B-cell transcriptional activator SPI1, and AKAP9 (A-Kinase Anchoring Protein 9). Transcriptome analysis showed shared expression patterns between BCWM.2 and patient tumor samples, including diminished gene expression of chromosome 6q. Drug response testing revealed that BCWM.2 cells did not respond to the BTK-inhibitors ibrutinib, zanubrutinib or pirtobrutinib but were sensitive to the BCL2 inhibitor venetoclax. Furthermore, BCWM.2 cells readily engrafted in NOD-SCID mice by direct subcutaneous injection of 2 x 10^6 cells/mouse. Tumor growth (~500 mm^3) was observed in the mice four weeks post-inoculation. Importantly, follow-up evaluations demonstrated serial increases in human IgM levels in the mouse sera post-engraftment. Moreover, BCWM.2 engraftment was confirmed by immunohistochemistry with tumors exhibiting intermediate lymphoid cell morphology with plasmacytoid differentiation, CD20 and MUM1 expression, clonality for lambda light chain and IgM heavy chain, 60% KI67 staining. Whole exome sequencing of engrafted tumors further confirmed the presence of the MYD88 S243N and LYN I297N mutations detected in the cell line and the patient at the time of ibrutinib progression. Conclusions: BCWM.2 represents a novel, BTK-inhibitor resistant, BCL2 inhibitor sensitive WM cell line that demonstrates MYD88 (S243N) and LYN (I297N) somatic activating mutations, and deletions of 6q. BCWM.2 thereby provides an important new disease appropriate cell model for the in vitro and in vivo study of WM, including the development of targeted therapies for this disease.

Polytypic B-Cells, B-Cell Lymphoproliferative Disorders/Lymphomas, and Neoplastic T-Cells Divergently Differentiate from TET2-/ DNMT3A-Mutated Clonal Hematopoiesis in Patients with Follicular Helper T-Cell Lymphomas/Lymphoproliferative Disorders

Introduction: Patients with follicular helper T-cell lymphomas/lymphoproliferative disorders (TFH-LPDs) commonly develop B-cell lymphoproliferative disorders and lymphomas (B-LPDs), but the pathophysiology is incompletely understood. TFH-LPDs often arise from TET2-/ DNMT3A-mutated clonal hematopoiesis (CH). Here we show TET2/ DNMT3A mutations are commonly shared by polytypic B-cells (PBCs), B-LPDs, and TFH-LPDs, with the latter two often carrying additional unique mutations. Methods: Pathology database search at Memorial Sloan Kettering identified patients with TFH-LPDs and PBCs or B-LPDs available for genotyping. If available, myeloid cells were also genotyped. PBCs lacked aberrancies by morphology and flow cytometry (FC) immunophenotyping (including polytypic light chain expression). TFH-LPDs and B-LPDs were defined using International Consensus Classification criteria. Genotyping assays included targeted next-generation sequencing (NGS) with (n=13) or without (n=8; cases 1, 4, 9, 10, 15, 16, 17, 22) a matched germline control and droplet digital PCR (n=4; cases 2, 3, 5, 6). Genotyping was performed on bulk samples or FC-sorted cells, including T-, B-, and myeloid as available. In bulk samples, mutations with allele frequencies (VAFs) &amp;gt;2x the percentage of neoplastic T- and myeloid cells combined in B-LPD samples and of neoplastic T- and total B-cells in bulk blood (PB)/bone marrow (BM) samples were considered present in B-LPD or myeloid compartments, respectively. A &amp;gt;2% VAF cutoff was utilized. EBV status was assessed using EBER in situ hybridization on tissues, serum EBV DNA by quantitative PCR, and/or evaluation of off target EBV reads by NGS. Statistical analyses utilized Mann-Whitney U tests. Results: In total, 25 patients (10 female, 15 male; median age 70 [range 38-82] years) with TFH-LPDs (19 TFH lymphoma, angioimmunoblastic type, 4 TFH lymphoma, NOS, 2 T-LPD with TFH phenotype [defined by morphologically and/or immunophenotypically atypical T-cells with both TFH-like phenotypes and genomic alterations that did not fulfill morphologic criteria for diagnosis of lymphoma]) were identified. The TFH-LPDs harbored TET2 (25/25, 100%), DNMT3A (12/25, 48%), RHOA p.G17 (15/25, 60%), and IDH2 p.R172 (9/25, 36%) mutations (Figure 1A). PBCs from 11 patients were evaluated (7 EBV+, 3 EBV-, 1 EBV unknown; 5 from lymph node, 4 PB, 2 BM). PBCs from 7/11 (64%) shared identical TET2 (n=6; median VAF 0.11 [range 0.02 - 0.27]) and/or DNMT3A (n=3; median VAF 0.05 [range 0.03-0.49]) mutations with corresponding TFH-LPDs. PBCs in 1 patient harbored a unique mutation ( TET2). Four of 8 (50%) patients in which myeloid cells were assessed harbored identical TET2 and/or DNMT3A mutations in the myeloid, PBC, and TFH-LPD compartments. B-LPDs were evaluated from 14 patients (5 diffuse large B-cell lymphoma, 5 polymorphic B-LPD, 2 follicular lymphoma, 1 B-LPD, NOS, 1 plasma cell myeloma; 11 EBV+, 3 EBV-; 9 from tissue, 4 PB, 1 BM). Polymorphic B-LPDs showed atypical polymorphic morphology and an abnormal B-cell immunophenotype by FC and/or a clonal IgH gene rearrangement. The B-LPD, NOS showed an abnormal CD5-/CD10- immunophenotype with light chain restriction by FC without available tissue for morphologic evaluation. Nine of 14 (64%) B-LPDs shared identical mutations with corresponding TFH-LPDs ( TET2 [n=9; median VAF 0.42 (range 0.10- 0.56)], DNMT3A [n=5; median VAF 0.27 (range 0.05-0.50)], TET3 [n=1; VAF 0.32]). Myeloid compartments in 9/12 (75%) patients shared identical TET2 and/or DNMT3A mutations with B- and/or T-LPDs. B-LPDs more commonly harbored additional mutations absent in corresponding TFH-LPDs than PBCs (12/14 [86%] vs 1/8 [13%] evaluated by NGS; p=0.001). Unique mutations in B-LPDs involved epigenetic/transcriptional regulation (n=14, e.g. TET2, KMT2D, SETD5), signaling pathways (n=22, e.g. DTX1, KRAS, EPHA5), and DNA damage response (n=3, e.g. ATM, CHEK2, BRCA2). Incidences of shared and unique mutations did not significantly differ among EBV+ and EBV- cases. Conclusions: Both PBCs and B-LPDs in TFH-LPD patients are commonly EBV+ and share TET2/ DNMT3A mutations with TFH-LPDs, consistent with common origin from CH. B-LPDs more often harbor additional unique mutations than PBCs. Thus, CH mutations and EBV activation in B-cells may predispose TFH-LPD patients to B-LPDs, which may develop with additional private genomic aberrations aiding transformation (Figure 1B).

