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The clinical significance of an abnormal free light chain (FLC) test, performed due to unspecific complains in the absence of a known plasma cell dyscrasia (PCD) or lymphoproliferative disease (LPD), is not fully elucidated. We investigated the importance of an abnormal FLC ratio (FLC-R) in this setting. Patients registered in the Maccabi Healthcare Services database, tested for FLC during 2007-2023 without previously documented PCD/LPD or increased total protein (TP) level, were reviewed. Demographics, co-morbidities, and laboratory tests were recorded. FLC-R was defined as normal (0.26-1.65) or slightly (slAb 0.1-0.26/1.65-4), moderately (mAbn 0.1-0.05/4-8) and significantly abnormal (sigAb- < 0.05 or > 8). Factors associated with PCD/LPD and overall survival were identified. In total, 8,661 patients, 2,215 (25.6%) with abnormal FLC-R [2,090 (24.1%)-slAb, 65 (0.75%)-mAbn and 60 (0.7%)-sigAb], were analyzed. Almost none had anemia nor acute renal failure. 14% had concomitant increased immunoglobulins. Within a median follow-up of 52 months, 943 were diagnosed with PCD (816-MGUS, 127-MM/Amyloidosis/plasmacytoma) and 48 with LPD. Median time to PCD and LPD were 19 and 28 months. Multivariate analysis found slAb (HR = 1.8, CI95%:1.53-2.12, p < 0.001), mAbn (HR = 6.3, CI95%:4.16-9.53, p < 0.001), and sigAb FLC (HR = 10.4, CI95%:7.0-15.35, p < 0.001), to be associated with PCD/LPD diagnosis. Decreased IgG, increased IgA, and concomitant comorbidities predicted PCD, whereas increased IgM predicted LPD. Older age, male gender, anemia, decreased albumin, increased IgG and concomitant comorbidities, predicted shorter survival. Our large study emphasizes the independent clinical significance of abnormal FLC-R as a predictor of PCD/LPD diagnosis even in patients with normal TP level, promoting early detection of PCD/LPD., (© 2024. The Author(s).)

Assessment of T-cell receptor γ gene (TRG) rearrangements is an importants consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard of such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment size-based methods, given that they generate results with specific clonotype sequence information and allow for more accurate quantitation. The present study evaluated various test parameters and performance characteristics of a commercially available NGS-based TRG gene-rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%. The concordance rates were 88% to 95% in the V γ 1-8, V γ 10, and V γ 11 gene families, but lower in the V γ 9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing clonotypes of the same fragment size. This clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice., Methods: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls., Results: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good., Conclusions: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies., (© 2020 International Clinical Cytometry Society.)

Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing (NGS) has provided unprecedented assay specificity, with a sensitivity matching that of polymerase chain reaction-based measurable residual disease (MRD) detection down to the 10 -6 level. Although reaching 10 -6 to 10 -7 is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is placed in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold-in terms of both the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency to enable critical assessment of the MRD assays for clinical implementation and feasibility., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: ABO-incompatible heart transplantation increases donor availability in young children and is evolving into standard of care in children younger than 2 years. Previous smaller studies suggest similar outcomes to ABO-compatible heart transplantation, but persisting alterations of the immune system in ABO-incompatible recipients might increase the risk of some infections or benefit the graft owing to reduced HLA reactivity. We aimed to assess long-term outcomes in young children after they received ABO-incompatible or ABO-compatible heart transplantation., Methods: In this multicentre, prospective cohort study, we analysed data from the Pediatric Heart Transplant Society registry to compare children who received ABO-incompatible or ABO-compatible heart transplantation before age 2 years between Jan 1, 1999, and June 30, 2018. Given significantly different clinical demographics between the two groups, we also matched each ABO-incompatible recipient to two ABO-compatible recipients using propensity score matching. We assessed patient and graft survival, coronary allograft vasculopathy, malignancy, acute rejection (any episode resulting in augmentation of immunosuppression), and infections (requiring intravenous antibiotic or antiviral therapy or life-threatening infections treated with oral therapy)., Findings: We included 2206 children who received a heart transplant before age 2 years, with 11 332·6 patient-years of cumulative observation time. Children who received an ABO-incompatible transplant (n=364) were younger and a larger proportion had congenital heart disease and ventilator and mechanical circulatory support than the ABO-compatible recipients (n=1842). After matching, only differences in blood group (more O in ABO-incompatible and more AB in ABO-compatible groups) and use of polyclonal induction therapy with anti-thymocyte globulins persisted. The two matched groups had similar post-transplantation graft survival (p=0·74), freedom from coronary allograft vasculopathy (p=0·75), and malignancy (p=0·51). ABO-incompatible recipients showed longer freedom from rejection (p=0·0021) in the overall cohort, but not after matching (p=0·48). Severe infections (p=0·0007), bacterial infections (p=0·0005), and infections with polysaccharide encapsulated bacteria (p=0·0005) that share immunological properties with blood group antigens occurred less frequently after ABO-incompatible heart transplantation., Interpretation: ABO-incompatible heart transplantation for children younger than 2 years is a clinically safe approach, with similar survival and incidences of rejection, coronary allograft vasculopathy, and malignancy to ABO-compatible recipients, despite higher-risk pre-transplant profiles. ABO-incompatible transplantation was associated with less bacterial infection, particularly encapsulated bacteria, suggesting that the acquired immunological changes accompanying ABO tolerance might benefit rather than jeopardise transplanted children., Funding: Pediatric Heart Transplant Society., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: To identify predictive factors for lymphoproliferative disorders (LPDs) that persist after methotrexate (MTX) withdrawal (Persistent-LPD) and the optimal treatment for rheumatoid arthritis (RA) after LPD regression., Methods: Among 3666 patients with RA treated with MTX in our department from 2006 to 2017, 26 cases of LPD that regressed after MTX withdrawal (Regressive-LPD) and 25 cases of Persistent-LPD were compared. Multivariate logistic analysis was performed to identify predictive factors for Persistent-LPD. Retention rates of biological disease-modifying antirheumatic drugs (bDMARDs) were calculated using the Kaplan-Meier Method., Results: In Persistent-LPD, the incidence of diffuse large B-cell lymphoma was higher (76%). The overall 2-year survival rate was 83.9%: 95.8% for Regressive-LPD and 71.0% for Persistent-LPD. The International Prognostic Index (IPI) risk classification was useful for predicting Persistent-LPD. bDMARDs were introduced in 38 RA patients after LPD regression. Unadjusted retention rate of bDMARDs in the 51 LPD patients was significantly lower than that in the 1668 non-LPD RA patients in our bDMARD cohort (controls) ( p  = 0.029). The 1-year retention rates for bDMARDs were 69% and 64% for tocilizumab and abatacept, respectively vs. 46% for TNF-inhibitor (TNFi)., Conclusion: Risk assessment using IPI predicted Persistent-LPD. After LPD regression, non-TNFi tended to have higher retention rates.

