IgM paraprotein-associated peripheral neuropathy: small CD20-positive B-cell clones may predict a monoclonal gammopathy of neurological significance and rituximab responsiveness.

IgM paraprotein-associated peripheral neuropathy (PN) in patients without overt evidence of lymphoma is a recognised clinical entity of unknown aetiology. Interrogating the bone marrow B-cell or plasma cell clones underlying paraproteinemic neuropathies may improve our understanding of both pathogenesis and treatment options. This retrospective observational analysis of IgM paraprotein-associated PN identified five patients with small pathological MYD88 L265P and CD20-positive B-cell clones in their bone marrow using multi-parametric flow cytometry, who have shown durable neurological response to rituximab. We posit that multi-parametric flow cytometry may be instrumental in identifying the cellular source of the paraprotein in IgM paraprotein-associated PN, and thus directing appropriate immunomodulatory therapy. Further understanding of these small pathological B-cell clones may also provide additional insight into mechanisms of monoclonal gammopathy of clinical significance overall.

Proinflammatory CD20+ T cells in the pathogenesis of multiple sclerosis.

With the discovery that the highly effective anti-CD20 antibody therapies developed to deplete CD20+ B cells deplete CD20+ T cells equally well, a great interest in the biological properties of CD20+ T cells has emerged. In this study we show that CD20+ T cells have a proinflammatory Th1/Tc1 phenotype with a high proliferative capacity to CNS antigens. We also found that the percentage of CD20+ T cells is increased in the blood of patients with multiple sclerosis and are enriched in the CSF of the patients. Furthermore, we found a positive correlation between CD20+ T cells in the CSF and multiple sclerosis disease severity and see that regulation of CD20+ T cells likely contributes to the positive treatment effect of the multiple sclerosis treatment alemtuzumab. These data represent an important contribution to the understanding of the nature of CD20+ T cells and strongly suggests a role of CD20+ T cells in the pathogenesis of multiple sclerosis.

Diffuse large B-cell lymphoma: 2019 update on diagnosis, risk stratification, and treatment.

DISEASE OVERVIEW: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive non-Hodgkin lymphoma originating from the germinal center, and it represents a heterogeneous group of diseases with variable outcomes that are differentially characterized by clinical features, cell of origin (COO), molecular features, and most recently, frequently recurring mutations. DIAGNOSIS: DLBCL is ideally diagnosed from an excisional biopsy of a suspicious lymph node, which shows sheets of large cells that disrupt the underlying structural integrity of the follicle center and stain positive for pan-B-cell antigens, such as CD20 and CD79a. COO is determined by immunohistochemical stains, while molecular features such as double-hit or triple-hit disease are determined by fluorescent in situ hybridization analysis. Commercial tests for frequently recurring mutations are currently not routinely used to inform treatment. RISK STRATIFICATION: Clinical prognostic systems for DLBCL, including the rituximab International Prognostic Index, age-adjusted IPI, and NCCN-IPI, use clinical factors for the risk stratification of patients, although this does not affect the treatment approach. Furthermore, DLBCL patients with non-germinal center B-cell (GCB)-like DLBCL (activated B-cell like and unclassifiable) have a poorer response to up-front chemoimmunotherapy (CI) compared to patients with GCB-like DLBCL. Those with c-MYC-altered disease alone and in combination with translocations in BCL2 and/or BCL6 (particularly when the MYC translocation partner is immunoglobulin) respond poorly to up-front CI and salvage autologous stem cell transplant at relapse. RISK-ADAPTED THERAPY: This review will focus on differential treatment of DLBCL up-front and at the time of relapse by COO and molecular features.

Which Markers Should the used for Diagnostic Chronic Lymphocytic Leukemia Immunophenotyping Scoring System by Flow Cytometry?