Investigating the Influence of Germline ATM Variants on Susceptibility to Chronic Lymphocytic Leukemia and Other Malignancies

Introduction: Chronic lymphocytic leukemia (CLL) patients (pts) face an increased susceptibility to secondary cancers. Furthermore, the hereditary predisposition to CLL is influenced by factors that have yet to be fully elucidated by GWAS-identified common variants. Among the hereditary cancer risk mutations observed, germline Ataxia-telangiectasia mutated ( ATM) aberrations are among the most prevalent. Rare germline ATM variants are detected in 24% of CLL pts, surpassing the frequency observed in other hematologic neoplasms and the general population. In CLL, loss of ATM via 11q23 deletion is linked to an unfavorable prognosis and a shortened progression-free survival. The extent to which germline ATM variants predispose CLL pts to other malignancies remains unclear; to examine this potential risk we conducted a retrospective study to assess the influence of germline ATM variants on the predisposition to secondary neoplasms in CLL pts and their relatives. Methods: Five hundred and eighty-five pts seen at Dana-Farber Cancer Institute and who had NGS performed to evaluate germline ATM status, either through direct germline sequencing of saliva or by inference according to the hierarchical algorithm we have previously published (Lampson, 2022), were mailed a questionnaire that investigated: demographics; personal and family history of any cancer; non-medical radiation and Agent Orange exposure; and Ataxia-Telangiectasia syndrome (AT). Of these, 333 replied (57%). Information collected from our CLL database included FISH, IGHV status and treatment status. European ancestry was categorized based on the United Nations geoscheme (UNSD, 2019). Pt characteristics are in Table 1. Pts were stratified into 4 groups, based on ATM mutational status and del(11q): Group-1, ATM germline variants alone (N=63); Group-2, ATM germline with somatic ATM mutation and/or del(11q)(N=22); Group-3, ATM somatic aberration alone (including del(11q))(N=41); and Group-4, no ATM aberration (N=207). Results: Of the 333 pts in our cohort, baseline demographic and clinical characteristics among the 4 groups were balanced, except for unmutated IGHV status. Nineteen (86%) and 36 (86%) pts of groups 2 and 3, carrying somatic ATM aberrations, had unmutated IGHV, compared to 21 (33%) and 76 (37%) of groups 1 and 4, without somatic ATM aberrations(p&amp;lt;0.01). Regarding treatment, 60% of our cohort had received at least one line of therapy (median: 1 (1-7)). Among the 164 pts (49%) with history of an additional non-CLL neoplasm, 221 cancers were reported. Excluding pts with only non-melanoma skin cancers, the incidence is reduced to 31% (104 pts). The median number of second cancers per pt is 1 (1-4). Of the 134 pts with available age of onset data for the secondary malignancy, 80 (60%) were diagnosed with the secondary cancer after CLL. Comparing pts with germline ATM variants (N=85) to the ones without germline ATM variants (N=248), no significant difference in the incidence of secondary cancer was found (p=0.73). The median age of secondary cancer onset was also similar between the groups (median: 63 and 64 years in germline ATM present vs absent, p=0.28). Of the whole cohort, 283 pts (85%) reported at least one relative with a cancer history (median 2 relatives affected (1-17)). The incidence of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in pts with germline ATM variants (32%) compared to those without germline ATM variants (21%), including familial CLL (25% vs18%) (p=0.04). Familial CLL was also strongly associated with Ashkenazi Jewish ethnicity (39% incidence in Ashkenazi pts vs 16% in non-Ashkenazi, p=0.004). No difference in the incidence of other hematologic or solid malignancies was found between the relatives in the two groups. No pts reported a familial or personal history of AT. Compared to pts without any ATM aberration, time to first treatment (TTFT) was shorter in pts harboring somatic ATM events, while no difference was observed between pts with germline ATM variants (median TTFT: 82, 59, 52, 90 months for group 1, 2, 3 and 4 respectively, p=0.01) (Figure 1). Conclusions: Our results suggest a higher incidence of B-cell lymphoproliferative disorders, including familial CLL, in the relatives of CLL pts carrying germline ATM variants. The presence of these germline variants did not impact TTFT, compared to pts harboring somatic ATM mutations.