Objectives: To determine the clinical characteristics of methotrexate-associated lymphoproliferative disorder (MTX-LPD)., Methods: In this study, 12 RA patients who developed MTX-LPD were assessed. The peripheral blood lymphocyte (PBL) count at the onset of MTX-LPD was compared to that 6 months before the onset, in Epstein-Barr virus-encoded RNA (EBER)-positive and -negative subgroups. We examined the change in the PBL count after MTX withdrawal. In patients with relapsed LPD, changes in the PBL count before relapse were also examined., Results: Regression of LPD after MTX withdrawal was noted in eight patients. In these patients, the PBL count was decreased at the onset of MTX-LPD compared to 6 months before the onset; the decrease was significantly more prominent in EBER-positive patients. In cases of spontaneous regression of LPD, the PBL count recovered quickly after MTX withdrawal. Four of eight patients showed a recurrence of LPD after they improved following MTX withdrawal. These patients also exhibited a decreased PBL count at recurrence compared to 6 months before recurrence., Conclusion: A decrease in the PBL count might be involved in the pathogenesis of MTX-LPD, especially in EBER-positive cases and in patients with LPD relapse after MTX withdrawal following initial improvement.

Next-generation sequencing (NGS) of immunoglobulin (IG) and T cell receptor (TR) rearrangements represents a modern alternative to classical RQ-PCR-based minimal residual disease (MRD) detection. The same primer sets and conditions can be used for all patients, which is undoubtedly one of the most important benefits of NGS, not only reducing the labor required to perform the analysis but also enabling the assay to comply with the upcoming EU IVD regulation. So far, only one standardized academic protocol for this task has been published, developed, and validated within the EuroClonality-NGS working group. In this chapter we describe the materials and methods for amplicon library preparation for sequencing on Illumina MiSeq, and the bioinformatic pipeline for this protocol.

Epstein-Barr virus (EBV) preferentially infects epithelial cells and B lymphocytes and sometimes T and NK lymphocytes. Persistence of EBV-infected cells results in severe lymphoproliferative disorders (LPDs). Diagnosis of EBV-driven T or NK cell LPD and chronic active EBV diseases (CAEBV) is difficult, often requiring biopsies. Herein, we report a flow-FISH cytometry assay that detects cells expressing EBV-encoded small RNAs (EBERs), allowing rapid identification of EBV-infected cells among PBMCs. EBV-infected B, T, and/or NK cells were detectable in various LPD conditions. Diagnosis of CAEBV in 22 patients of Caucasian and African origins was established. All exhibited circulating EBV-infected T and/or NK cells, highlighting that CAEBV is not restricted to native American and Asian populations. Proportions of EBV-infected cells correlated with blood EBV loads. We showed that EBV-infected T cells had an effector memory activated phenotype, whereas EBV-infected B cells expressed plasma cell differentiation markers. Thus, this method achieves accurate and unambiguous diagnoses of different forms of EBV-driven LPD and represents a powerful tool to study their pathophysiological mechanisms., Competing Interests: Disclosures: B. Terrier reported personal fees from AstraZeneca, GlaxoSmithKline, Roche/Chugai, and Vifor Pharma outside the submitted work. L. Galicier reported personal fees from Lilly and VIREOTEAM and nonfinancial support from EUSA Pharma, Overcome, and Janssen-Cilag outside the submitted work. No other disclosures were reported., (© 2020 Fournier et al.)

Background: Within the hematopoietic compartment, fibromodulin (FMOD) is almost exclusively expressed in chronic lymphocytic leukemia (CLL) lymphocytes. We set out to determine whether FMOD could be of help in diagnosing borderline lymphoproliferative disorders (LPD)., Methods: We established 3 flow cytometry-defined groups (CLL [n = 65], borderline LPD [n = 28], broadly defined as those with CLLflow score between 35 and -20 or discordant CD43 and CLLflow, and non-CLL LPD [n = 40]). FMOD expression levels were determined by standard RT-PCR in whole-blood samples. Patients were included regardless of lymphocyte count but with tumor burden ≥40%., Results: FMOD expression levels distinguished between CLL (median 98.5, interquartile range [IQR] 37.8-195.1) and non-CLL LPD (median 0.012, IQR 0.003-0.033) with a sensitivity and specificity of 1. Most borderline LPDs were CD5/CD23/CD200-positive with no loss of B-cell antigens and negative or partial expression of CD43. 16/22 patients with available cytogenetic analysis showed trisomy 12. In 25/28 (89%) of these patients, FMOD expression levels fell between CLL and non-CLL (median 3.58, IQR 1.06-6.21)., Discussion: This study could suggest that borderline LPDs may constitute a distinct group laying in the biological spectrum of chronic leukemic LPDs. Future studies will have to confirm these results with other biological data. Quantification of FMOD can potentially be of help in the diagnosis of phenotypically complex LPDs., (© 2020 International Clinical Cytometry Society.)