BACKGROUND: The scoring system used for chronic lymphocytic leukemia (CLL) cannot make an accurate diagnosis in some cases. Novel markers are available for the differential diagnosis of CLL, especially from MCL. However, these markers are still not incorporated into diagnostic algorithms. We investigated the role of CD43, CD81, CD200, and ROR1 in the differential diagnosis of CLL and their expression in non-CLL cases. METHODS: We investigated the role of CD43, CD81, CD20, and ROR1 in the differential diagnosis of CLL by incorporating them into the diagnostic panel after studying peripheral blood or bone marrow samples of 165 patients with 8-color flow cytometry. RESULTS: CD43 positivity was a sensitive marker but had a lower specificity for CLL. CD43 had high diagnostic value for CLL (sensitivity 100%, specificity 88.5%, AUC 98.0%). CD200 was a specific marker for CLL (sensitivity 98%, specificity 90%, AUC: 96%). CD81 expression was highest in the MCL cases, with a median expression rate of 68.5% (range: 54 - 82.5%). It was negative in all the CLL cases. For CLL, CD81 negativity had a sensitivity of 95%, a specificity of 82% and an AUC of 92%. ROR1 was positive in all CLL and MCL cases. CD79b, on the other hand, was a fairly sensitive and specific marker for MCL. CONCLUSIONS: CD43, CD81, CD200, and ROR1 should be incorporated into diagnostic algorithms for the differential diagnosis of CLL, especially from MCL.

Evaluation of a 10color protocol as part of a 2tube screening panel for flow cytometric assessment of peripheral blood leukocytic subsets.

Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16- granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.

Antigenic burden and serum IgG concentrations influence rituximab pharmacokinetics in rheumatoid arthritis patients.

AIMS: Rituximab is a monoclonal antibody directed against CD20, which is approved in rheumatoid arthritis (RA). This study aimed at assessing the influence of CD19+ cell counts as target-antigen amount, and of immunoglobulin G (IgG) serum concentrations on rituximab pharmacokinetics in RA patients. METHODS: In a cohort of 64 RA patients who had received repetitive courses of rituximab, the influence of CD19+ cell count, IgG serum concentration, body surface area, sex and disease activity score in 28 joints on rituximab pharmacokinetic parameters was assessed using a population pharmacokinetic analysis. RESULTS: A two-compartment model, with first-order distribution and elimination best described the data. The volume of distribution of central compartment and clearance of rituximab were estimated at 4.7 l and 0.56 l day-1 , respectively. Distribution and elimination half-lives were 0.9 days and 17.3 days, respectively. As expected, the central volume of distribution increased with body surface area (P = 0.012) and was higher in male than in female (P = 0.004). We found that the elimination rate constant (k10 ) increased with CD19+ count (P = 0.00022) and IgG concentration (P = 7.4 × 10-8 ), and that k10 decreased with time (P = 0.00015), partly explained by a change in target-antigen amount. CONCLUSIONS: The association between CD19+ count and k10 may be explained by target-mediated drug disposition, while the association between IgG serum concentration and k10 may be explained by a saturation of the neonatal Fc receptor at high IgG concentrations, resulting in decreased recycling of rituximab.

Induced resistance to ofatumumab-mediated cell clearance mechanisms, including complement-dependent cytotoxicity, in chronic lymphocytic leukemia.

Ofatumumab (OFA), a human CD20-targeting mAb, kills B lymphocytes using the innate immune system including complement-dependent cytotoxicity (CDC). The efficacy of OFA in patients with chronic lymphocytic leukemia (CLL) is limited by drug resistance, which is not well characterized. To better understand mechanisms of resistance, we prospectively studied CLL cells isolated from blood samples collected before and after in vivo exposure to the initial dose of OFA therapy in 25 patients undergoing their first treatment for progressive CLL. As previously reported, OFA therapy rapidly decreased the absolute lymphocyte count, CD20 expression by CLL cells, and serum complement levels. We now show that after administration of the first dose of OFA, there was a modest rebound in the absolute lymphocyte count and serum complement levels, but substantial ongoing loss of CD20 expression by CLL cells. These post-OFA treatment CLL cells were highly resistant to OFA-mediated CDC but retained sensitivity to alemtuzumab-mediated CDC in vitro. Posttherapy serum OFA levels correlated inversely with both the amount of pretreatment circulating cell-bound CD20 and with the decrease in this value following treatment. In vitro OFA-mediated CDC did not predict clinical responses, and the patients with first-dose reactions to OFA did not have markers of increased complement activation in vivo. We propose that optimal efficacy of CD20- targeted therapy for CLL requires determining an mAb dose size and frequency that optimizes CLL killing without exceeding the capacity of the cytotoxic mechanisms and thus minimizes loss of CD20 expression in the surviving CLL cells.