Leukostasis Twist in Mantle Cell Lymphoma: Systematic Review and Case Report

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma. Patients often present with lymphadenopathy, early satiety, and B-symptoms. Despite up to 92% of MCL patients having peripheral blood involvement by flow cytometry, presentation with leukemic involvement and hyperleukocytosis are uncommon. Hyperleukocytosis, marked by a white blood count (WBC) &amp;gt;100 x 10 9/L, often leads to symptomatic leukostasis due to tissue hypoxia, generally found in acute leukemias. We conducted a systematic review of leukostasis in MCL, and present a case we encountered in our clinical practice of a non-nodal MCL variant showing leukostasis, spontaneous tumour lysis syndrome (TLS), and severe anemia to identify treatment strategies and outcomes. Methods: We searched PubMed for peer-reviewed articles and abstracts on “mantle cell lymphoma,” “hyperleukocytosis,” and “leukostasis.” All articles and abstracts that discuss hyperleukocytosis leading to leukostasis in MCL were included. Results: The search yielded eight results. After exclusions, five case reports and abstracts were included. These studies featured different MCL variants, presenting WBCs ranging from 121 to 1227 x 10 9/L, and various leukostasis symptoms. Leukapheresis was used in all five published cases, leading to lower WBCs and symptom improvement. Results are summarized in Table 1. Our patient was found to have the small lymphocyte variant of MCL, with WBC of 543.3 x 10 9/L on presentation. Our facility does not have leukapheresis capability. As a result, our initial treatment consisted of only supportive care and escalating doses of corticosteroids, with close monitoring for TLS. To our knowledge, this report is the first demonstration that IV methylprednisolone, without leukapheresis, is effective for acute management of leukostasis. Our patient's WBC reduced from 543.3 x 10 9/L to 230 x 10 9/L, leading to improved neurological function and oxygenation. Steroids were particularly useful in our patient with secondary, MCL-related warm autoimmune hemolytic anemia. Conclusion: This review highlights the importance of recognizing non-specific leukostasis symptoms, even in patients with mature B-cell lymphoproliferative disorders. Leukapheresis is effective in treating leukostasis in MCL patients. In hospitals without leukapheresis, escalating doses of IV methylprednisolone can be considered as a safe and effective strategy.