Regressive lymphoproliferative disorders (R-LPD) after methotrexate (MTX) withdrawal are one of the specific features of methotrexate - associated lymphoproliferative disorders (MTX-LPD). Although the impact of the absolute lymphocyte count (ALC) on the pathogenesis of R-LPD has been recently emphasized, understanding relapse/regrowth events (RRE) and differences among LPD subtypes is necessary. In this study, we confirmed ALC recovery in the regressive group (R-G; R-LPD without RRE) and relapse/regrowth group (R/R-G; R-LPD with RRE). The increase in ALC lasted at least 2 years in R-G, whereas it decreased within 3 years in R/R-G, supporting the better overall survival (OS) in R-G, as previously reported. In addition, our study suggested that an ALC of 1000/µL at the time of development of LPD is a significant predictor for treatment-free survival (TFS). Furthermore, an ALC of 1000/µL at 6 months after MTX withdrawal was found to be a significant indicator of TFS and OS for R-G and R/R-G. The ALC decreased gradually before LPD development in R/R-G, whereas it decreased 6 months before LPD development in R-G, confirming the important role of ALC in the pathogenesis of MTX-LPD such as regressive events and RRE. In addition to ALC, other predictive factors, such as serum C-reactive protein and soluble interleukin-2 receptors, may be helpful in the management of MTX-LPD, including the decision making for an additional chemotherapy for regressive LPD after MTX withdrawal.

Background/aim: CD38 is a cell surface marker commonly present in plasma cells and activated T cells, while CD138 is a representative plasma cell marker. The aim of this study was to describe the expression of cell surface markers including CD38 and CD138, in the tumors of patients with IgG4-related ophthalmic disease (IgG4-ROD) and extranodal marginal zone B-cell lymphoma (EMZL) of the ocular adnexa., Materials and Methods: Twenty-four consecutive patients of whom 12 had IgG4-ROD and 12 EMZL were enrolled in this study. Medical records were reviewed for flow cytometry (FCM) results on conventional T-cell markers, B-cell markers, CD38 and CD138., Results: Positive rates of T-cell markers, CD38 and CD138 were significantly higher in IgG4-ROD than in EMZL (p<0.01 and p<0.05, respectively)., Conclusion: Our FCM results on CD38 and CD138 showed that the lymphocyte populations were different between IgG4-ROD and EMZL, which may reflect the different pathophysiology of the two diseases., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

EBV-associated PTLD following allogeneic HSCT is a serious complication associated with significant mortality. In this retrospective study, we evaluated whether lymphocyte subset numbers and CD8:CD20 ratio at time of EBV viremia in children undergoing allogeneic HSCT could predict development of PTLD. Absolute lymphocyte count, lymphocyte subsets, and CD8:CD20 ratio at the time of EBV viremia were analyzed. Patients who were treated preemptively with rituximab for high blood EBV viral load were excluded. Out of 266 patients transplanted during the study period, 26 patients were included in the analysis. Patients were divided into two cohorts; cohort 1 included patients with EBV-associated PTLD (n = 5; four with proven, one with probable PTLD). Cohort 2 included patients with EBV viremia without PTLD (n = 21). Lymphocyte recovery was slower in the PTLD group. CD8:CD20 ratio was significantly lower in the PTLD group (median 0.15) compared to the non-PTLD group (median 2.4, P = .012). Using the ROC curve and 1 as the cutoff value, CD8:CD20 ratios were analyzed. In the PTLD group, 4/5 patients (80%) had a ratio <1 whereas in the non-PTLD group, all 21 patients had a ratio >1. Sensitivity and specificity were 80% and 100%, respectively. Negative and PPVs were 95% and 100%, respectively. Profoundly low T-cell count and CD8:CD20 ratio may be used to predict development of PTLD in the context of EBV viremia in children post-allogeneic HSCT. Further studies are needed to validate this finding., (© 2019 Wiley Periodicals, Inc.)

To evaluate diagnostic values for Epstein-Barr virus (EBV) DNA loads in different blood components of patients with EBV-positive T-cell/natural killer cell lymphoproliferative diseases, EBV DNA loads were compared among disease categories in each blood component from 59 patients. Plasma viral loads were significantly higher in "active" disease in chronic active EBV infection. EBV DNA was not detected in the plasma from 7 patients in whom EBV DNA was detected in peripheral blood mononuclear cells and whole blood. Diagnostic cutoff values for whole blood EBV DNA loads of patients with chronic active EBV infection compared with those of infectious mononucleosis was 104.2 (15 800) IU/mL., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objectives: Lymphocytosis may represent either a lymphoproliferative disorder (LPD) or a reactive process. The absolute lymphocyte count (ALC) threshold for further evaluation of lymphocytosis is not well established., Methods: We prospectively performed flow cytometry on blood samples from patients 50 years or older with ALCs of 4.0 × 109 cells/L or greater without a history of an LPD., Results: Monoclonal B-cell populations were found in 34 (19.1%) of 178 cases, with incidence increasing with age. In patients younger than 75 years, no monoclonal B-cell population was identified in patients with ALCs less than 4.4 × 109 cells/L, while such clones were found below and above this threshold in patients 75 years and older., Conclusions: These findings support a threshold for smear review and flow cytometry no lower than 4.4 × 109 cells/L in patients younger than 75 years and a threshold as low as 4.0 × 109 cells/L in patients 75 years and older., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In allogeneic hematopoietic stem cell transplantation recipients, reactivation of Epstein-Barr virus (EBV) can cause post-transplantation lymphoproliferative disorder (PTLD), which may rapidly progress to multiorgan failure and even death. Development of EBV PTLD correlates very closely with use of anti-thymocyte globulin (ATG) and type of transplant. To assess the incidences and clinical features of EBV DNAemia and PTLD in the setting of stem cell transplantation using unmanipulated G-CSF-primed allogeneic peripheral blood stem cells as graft, we performed a retrospective analysis of stem cell transplantation from HLA-matched sibling donors (MSD-SCT, n = 90) or HLA-haploidentical related donors (HID-SCT, n = 110) in patients with hematological malignancies. All of HID-SCT recipients and 27.8% of MSD-SCT recipients received an ATG-containing conditioning regimen. One-year cumulative incidence of EBV DNAemia was 44.1%, ranging from 4.8% in MSD-SCT recipients not using ATG to 20.0% in MSD-SCT recipients using ATG, and 73.7% in HID-SCT recipients. Risk factors for EBV reactivation included use of ATG (p = 0.008), male donor (p = 0.034), and cytomegalovirus DNAemia (p < 0.001). One-year incidence of EBV PTLD was 11.9%, ranging from 1.8% in recipients of MSD-SCT not using ATG to 4.4% in recipients of MSD-SCT using ATG, and 23.5% in recipients of HID-SCT. Risk factors for PTLD after HID-SCT included in fludarabine-containing conditioning regimen (p = 0.010), cytomegalovirus DNAemia (p = 0.036), and patient's age < 40-yr (p = 0.032). Two-year non-relapse mortality was higher for patients with EBV DNAemia than those without EBV DNAemia (35.8% vs. 15.3%, p = 0.002). One-year relapse-free survival and overall survival among patients with PTLD were 40.2% and 44.9%, respectively, as opposed to 63.4% and 68.4% among patients without PTLD (both p < 0.05). In multivariate analyses, EBV DNAemia predicted a lower risk of relapse (p = 0.025), while PTLD was a marginally significant predictor of relapse (p = 0.092). This study identified patients at risk of EBV reactivation and PTLD after unmanipulated allogeneic peripheral blood stem cell transplantation.