Chemotherapy-induced B-cell depletion in hepatoblastoma patients undergoing ABO-incompatible living donor liver transplantation.

LT from ABO-I donors requires preconditioning regimens to prevent postoperative catastrophic AMR. NAC for HBL is known to cause myelosuppression leading to a reduction in the number and function of lymphocytes. We investigated this chemotherapy-induced myelosuppression in HBL patients listed for LT from ABO-I donors with reference to the kinetics of B, T cells, and anti-ABO blood type isoagglutinin titers. Between 2005 and 2015, of the 319 patients who underwent LDLT at our institute, 12 were indicated for unresectable HBL. Three patients with unresectable HBL who underwent LDLT from ABO-I donors are included in this study. Immunosuppression consisted of a standard regime of tacrolimus and low-dose steroids as in ABO compatible/identical LDLT. No additional preoperative therapies for B-cell depletion were used. Absolute lymphocyte counts, lymphocyte subsets (including CD20+ B cells, CD3+CD4+ T cells and CD3+CD8+ T cells), and anti-ABO blood type isoagglutinin titers were measured before LDLT and postoperatively. The median age at diagnosis was 19 months (range, 3-31 months). The median follow-up was seven months (range, 6-15 months). The median interval from the last NAC to LDLT was 33 days (range, 25-52 days). The median interval from LDLT to adjuvant chemotherapy was 28 days (range, 22-36 days). The counts of CD20+ B cells before LDLT were depleted to median 5 cells/mm(3) (range, 0-6 cells/mm(3)). There was a transient rebound in the CD20+ B cell counts on day seven (maximum of 82 cells/mm(3)) followed by a decline starting at 14 days after LDLT that was sustained for the duration of adjuvant chemotherapy. Anti-ABO blood type isoagglutinin titers were lowered to between 1:1 and 1:16 before LDLT and remained low for the duration of follow-up in this study. All of the three patients remained in good health without either acute cellular or AMR after LDLT. The B-cell depletion that occurs after cisplatin-based chemotherapy for HBL may help accomplish safe ABO-I LDLT in children without the use of additional conditioning regimens for prevention of AMR.

[The case of hairy cell leukosis in pediatrics].

According publications' data, hairy cell leukosis encounters in humans aged from 26 to 70 years and in males four times more often than in females. The disease is manifested by impotence, splenomegaly, and presence in blood and bone marrow clone of lymphoid cells with particular morphology, cytochemic markers and immune phenotype (sIg+, CD19+, CD20+, CD5-, CD10-, with marked expression of CD25+, CD103+). The presented clinical case of illness with hairy cell leucosis is the first one detected in pediatric practice in adolescent of 16 years old.

Comparative analysis of portal cell infiltrates in antimitochondrial autoantibody-positive versus antimitochondrial autoantibody-negative primary biliary cirrhosis.

UNLABELLED: Substantial evidence supports dysregulated B-cell immune responses in patients with primary biliary cirrhosis (PBC), including the presence of serum antimitochondrial antibodies (AMAs). However, recent reports from murine models of PBC suggest that B cells may also provide regulatory function, and indeed the absence of B cells in such models leads to exacerbation of disease. The vast majority of patients with PBC have readily detectable AMAs, but a minority (<5%) are AMA negative (AMA(-)), even with recombinant diagnostic technology. This issue prompted us to examine the nature of B-cell infiltrates surrounding the portal areas in AMA-positive (AMA(+)) and AMA(-) patients, because they display indistinguishable clinical features. Of importance was the finding that the degree of bile duct damage around the portal areas was significantly milder in AMA(+) PBC than those observed in AMA(-) PBC patients. The portal areas from AMA(-) patients had a significant increase of cluster of differentiation (CD)5(+) cells infiltrating the ductal regions, and the levels of B-cell infiltrates were worse in the early phase of bile duct damage. The frequency of positive portal areas and the magnitude of CD5(+) and CD20(+) cellular infiltrates within areas of ductal invasion is associated with the first evidence of damage of biliary duct epithelia, but becomes reduced in the ductopenia stage, with the exception of CD5(+) cells, which remain sustained and predominate over CD20(+) cells. CONCLUSION: Our data suggest a putative role of B-cell autoimmunity in regulating the portal destruction characteristic of PBC.