A RESPON KEMOTERAPI FLUDARABINE PADA HAIRY CELL LEUKEMIA

HCL is part of a rare B-cell lymphoproliferative disorder and is a subtype of Chronic Lymphocytic Leukemia. It is characterized by progressive pancytopenia, splenomegaly, and bone marrow hypercellularity. Patients often come because of abnormal complete blood count results or complications such as infection. This case report explains a case of a 41-year-old woman who had recurrent shortness of breath and cough as symptoms, where the patient had a history of acute myeloblastic leukemia (AML) with non-routine fludarabine chemotherapy. After receiving treatment and underwent further investigations in the form of peripheral blood smear and bone marrow aspiration examination, it was found that the patient had hairy cell leukemia. Fludarabine was given to the patient and it is reported to give effect partial remission (based on last bone marrow aspiration) along with pancytopenia as a clinical manifestation from myelosuppression as a side effect for long-term used fludarabine.&#x0D; HCL is part of a rare B-cell lymphoproliferative disorder and is a subtype of Chronic Lymphocytic Leukemia. It is characterized by progressive pancytopenia, splenomegaly, and bone marrow hypercellularity. Patients often come because of abnormal complete blood count results or complications such as infection. This case report explains a case of a 41-year-old woman who had recurrent shortness of breath and cough as symptoms, where the patient had a history of acute myeloblastic leukemia (AML) with non-routine fludarabine chemotherapy. After receiving treatment and underwent further investigations in the form of peripheral blood smear and bone marrow aspiration examination, it was found that the patient had hairy cell leukemia. Fludarabine was given to the patient and it is reported to give effect partial remission (based on last bone marrow aspiration) along with pancytopenia as a clinical manifestation from myelosuppression as a side effect for long-term used fludarabine.

Effective Neutralizing Antibody Response Against SARS-CoV-2 Virus and Its Omicron BA.1 Variant in Fully Vaccinated Hematological Patients

SARS-CoV-2 and its variants cause CoronaVIrus Disease 19 (COVID-19), a pandemic disease. Hematological malignancies increase susceptibility to severe COVID-19 due to immunosuppression. Anti-SARS-CoV-2 neutralizing antibodies protect against severe COVID-19. This retrospective real-life study aimed to evaluate seropositivity and neutralizing antibody rates against SARS-CoV-2 and its Omicron BA.1 variant in hematological patients. A total of 106 patients with different hematologic malignancies, who have mostly received three or more vaccine doses (73%), were included in this study. Serum was collected between May and June 2022. The primary endpoint was anti-SARS-CoV-2 antibody response against ancestral (wild type; wt) and Omicron BA.1 virus, defined as a neutralizing antibody titer ≥ 1:10. Adequate neutralizing antibody response was observed in 75 (71%) and 87 (82%) of patients for wt and Omicron BA.1 variants, respectively.However, patients with B-cell lymphoproliferative disorders and/or those treated with anti-CD20 monoclonal antibodies in the prior 12 months showed a lower seropositivity rate compared to other patients against both Omicron BA.1 variant (73% vs 91%; P = 0.02) and wt virus (64% vs 78%; P = 0.16). Our real-life experience confirmed that full vaccination against SARS-CoV-2 induces adequate neutralizing antibody protection for both the wt virus and Omicron BA.1 variants, even in hematological frail patients. However, protective measures should be maintained in hematological patients, especially those with B-cell lymphoproliferative diseases treated with anti-CD20 monoclonal antibodies, because these subjects could have a reduced neutralizing antibody production.

Nonviral cryoglobulinemic vasculitis: an updated review for clinical practice

The clinical spectrum of cryoglobulinemic-associated diseases is broad and heterogeneous, with manifestations ranging from mild symptoms (e.g., isolated palpable purpura) to organ- and life-threatening involvement (e.g., membranoproliferative glomerulonephritis). Cryoglobulins are classified into three types. Type I cryoglobulinemia consists of one monoclonal immunoglobulin (Ig) and is practically always associated with B-cell lymphoproliferative disorders. In contrast, type II/III (mixed) cryoglobulinemia is composed of mono- or polyclonal IgM with rheumatoid factor activity bound to polyclonal IgG. Since the introduction of more efficient therapies for chronic hepatitis C virus (HCV), other diseases such as systemic autoimmune disorders and lymphoproliferative neoplasms have been established as the main causes of mixed cryoglobulinemic vasculitis. The pathogenesis of cryoglobulinemic vasculitis is a complex multifactorial process that involves B-cell aberrant lymphoproliferation and autoantibody production. Therefore, treatment of these patients may involve not only measures aimed to mitigate the severity of clinical manifestation but also those that address the associated underlying disease responsible for Ig production. The treatment of patients with type I cryoglobulinemia is primarily focused on controlling B lymphocyte clones responsible for cryoglobulin production, mostly with chemotherapy drugs. Treatment of mixed cryoglobulinemia syndrome is based on rituximab plus glucocorticoids, which induces remission in the vast majority of cases. In the rare patients that do not respond to rituximab administration, potential rescue approaches include alkylating agents, biologic therapies, conventional immunosuppression, and plasma exchange, although with partial efficacy. This narrative review explores the etiology, pathophysiology, clinical manifestations, treatment, and prognosis of nonviral cryoglobulinemic disease. A special focus is placed on the treatment of type I cryoglobulinemia and rituximab-resistant non-HCV cryoglobulinemic vasculitis.