Deficiency of Adenosine Deaminase 2 (DADA2) is a monogenic autoinflammatory disorder characterized by livedo reticularis, skin ulcers, subcutaneous rash, aphthous ulcers, and leukocytoclastic vasculitis, neurological signs such as early onset stroke and polyneuropathy. A minority of DADA2 patients suffer from severe cytopenia and lymphoproliferation. Herein, we report an adolescent patient, followed up as having a hematological disorder for many years, eventually diagnosed as having DADA2. In view of the presence of elevated acute phase reactants, hepatosplenomegaly, low IgM level, lymphopenia, anemia, and neutropenia, and a subtle neurological involvement we considered DADA2 diagnosis. The diagnosis was confirmed by identification of a novel L451W mutation in CECR1 gene. The patient has been successfully treated with etanercept, monthly intravenous immunoglobulin replacement, and low-dose methylprednisolone. In conclusion, although the absence of skin and neurological findings, low IgM levels, and persistent lymphopenia should lead the physicians to consider DADA2 in patients with particularly complicated hematological abnormalities.

Background: Hydroa vacciniforme-like lymphoproliferative disorder (HVLLPD) is a rare Epstein-Barr virus (EBV)-related disease that is usually found in East Asians and Latin Americans., Objective: To report the characteristics of HVLLPD in Chinese patients., Methods: Retrospective analysis of patients with HVLLPD from a single institute., Results: A total of 41 patients were enrolled. All patients presented with papulovesicular lesions, mainly distributed on sun-exposed areas, with 26 patients showing systemic symptoms. Follow-up data were available for 20 patients, 16 patients were alive, and 4 patients died. Of the 4 deceased patients, 3 had taken a serum EBV DNA test that showed high viral loads. These 3 patients also received chemotherapy. Histopathology was characterized by dense proliferation of lymphocytes in the dermis. Angiotropism or angiodestruction was found in the majority of patients, whereas prominent cellular polymorphism was noticed in only 4 patients. All patients were positive for CD3, TIA1 cytotoxic granule associated RNA binding protein, and EBV-encoded RNA in situ hybridization., Limitations: This was a retrospective study., Conclusions: HVLLPD in Chinese patients showed indolent behavior in the majority of cases, which differed from the characteristics of HVLLPD in Latin Americans. Patients with high serum EBV DNA loads had an increased risk of their disease evolving into aggressive disease. Chemotherapy should not be considered as first-line treatment for most Chinese patients., (Copyright © 2019 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Peripheral gamma-delta T-cell proliferations are encountered in reaction to certain infections and in primary malignancies. Identifying sources of benign reactions is key in avoiding unnecessary workup and surveillance of these aggressive malignancies. Borrelia infections have been implicated in a number of lymphoproliferative disorders, but rarely, if ever, in this setting. While gamma-delta T-cells are known to play a prominent role in the immune response to Borrelia infection, B-cell differentiation is encountered in the majority of Borrelia-associated proliferations. We present here a unique case of benign-appearing peripheral gamma-delta T-cell lymphoid proliferation in the setting of a tick-bite with subsequent erythema migrans-like skin findings.

