Peripheral Blood B-cell Counts Research Articles

Humoral Responses Against SARS-CoV-2 and Variants of Concern After mRNA Vaccines in Patients With Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia.

Patients with non-Hodgkin lymphoma including chronic lymphocytic leukemia (NHL/CLL) are at higher risk of severe SARS-CoV-2 infection. We investigated vaccine-induced antibody responses in patients with NHL/CLL against the original SARS-CoV-2 strain and variants of concern including B.1.167.2 (Delta) and B.1.1.529 (Omicron). Blood from 121 patients with NHL/CLL receiving two doses of vaccine were collected longitudinally. Antibody binding against the full-length spike protein, the receptor-binding, and N-terminal domains of the original strain and of variants was measured using a multiplex assay. Live-virus neutralization against Delta, Omicron, and the early WA1/2020 strains was measured using a focus reduction neutralization test. B cells were measured by flow cytometry. Correlation between vaccine response and clinical factors was determined. Mean anti-SARS-CoV-2 spike immunoglobulin G-binding titers were 85-fold lower in patients with NHL/CLL compared with healthy controls, with seroconversion occurring in only 67% of patients. Neutralization titers were also lower and correlated with binding titers (P < .0001). Treatment with anti-CD20-directed therapies within 1 year resulted in 136-fold lower binding titers. Peripheral blood B-cell count also correlated with vaccine response. At 3 months from last anti-CD20-directed therapy, B-cell count ≥ 4.31/μL blood around the time of vaccination predicted response (OR 7.46, P = .04). Antibody responses also correlated with age. Importantly, neutralization titers against Delta and Omicron were reduced six- and 42-fold, respectively, with 67% of patients seropositive for WA1/2020 exhibiting seronegativity for Omicron. Antibody binding and live-virus neutralization against SARS-CoV-2 and its variants of concern including Delta and Omicron were substantially lower in patients with NHL/CLL compared with healthy vaccinees. Anti-CD20-directed therapy < 1 year before vaccination and number of circulating B cells strongly predict vaccine response.

The Role of Imaging in Predicting Time to First Treatment and Overall Survival in Individuals with CLL-like High Count Monoclonal B-Cell Lymphocytosis

Background: The 2008 International Working Group for CLL (IWCLL) criteria define MBL as a clonal B-cell disorder with a peripheral blood B-cell count of &lt;5 x 109/L, in the absence of cytopenias(s), lymphadenopathy and/or organomegaly on physical examination, and no other concomitant lymphoproliferative disorder. In the presence of palpable lymphadenopathy and/or organomegaly, and a B-cell count of &lt; 5 x 109/L, a diagnosis of small lymphocytic lymphoma (SLL) is established. A study by Gentile et al (Am J Hematol. 2013) reported that 29 out of 62 individuals with clinically identified MBL (42%) had lymphadenopathy identified on CT imaging. Notably, however, after a median follow-up of 35 months, the rate of progression among cases of MBL with lymphadenopathy identified on CT imaging was only 6.9% (2/29 patients), which was no different than in cases of MBL without lymphadenopathy on CT imaging (rate of progression, 9%; 3/33 patients). Our study sought to 1) determine if lymphadenopathy and/or organomegaly detected by imaging studies performed for unrelated reasons in individuals with MBL can identify a subset with shorter time to first CLL therapy (TFT) and overall survival (OS); and 2) compare outcomes of individuals with MBL who have incidentally detected lymphadenopathy/splenomegaly and SLL patients. Methods: We used the Mayo Clinic CLL Database to identify individuals with high-count MBL (absolute B-cell count: 0.5 - 4.9 x 109/L) and divided them into the following three cohorts: a) Cohort A: no imaging studies available at MBL diagnosis; b) Cohort B: imaging studies done within one year of MBL with no evidence of lymphadenopathy and/or organomegaly; and c) Cohort C: imaging studies done within one year of MBL with lymphadenopathy and/or organomegaly, attributable to MBL. All individuals with MBL were first seen at Mayo Clinic after 1/1/2002; those with a concomitant lymphoproliferative disorder within 3 months of diagnosis were excluded. Patients with SLL seen during the same time interval were identified as a comparison cohort. We compared the baseline characteristics (Kruskal Wallis for continuous and Chi-square for categorical variables) and TFT and OS across the three MBL cohorts, and patients with SLL. Cumulative incidence of TFT was adjusted for the competing risk of death. Age- and sex-adjusted OS was analyzed using inverse weights methods. Results: 657 individuals with high-count MBL with a median follow-up of 6.7 years were identified: 415 (63%) individuals were in Cohort A, 142 (22%) in Cohort B, and 100 in (15%) Cohort C. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated IGHV, high expression of CD38, and higher beta-2 microglobulin level (Table 1). SLL patients were younger, had higher rate of unmutated IGHV, and had high expression of CD49d compared to MBL individuals in Cohort C (Table 1). There was suggestive evidence of a difference in TFT between Cohorts B and C (estimated 10-year treatment rate 28.3% vs. 41.6%; p=0.16; HR 1.62 95% CI 0.84-3.15). TFT was significantly shorter in SLL patients compared to those with Cohort C (estimated 10-year treatment rate 71% vs. 41.6%, respectively, P&lt;0.001; Figure 1A). There was a trend toward shorter OS in Cohort C compared to Cohort B (estimated 10-year OS rate 53.7% vs. 64.2%, respectively, P=0.09; Figure 1B). In contrast, there was no difference in OS among MBL individuals in Cohort C compared to those with SLL (estimated 10-year OS rate 56.9% vs. 62.6%, P=0.72). Summary/Conclusion: Our findings demonstrate that individuals with MBL who have incidentally identified lymphadenopathy/splenomegaly at diagnosis are more likely to have an unfavorable risk profile, shorter TFT and OS compared to patients who did not have lymphadenopathy/splenomegaly at MBL diagnosis. Additionally, these individuals have a longer TFT but similar OS compared to SLL patients seen during the same time interval. If externally validated, these findings have significant implications for individuals with newly diagnosed MBL. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding.