Advances in Treatment of Waldenström Macroglobulinemia.

The discovery of recurring somatic mutations, in particular MYD88 and CXCR4 mutations, in Waldenström macroglobulinemia (WM), a rare B-cell lymphoproliferative disorder, led in the last decade to the development of several therapeutic agents with high efficacy. This review aims to provide an overview of available treatments in WM and novel agents, focusing on studies published over recent years. There is no international consensus on the best first-line option in treatment-naïve patients. Randomized clinical trials are rare in WM and there has been no prospective comparison of chemoimmunotherapy and BTK inhibitors in the frontline setting. Chemoimmunotherapy and BTK inhibitors, the two feasible and most widely used treatments in first-line treatment, represent very different options in terms of duration of therapy, route of administration, cost, and adverse effect. In addition to tumor genotype and patient comorbidities, choice of therapy in WM should take into account these parameters. Results of ongoing and future clinical trials evaluating fixed-duration combinations with BTK inhibitors and novel agents are awaited.

A-303 Validation of a Quantitative and Qualitative Molecular Test for the Detection of Epstein-Barr Virus by Real-Time qPCR

Abstract Background The Epstein-Barr virus (EBV, HHV-4) is a human virus of the Herpesviridae family, mainly associated with infectious mononucleosis in adolescence, but it can also be related to other tumors. About 90% of the world's population has been infected by this virus, although the vast majority of these people are asymptomatic. In immunocompromised individuals, such as transplant recipients and HIV-infected patients, high EBV replication is a major predisposing factor to the development of a wide range of B-cell lymphoproliferative disorders such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's and non-Hodgkin lymphoma. Early detection of EBV is crucial for the effective management of patients on immunosuppressive therapy after transplantation and immunoproliferative disease. EBV-related diseases, such as in suspected cases of chronic fatigue syndrome and after transplants can be identified by EBV-DNA quantification. Molecular assays, such as Real Time qPCR, have high sensitivity and specificity and are a useful tool for the early diagnosis of EBV infection. This assay differentiates healthy carriers, with low levels of viral load, from ill carriers with a high rate of viral replication. In this way, this work aimed to validate and verify the performance of the alinity m EBV assay. Methods To evaluate the molecular detection of the EBV virus, we used the automated EBV alinity m assay (Abbott). Were evaluated 141 samples from the Molecular Genetics routine of the Pardini Group. Of those, 91 samples (63 plasma and 28 cerebrospinal fluid) were previously tested by in-house PCR for qualitative EBV detection and 50 samples (plasma) were previously tested for quantitative EBV detection by Real-Time PCR (Seegene platform). At the end of the tests, the previous qualitative and quantitative results were compared with the Alinity m EBV test results. Results The range of Alinity m EBV assay detection was &amp;lt;20 IU/mL or &amp;lt;1.30 log(IU/mL) to 200 000 000 IU/ml or 8.30 log(IU/ml). For qualitative analysis, the positive agreement was 100% in both plasma (7/7) and cerebrospinal fluid (6/6) samples, with quantification variation from 2.07 to 4.60 Log(IU/mL) and &amp;lt;1 .30 to 6.30 Log(IU/mL), respectively. The negative agreement was 89% (50/56) for plasma samples, with 6 discordant samples between 1.13 to 3.39 Log(IU/mL) and 91% for cerebrospinal fluid samples (21/23), with two disagreements (1.69 and 2.23 Log(IU/mL), considering the results below the detection range of the assay (&amp;lt;20 IU/mL or &amp;lt;1.30 Log (IU/mL)) as negative because they did not clinical relevance. In quantitative analysis, the negative agreement between comparative methodology (Seegene platform) was 81.0% (33/42), considering as discordant only the samples with a quantifiable detected result. Discordant samples detected by Alinity—and not detected by Seegene platform—presented quantification between 21–791 IU/mL, indicating greater sensitivity of the analyzed methodology. Positive agreement was 100% (5/5). Conclusion The Alinity m EBV Assay demonstrated good performance for molecular detection of EBV. This test can be used as an important tool for clinicians to identify patients with infection, especially in immunosuppressed cases that require a fast, accurate, and reliable diagnosis.