Introduction: Bruton tyrosine kinase (BTK) is a key component of B-cell receptor signalling, critical for cell proliferation. Acalabrutinib, a selective, covalent BTK inhibitor, recently received an accelerated approval in relapsed/refractory mantle cell lymphoma. This analysis characterized the population pharmacokinetics (PK) of acalabrutinib and its metabolite ACP-5862., Methods: Data were obtained from six phase I/II trials in adult patients with B-cell malignancy and seven phase I trials in healthy volunteers. Pooled concentration-time data, at dose levels ranging from 15 to 400 mg, were analysed using non-linear mixed-effects modelling. Base model parameters were scaled with body weight and normalized to 70 kg (fixed exponents: 0.75 and 1 for clearance and volumes, respectively). A full covariate approach was used to evaluate any relevant effects of dose, health group/disease status, hepatic and renal impairment, use of acid-reducing agents, race and sex., Results: A total of 11,196 acalabrutinib and 1068 ACP-5862 concentration-time samples were available. The PK of both analytes were well described using two-compartment disposition models. Acalabrutinib absorption was characterized using sequential zero- and first-order constants and a lag time. Apparent clearance (CL/F) of acalabrutinib was 169 L/h (95% CI 159-175). Relative to the 100 mg dose group, the 15 and 400 mg dose groups showed a 1.44-fold higher and 0.77-fold lower CL/F, respectively. The clearance for ACP-5862 was 21.9 L/h (95% CI 19.5-24.0). The fraction metabolized was fixed to 0.4. The central and peripheral volumes of distribution were 33.1 L (95% CI 24.4-41.0) and 226 L (95% CI 149-305) for acalabrutinib, and 38.5 L (95% CI 31.6-49.2) and 38.4 L (95% CI 32.3-47.9) for ACP-5862. None of the investigated covariates led to clinically meaningful changes in exposure., Conclusion: The PK of acalabrutinib and its metabolite ACP-5862 were adequately characterized. Acalabrutinib CL/F decreased with increasing dose, but the trend was small over the 75-250 mg range. No dose adjustment was necessary for intrinsic or extrinsic covariates.

Post-transplant lymphoproliferative disease (PTLD) is a serious complication of solid organ transplantation. As early diagnosis remains challenging, we investigated the utility of serum-free light chain (FLC) and heavy chain/light chain pairs (HLC) as diagnostic biomarkers. Pre-treatment serum FLC and HLC levels were measured in 20 patients at their first diagnosis of B cell PTLD and in 14/20 patients during follow-up. Results were compared to serum FLC/HLC levels of 90 matched PTLD-free transplanted controls. Renal dysfunction was common in both cohorts, and combined FLC levels were often elevated above the conventional upper limit of normal (45.7 mg/L). Combined FLC levels were higher in patients with PTLD than in transplant controls (p = 0.013), and levels above the conventional ULN were associated with PTLD (OR 3.2, p = 0.05). Following adjustment to cystatin C as a marker of renal function an even stronger association was found for a (dimensionless) threshold value of 37.8 (OR 8.9, p < 0.001). In addition, monoclonal proliferation (abnormal FLC ratio, using an established renal range cutoff) was more common in PTLD than in controls (3/20 vs. 2/90, p = 0.04). Following therapy, at the time of protocolised restaging, patients experiencing subsequent sustained complete remission displayed lower FLC levels than those not experiencing such remission (p = 0.053). No relationship with HLC results was seen. Elevated polyclonal FLC levels (especially when adjusted for renal function) and monoclonal proliferation are a potential biomarker for PTLD diagnosis and disease surveillance. However, prospective validation is necessary before FLC measurement should be incorporated in follow-up of transplant recipients and PTLD management.

We describe the case of a 23-month-old male infant with Epstein-Barr virus (EBV)-associated lymphoproliferative disorder, which mimicked the recurrence of EBV-associated hemophagocytic lymphohistiocytosis. Chemotherapy with dexamethasone, etoposide, and cyclosporine resolved fever, hepatosplenomegaly, and pancytopenia. However, on day 81 of illness, the patient developed similar symptoms. Plasma EBV-DNA levels markedly increased again, but no T-cell clonality was observed. B cells were identified to be infected with EBV. He was successfully treated with rituximab, dexamethasone and etoposide. When recurrence of EBV-associated hemophagocytic lymphohistiocytosis is suspected, performing tests to identify the infected cells will enable accurate understanding of the clinical condition, resulting in proper treatments.

Hypereosinophilic syndrome (HES) is a group of rare blood disorders characterized by a persistent elevation of blood eosinophil count ⩾1.5×10 9 /L and clinical manifestations attributable to eosinophilia or tissue hypereosinophilia. Lymphocytic variant of HES (HES-L) is a known subtype according to World Health Organization classification. It is well documented in the literature that patients with HES-L are predisposed to develop T-cell lymphoma. We report a case of T-cell lymphoproliferation associated with hypereosinophilia, which has been successfully treated with mycophenolate mofetil, with resolution of skin lesions and normalization of eosinophil count and immunoglobulin E level. We believe this is a clinically relevant case since this is a rare disease with little known knowledge on its best treatment modality., (Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.)

Purpose: Emerging EBV DNAemia in plasma is considered an early sign of post-transplant lymphoproliferative disorder (PTLD). The aim of this study was to quantify the extent of benefit from screening for EBV DNAemia to detect emerging PTLD among solid organ (SOT) or hematopoietic stem cell transplant recipients (HSCT)., Methods: We used receiver operating characteristic (ROC) curves for assessing ability of models to predict PTLD. Among 2642 recipients transplanted between January 2004 and December 2014, 79 (3%) developed PTLD., Results: EBV DNAemia was observed in 331/1784 recipients (18.6%, 95% CI 16.8-20.4) with measured EBV DNA. The area under the curve (AUC) of the ROC of EBV DNAemia to identify persons with subsequent PTLD was 72% (95% CI, 64-79%) among SOT and 59% (51-68%) among HSCT. Including clinical predictors such as age, gender, transplant year and type, high-risk EBV serostatus, and routine biochemistry in addition to EBV DNAemia increased AUC to 83% (75-90%) among SOT and 84% (79-89%) among HSCT. Among HSCT, including additional factors such as T-cell-depleting treatment, acute graft vs. host disease and donor match increased AUC to 85% (78-91%)., Conclusions: We constructed a model to better predict PTLD compared to EBV DNA screening alone which could have clinical implications.