Association of CD20 levels with clinicopathological parameters and its prognostic significance for patients with DLBCL.

Diffuse large B-cell lymphomas (DLBCL) express CD20. CD20 expression is described as negative, weak, or normal as determined by flow cytometry (FCM) and is an important target for the treatment of DLBCL. However, the impact of CD20 levels at onset of the disease on patient prognosis has not been fully elucidated. We analyzed 174 DLBCL cases newly diagnosed between January 1998 and April 2010. The relationship of the association between CD20 levels and patients' backgrounds and prognoses was analyzed using the Kaplan-Meier method and Cox proportional hazard regression. Of the 174 patients, three cases (1.7%) were defined as CD20 negative based on immunohistochemistry (IHC). Although the other 171 cases were positive by IHC, eight cases (4.7%) were defined as negative and 33 cases (19.3%) were defined as weak when analyzed by FCM. Of the 105 patients who received rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone therapy, those who were CD20 negative (FCM) showed significantly inferior overall (hazard ratios (HR): 6.79, 95% CI: 1.32-34.96, p = 0.04) and progression-free survival (HR: 7.3, 95% CI: 1.49-35.8, p = 0.04) compared to patients who were CD20 normal. Our findings indicate that the CD20 level (FCM) at onset is an independent predictor of the prognosis of patients with DLBCL.

Elevated frequency of CD1c+ myeloid dendritic cells in the peripheral blood mononuclear cells of simian/human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV) repeatedly infected Chinese rhesus macaques.

CD1c+ myeloid dendritic cells (mDCs) in the peripheral blood of 30 SHIV-SF162p4 and SIVmac251 sequentially infected Chinese rhesus macaques were examined by flow cytometry to obtain further insight into mDC alterations in HIV/AIDS. The CD1c+ cells were found to be mononuclear leukocytes rather than granulocytes, and most of them expressed CD20. CD1c+mDCs (CD1c+CD20-) consisted of two morphological subsets: the granular and the large CD1c+mDCs. The expression of HLA-DR, CD86, and CD11b, but no CCR7, CD83 and CD123, together with their endocytotic capacity indicated that they were immature mDCs. Their frequency at weeks 10 and 12 post-infection was significantly higher than that of un-infected ones; the large CD1c+mDC level was significantly different between time points and almost absent from un-infected rhesus monkeys; significant correlations between CD1c+mDCs and plasma viral load levels were also observed. These data indicated a possible role for CD1c+mDCs in the pathophysiological process of SIV/HIV infection.

Maintenance therapy with mycophenolate mofetil after rituximab in pediatric patients with steroid-dependent nephrotic syndrome.

Rituximab (RTX) has a significant steroid-sparing effect in children with steroid-dependent nephrotic syndrome (SDNS). However, patients are likely to relapse with the recovery of CD20+ cells. We conducted a small prospective cohort study with a historical control to evaluate the effect of RTX infusion followed by mycophenolate mofetil (MMF) as a maintenance therapy. Nine patients with SDNS who stopped their steroid treatment but were treated with MMF after RTX infusion were prospectively observed (group A). Seven patients with SDNS who discontinued steroid and immunosuppressive agents after RTX administration served as a control (group B). During the first year after the administration of RTX, six patients in group A and one patient in group B did not suffer a relapse (p < 0.05). The number of patients who relapsed during the 1 year preceding RTX treatment did not differ between the two groups [4.1 (A) vs. 5.7 (B)], but it was significantly lower in the MMF-treated group 1 year after the RTX treatment [0.4 (A) vs. 2.3 (B), p < 0.005]. The daily amount of prednisolone after the RTX treatment was lower in group A than in group B (0.11 vs. 0.46 mg/kg/day, respectively; p < 0.05). Three patients in group A and five patients in group B relapsed to SDNS and needed additional RTX treatment(s) within 1 year (odds ratio 5.0). Based on these results, we conclude that maintenance therapy with MMF after RTX is a good clinical option.

Diagnostic performance of the ClearLLab 10C B cell tube.

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.

Circulating CD52 and CD20 levels at end of treatment predict for progression and survival in patients with chronic lymphocytic leukaemia treated with fludarabine, cyclophosphamide and rituximab (FCR).