Background: Serial monitoring of Epstein-Barr virus (EBV) reveals that certain pediatric liver transplant (LT) recipients exhibit high EBV loads for long periods. We investigated the incidence and risk factors of chronic high EBV (CHEBV) loads (continuous EBV DNA >10 000 IU/mL of whole blood for ≥6 months) and long-term outcomes., Methods: This single center, retrospective observational study investigated pediatric LT recipients who survived ≥6 months. We quantitated EBV DNA weekly during hospitalization and subsequently every 4 or 6 weeks at the outpatient clinic. Tacrolimus was maintained at a low trough level (<3 ng/mL, EBV DNA load >5000 IU/mL)., Results: Thirty-one of 77 LT recipients developed CHEBV. Univariate analysis revealed that age <2 years and body weight <10 kg upon LT, operation time <700 minutes, warm ischemia time (WIT) >35 minutes, graft-to-recipient weight ratio (GRWR) >2.7%, and preoperative EBV seronegativity were significantly associated with the development of CHEBV loads. Multivariate analysis identified significant associations of CHEBV with WIT >35 minutes, GRWR >2.7%, and preoperative seronegative. None of the recipients developed post-transplantation lymphoproliferative disorder. Survival rates of patients with and without CHEBV loads were not significantly different., Conclusions: A significant number of pediatric LT recipients developed CHEBV loads. Long WIT, high GRWR, and preoperative EBV seronegativity were significantly associated with the development of CHEBV loads. Although the long-term outcomes of patients with or without CHEBV loads were not significantly different, further studies of more subjects are warranted., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: ACA-positive/primary Sjögren's syndrome (pSS) represents a distinct overlapping entity with intermediate features in between limited systemic sclerosis (lSSc) and pSS. Few data are available on their general risk for lymphoproliferative complications, specifically regarding adverse predictors at the level of minor salivary gland (MSG) histology. The objectives of this work are: a) to characterise, through a detailed immunohistochemistry study, the organisation of the lymphomonocitic infiltrates in ACA-positive/pSS patient vs. ACA-negative/pSS patients focusing on the presence of GC-like structures in minor salivary gland biopsies; b) to compare the frequency of traditional clinical and serological risk factors for lymphoma between the two subgroups., Methods: We analysed 28 MSG samples from ACA-positive/pSS patients and 43 consecutive MSGs from ACA-negative/pSS, using sequential IHC staining for CD3, CD20 and CD21 in order to define the T/B cell segregation within the periductal infiltrates and presence of ectopic GC-like on the detection of GC-like structures. Clinical and serological data of all the patients were retrieved and analysed., Results: Ectopic lymphoid structures (ELS) with GC-like structures were observed in 7 out of 28 ACA-positive/pSS patients (25%) and in 13 out of 43 ACA-negative/pSS patients (30.2%). Similarly, no statistical significant difference was found between the two groups as far as the classical pSS risk factors for lymphoproliferative complications was concerned (i.e. salivary gland enlargement, purpura, low C4, leukocytopenia, clonal gammopathy). Finally, the 3 cases of non-Hodgkin's lymphoma observed were equally distributed between the two subsets., Conclusions: Overall, this study indicates that ACA-positive/and ACA-negative pSS patients apparently present a similar risk for lymphoproliferative complications as suggested indirectly by the analogies between the two groups observed at the histopathology level.

Background: Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of allogeneic hematopoietic cell transplantation (HCT). Epstein-Barr virus (EBV) reactivation (detectable DNAemia) predisposes to the development of PTLD., Methods: We retrospectively studied 306 patients monitored for EBV DNAemia after Thymoglobulin-conditioned HCT to determine the utility of the monitoring in the management of PTLD. DNAemia was monitored weekly for ≥12 weeks post-transplantation., Results: Reactivation was detected in 82% of patients. PTLD occurred in 14% of the total patients (17% of patients with reactivation). PTLD was treated with rituximab only when and if the diagnosis was established. This allowed us to evaluate potential DNAemia thresholds for pre-emptive therapy. We suggest 100,000-500,000 IU per mL whole blood as this would result in unnecessary rituximab administration to only 4-20% of patients and near zero mortality due to PTLD. After starting rituximab (for diagnosed PTLD), sustained regression of PTLD occurred in 25/25 (100%) patients in whom DNAemia became undetectable. PTLD progressed or relapsed in 12/17 (71%) patients in whom DNAemia was persistently detectable., Discussion: In conclusion, for pre-emptive therapy of PTLD, we suggest threshold DNAemia of 100,000-500,000 IU/mL. Persistently detectable DNAemia after PTLD treatment with rituximab appears to have 71% positive predictive value and 100% negative predictive value for PTLD progression/relapse., (Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: The lymphocyte positional parameters included in Sysmex XN have been suggested as useful means to differentiate lymphoproliferative disorders (LPD), mononucleosis syndrome (MNS) and other lymphocytoses., Methods: We evaluated Sysmex XN analysers, which supply 6 lymphocyte positional parameters that can be measured in the WDF scattergram: LY-X, LY-Y, LY-Z, LY-WX, LY-WY and LY-WZ., Results: We collected 301 samples from normal controls, polyclonal lymphocytosis, MNS and LPD. MNS and monoclonal expansion of T granular lymphocyte (T-GL) diagnostic groups accumulated higher numbers of significant differences in the mean values in comparison with the other groups. We propose a new algorithm that can differentiate T-GL cases from other diagnostic groups with an SE of 67.5%, an SP of 98.2%, a PPV of 87.1% and an NPV of 94.3%. Another algorithm showed its efficiency to differentiate MNS cases from other diagnostic groups with an SE of 63.6%, an SP of 97.5%, a PPV of 70.0% and an NPV of 96.7%. In 38.5% of all cases, the analyser did not generate any morphologic flag. Abnormal results in lymphocyte positional parameters were useful to detect 72.5% of these samples., Conclusion: The lymphocyte positional parameters provided by Sysmex XN analysers are useful to differentiate expansions of T-GLs from other LPD and to differentiate MNS cases from other diagnostic groups. In addition, these parameters are very useful for detecting changes in the lymphocytes that make it necessary to review blood smears in samples without morphological flagging., (© 2017 John Wiley & Sons Ltd.)

Minimal residual disease (MRD) detection has a powerful prognostic relevance for response evaluation and prediction of relapse in hematological malignancies. Real-time quantitative PCR (qPCR) has become the settled and standardized method for MRD assessment in lymphoid disorders. However, qPCR is a relative quantification approach, since it requires a reference standard curve. Droplet digital TM PCR (ddPCR TM ) allows a reliable absolute tumor burden quantification withdrawing the need for preparing, for each experiment, a tumor-specific standard curve. We have recently shown that ddPCR has a good concordance with qPCR and could be a feasible and reliable tool for MRD monitoring in mature lymphoproliferative disorders. In this chapter we describe the experimental workflow, from the detection of the clonal molecular marker to the MRD monitoring by ddPCR, in patients affected by multiple myeloma, mantle cell lymphoma and follicular lymphoma. However, standardization programs among different laboratories are needed in order to ensure the reliability and reproducibility of ddPCR-based MRD results.

The ZEBRA protein (encoded by the BZLF1 gene), is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. Several studies highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders like post-transplant lymphoproliferative disease (PTLD). Here, we use an antigen-capture ELISA assay specifically designed to detecting the circulating soluble ZEBRA (sZEBRA) in serum samples (threshold value determined at 40ng/mL). We retrospectively investigated a population of 66 transplanted patients comprising 35 PTLD. All the samples from a control population (30 EBV-seronegative subjects and 25 immunocompetent individuals with EBV serological reactivation), classified as sZEBRA < 40ng/mL were assigned as negative. At PTLD diagnosis, EBV genome (quantified by qPCR with EBV DNA>200 copies/mL) and sZEBRA were detectable in 51% and 60% of cases, respectively. In the patients who developed a pathologically-confirmed PTLD, the mean sZEBRA value in cases, was 399 ng/mL +/- 141 versus 53ng/mL +/- 7 in patients who did not (p  < 0,001). This is the first report relating to the detection of the circulating ZEBRA in serum specimens, as well as the first analysis dealing with the lytic cycle of EBV in PTLD patients with this new biomarker.

Background: Post-transplant lymphoproliferative disorder (PTLD) is a severe complication of solid organ transplantation that can be classified into two major subtypes, namely, early lesions and non-early lesions, based on histopathological findings. In the vast majority of cases, proliferating cells are B lymphocytes and, most frequently, proliferation is induced by Epstein-Barr virus (EBV) infection., Methods: The aim of our study was to evaluate the natural history of EBV infection and its possible evolution toward PTLD in a pediatric cohort of patients who received a renal transplant between January 2000 and December 2013. A total of 304 patients were evaluated for this study, of whom 103 tested seronegative for EBV at transplantation., Results: Following transplantation, 50 of the 103 seronegative patients (48.5%) developed a first EBV infection, based on the results of PCR assays for EBV DNA, with 19 of these patients ultimately reverting to the negative state (<3000 copies/ml). Among the 201 seropositive patients only 40 (19.9%) presented a reactivation of EBV. Non-early lesions PTLD was diagnosed in ten patients, and early lesions PTLD was diagnosed in five patients. In all cases a positive EBV viral load had been detected at some stage of the follow-up. Having a maximum peak of EBV viral load above the median value observed in the whole cohort (59,909.5 copies/ml) was a significant and independent predictor of non-early lesions PTLD and all PTLD onset., Conclusions: A high PCR EBV viral load is correlated with the probability of developing PTLD. The definition of a reliable marker is essential to identify patients more at risk of PTLD and to personalize the clinical approach to the single patient.

An 84-year-old woman was referred to our hospital presenting anemia. Her hemoglobin level was 5.8 g/dL, and white blood cell count was 9400/μL, consisting of 82% lymphocytes. Given the lymphocyte phenotype (CD2+, CD3-, CD16+, and CD56-) and negative whole blood EBV viral load, we made a diagnosis of chronic lymphoproliferative disorder of NK cells (CLPD-NK). We suspected hemolytic anemia because of the high levels of reticulocytes in the peripheral blood and the low haptoglobin value. Although the direct Coombs test was negative and there was no cold agglutination, we examined her red-blood-cell-bound IgG (RBC-IgG), which was elevated. She was diagnosed as having as Coombs-negative autoimmune hemolytic anemia (AIHA). We report the effectiveness of oral cyclophosphamide for Coombs-negative autoimmune hemolytic anemia in CLPD-NK.

We have developed a lymphoproliferative disorder screening tube (LPD-ST) with the aim to provide comprehensive immunophenotyping of lymphocyte subsets with minimal need for additional testing. The LPD-ST consists of CD4/kappa FITC, CD8/lambda PE, CD3/CD14ECD, CD38PC5.5, CD20/CD56PC7, CD10APC, CD19APC-A700, CD5APC-A750, CD57/CD23PB and CD45KO. The LPD-ST was validated against previously used lymphocyte subset panels in Canada (n=60) and in Sweden (n=43) and against the OneFlow ™ LST (n=60). The LPD-ST panel was then implemented in clinical practice using dried monoclonal antibody reagents (Duraclone ® ) on 649 patient samples in Sweden. In 204 of 649 samples (31%), a monotypic B-cell population was found. Of these cases, a final diagnosis could be rendered in 106 cases (52%), and in the remainder, additional B-cell immunophenotyping was performed. In 20 (3%) samples, an aberrant T-cell population was confirmed by additional testing. Of 425 samples diagnosed as normal/reactive lymphoid tissue, 50 (12%) required additional immunophenotyping, mostly due to an abnormal CD4/CD8 ratio. The LPD-ST tube significantly minimizes the need for additional testing, improves the turn-around time, and reduces the cost of LPD immunophenotyping. It is also suitable for investigating paucicellular samples such as cerebrospinal fluid or fine needle aspirates., (© 2017 John Wiley & Sons Ltd.)