CD52 and CD20 antigens are important therapeutic targets for the monoclonal antibodies (mAbs) alemtuzumab and rituximab respectively. Circulating CD52 (cCD52) and CD20 (cCD20) have prognostic utility in lymphoid malignancies. The efficacy of mAb therapy in patients with chronic lymphocytic leukaemia (CLL) may be adversely affected by cCD52 or cCD20. In this report, blood and bone marrow (BM) cCD52 and cCD20 were measured at response assessment in previously treated (N = 235) patients with CLL who received fludarabine, cyclophosphamide, and rituximab (FCR). Univariate and multivariate statistical models evaluated correlations of pre- and response variables with progression-free (PFS) and overall survival (OS). Response variables included 1996 National Cancer Institute-Working Group (NCI-WG) response, polymerase chain reaction (PCR) for immunoglobulin heavy chain (IGHV) in BM, and cCD52 and cCD20 levels (blood and BM) at response assessment. Using multivariate analysis, response blood and BM cCD52, blood cCD20, and NCI-WG response were significant independent predictors of PFS. At the time of response assessment, BM cCD52 correlated with OS in univariate analysis. cCD52 and cCD20, therefore appear useful in predicting survival and may be important for monitoring patients following salvage FCR (fludarabine, cyclophosphamide, rituximab) therapy. These data further indicate that plasma may be a good target to evaluate for minimal residual disease using cCD52/cCD20 levels.

Severe idiopathic erythroblastic synartesis: successful treatment with the anti-CD20 monoclonal antibody rituximab.

Erythroblastic synartesis is a very rare disorder, considered to be caused by autoimmune mechanisms, leading to aggregation of erythroid precursor cells in the bone marrow and subsequently to acquired dyserythropoiesis with severe, transfusion-dependent anemia. An association with lymphoproliferative or autoimmune diseases has been reported or strongly suggested in all six published cases. Here, we report a young patient with severe idiopathic erythroblastic synartesis without an underlying disease, who was successfully treated with rituximab, an anti-CD20 monoclonal antibody. The patient received rituximab at a dose of 375 mg/m(2) once weekly for 4 wk after failure of both immunosuppressive therapies with corticosteroids and intravenous immunoglobulins. At a follow-up of 30 months after treatment, the patient is still in continuous complete remission without any further treatment, suggesting that rituximab may induce prolonged remissions and eventually cure in this rare disease.

Rituximab in childhood pemphigus vulgaris: a long-term follow-up case and review of the literature.

Pemphigus vulgaris is an infrequent but life-threatening autoimmune blistering disease that is rare in the pediatric age. Although the mainstay of therapy for childhood pemphigus vulgaris (CPV) is steroids, adjuvant immunosuppressive drugs are often needed to control the disease. Thus, an important part of CPV morbidity can be related to treatment. We report the youngest CPV patient described in the English literature, an 18-month-old boy, who has been followed up for 16 years. During this period, the patient has received several immunosuppressive therapies with variable response. He has finally achieved a long-lasting and complete remission with rituximab. Successful treatment with rituximab has previously been reported in 6 cases of CPV, in whom rituximab presented a good side effect profile. Our patient has experienced a chronic and severe clinical course with multiple flares eventually developing vegetative lesions. Since he presented refractoriness to multiple therapies, we administered rituximab. The introduction of this drug led to a dramatic clinical response and a long-term clinical remission. Based on the experience of this case and the data reported in the literature, we believe that rituximab may be a safe and efficacious agent for the treatment of severe CPV.

[Galavit-induced change of immunologic parameters in patients with non-small lung cancer].

Administration of an immunomodulator, galavit, for stage II-III non-small lung cancer along with standard therapy was followed by immunological vigor improvement by the end of the course. CD3, CD4, IgA, IgM and IgG indices were normal in more than 80% of the study group by day 51 after surgery; CD8, CD20 and HLA-DR--in more than 50%; CD16--in 45.2%. In control, by day 51, normal IgG and HLA-DR levels were reported in 60%. The remaining indices were normal in less than 50%. Our results point to immunological vigor improvement due to use of galavit. The drug is well tolerated, has neither side effects nor toxicity.