Background: Monoclonal immunoglobulins (paraproteins) are produced by B lymphocytes in lymphoproliferative disorders. A single monoclonal immunoglobulin is homogeneous in terms of its structure, and it can occur in human serum at high concentration and cause significant interference in laboratory assays., Case: We present a case of an 84-year-old man who was admitted to the hospital for progression of dyspnea. Basic laboratory tests showed a serum concentration of conjugated bilirubin, measured using the diazo spectrophotometric method, which was much higher than that of total bilirubin. The cause of the discrepancy was attributed to analytical interference by monoclonal immunoglobulins, which helped establish a diagnosis of lymphoproliferative disorder., Conclusion: Monoclonal immunoglobulins are relatively rare in serum but are an important cause of analytical interference. Monoclonal immunoglobulins should always be considered a source of interference when unexpectedly high, low, or contradictory data are encountered, and appropriate confirmatory tests (electrophoresis, imunofixation) should be performed in such circumstances. Failure to do so can result in errors in diagnosis and inadequate treatment. Conversely, when samples contain abnormal and especially high monoclonal immunoglobulin levels, the biochemical data should be carefully examined for any discrepancies, such as paraprotein interference, and the results should be taken into consideration in patient management.Key words: paraproteins - lymphoproliferative disorders - bilirubin - interferenceThe authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 22. 1. 2016Accepted: 1. 12. 2016.

Venous thromboembolism, usually entailing deep vein thrombosis, pulmonary embolism, or both, is a complex and multifactorial disorder, in which a number of putative conditions interplay and finally contribute to propel the individual risk over a certain degree, so ultimately culminating in the development of venous occlusive disorders. Thrombophilia is commonly defined as a propensity to develop venous thromboembolism on the basis of an underlying hypercoagulable state attributable to inherited or acquired disorders of blood coagulation or fibrinolysis. The thrombophilic conditions are conventionally classified as inherited (or genetically determined) and acquired. The former include deficiencies of natural anticoagulants such as antithrombin, protein C, protein S, increased values of clotting factors (especially factor VIII), as well as prothrombotic polymorphisms in genes encoding for factor V (i.e., factor V Leiden) and prothrombin. The latter conditions mainly entail antiphospholipid antibody syndrome, malignancy, acquired elevations of coagulation factors or acquired reduction of natural inhibitors, or hyperhomocysteinemia. Deepened knowledge of all potential risk factors, as well as the clear understanding of their role in the pathophysiology of venous thrombosis, are both essential to help achieve a faster and more efficient diagnosis of this condition as well as a more effective prophylaxis of patients at higher risk and treatment of those with manifest disease.

Primary chronic cold agglutinin disease (CAD) is a well-defined clinicopathologic entity in which a specific, clonal lymphoproliferative B-cell bone marrow disorder results in autoimmune hemolytic anemia. The immune hemolysis is entirely complement-dependent, predominantly mediated by activation of the classical pathway and phagocytosis of erythrocytes opsonized with complement protein C3b. Typical clinical features in CAD have diagnostic and therapeutic implications. Pharmacologic treatment should be offered to patients with symptom-producing anemia or disabling circulatory symptoms. CAD should not be treated with corticosteroids. Based on an individualized approach, rituximab monotherapy or rituximab-fludarabine in combination is recommended as first-line therapy. Rituximab-bendamustine is still an investigational therapy. Although complement-modulating agents are still to be considered experimental in CAD, therapy with the anti-C1s monoclonal antibody TNT009 seems promising., (© 2016 by The American Society of Hematology. All rights reserved.)

Waldenström macroglobulinemia (WM) is a low-grade incurable immunoglobulin M + (IgM + ) lymphoplasmacytic lymphoma for which a genetically engineered mouse model of de novo tumor development is lacking. On the basis of evidence that the pro-inflammatory cytokine, interleukin 6 (IL6), and the survival-enhancing oncoprotein, B cell leukemia 2 (BCL2), have critical roles in the natural history of WM, we hypothesized that the enforced expression of IL6 and BCL2 in mice unable to perform immunoglobulin class switch recombination may result in a lymphoproliferative disease that mimics WM. To evaluate this possibility, we generated compound transgenic BALB/c mice that harbored the human BCL2 and IL6 transgenes, EμSV-BCL2-22 and H2-L d -hIL6, on the genetic background of activation-induced cytidine deaminase (AID) deficiency. We designated these mice BCL2 + IL6 + AID - and found that they developed-with full genetic penetrance (100% incidence) and suitably short latency (93 days median survival)-a severe IgM + lymphoproliferative disorder that recapitulated important features of human WM. However, the BCL2 + IL6 + AID - model also exhibited shortcomings, such as low serum IgM levels and histopathological changes not seen in patients with WM, collectively indicating that further refinements of the model are required to achieve better correlations with disease characteristics of WM.

Patients receiving methotrexate (MTX) for the treatment of autoimmune disease are at a high risk of developing lymphoproliferative disorders (LPD), the so-called methotrexate-associated lymphoproliferative disorders (MTX-LPD). We recently performed abdominal ultrasonography (US) in a patient with rheumatoid arthritis (RA) who had developed hepatic dysfunction during the course of MTX therapy; the examination revealed multiple well-demarcated hepatic tumors with slightly irregular borders, the largest one measuring 9 cm in diameter. In view of the finding of portal and hepatic veins perforating the tumor, we suspected a diagnosis of malignant lymphoma and performed a hepatic tumor biopsy. Histopathological examination of the biopsy specimens revealed a diagnosis of diffuse large B-cell lymphoma, and we made a final diagnosis of MTX-LPD. MTX treatment was discontinued, which resulted in rapid resolution of the lesions. Resolution of MTX-LPD can be obtained just by discontinuation of MTX treatment. In patients receiving MTX therapy who are found to have hepatic tumors perforated by the portal vein and/or hepatic vein on abdominal US, it is advisable to perform hepatic tumor biopsy to facilitate differential diagnosis of MTX-LPD and enable a definite diagnosis.

B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.