Impaired B Lymphopoiesis Concurrent with Marrow Infiltration By Allogeneic Donor T-Cells: Further Evidence for Bone Marrow Graft-Versus-Host Disease

Background: The recently introduced concept of bone marrow graft-versus-host disease (BM-GvHD) representing destruction of the host hematopoietic niche in the marrow by allogeneic donor T-cells is loosely defined. Otherwise unexplained B lymphocytopenia and other cytopenias of unclear etiology that frequently occur in the early post-transplant period are often attributed to BM-GvHD. B lymphocytopenia can co-exist with systemic GvHD, supporting the theory of concurrent donor T-cell-induced damage to the marrow causing suppressed B lymphopoiesis. However, demonstration of such correlations has been difficult in humans due to the lymphotoxic effect of steroids which are the frontline therapy for GvHD. The best evidence comes from a recent study showing a correlation between delayed recovery of B lymphopoiesis and donor T-cell infiltration in the marrow 3-4 weeks post-transplant. The potential confounding effect of steroids was not evaluated. The purpose of the present study was to assess whether there is significant donor T-cell infiltration in the marrow at the time of B lymphocytopenia in carefully selected, fully chimeric allo-SCT recipients on minimal or no steroids and with minimal or no systemic GvHD.Methods: A total of 11 patients who underwent allo-SCT for myeloid malignancies were retrospectively studied. Inclusion criteria were: (i) bone marrow biopsy available on days 90-100 or 170-190 post-SCT concurrent with peripheral blood B-cell count using flow cytometry, (ii) full donor chimerism at the time of bone marrow biopsy, (iii) no B lymphodepleting therapy post-SCT, (iv) not on more than 15 mg/d of prednisone on the day of measurement, (v) no GvHD other than acute stage I skin GvHD, and (vi) delayed B-cell recovery defined as <10 CD19+ B-cells/µl on days 90-100 or <100 cells/µl on days 180-200. Peripheral B-cell count measurements were not due to specific clinical indications and were either based on the treating physician's routine practice or the protocols patients were enrolled to. Similar to previous studies, increased T-cell infiltration was defined as ≥5% of total nucleated cells in the core determined by anti-CD3 antibody labeling in a fully chimeric recipient. We determined the frequency of increased marrow T-cell infiltration (as a marker of acute BM-GvHD) on the same day when delayed B-cell recovery (as a marker of impaired B lymphopoiesis) was diagnosed.Results: 11 patients (10 males) with a median (range) age of 60 (32-67) years were studied. Measurements were made between days 90-100 and 170-190 post-SCT in 4 and 7 patients, respectively. The underlying diagnosis was acute myeloid leukemia (n = 7) or myelodysplastic syndrome (n = 4). The donor was a matched sibling (n = 1), matched unrelated donor (n = 4), or haploidentical donor (n = 6). Conditioning was ablative in 5 patients. Leukopenia, anemia, and thrombocytopenia were present in 3, 9, and 11 patients, respectively. The median (range) B-cell count on days 90-100 and 180-200 was 7 (0-9) and 19 (0-65) cells/µl, respectively. All patients had ≥5% T-cells/µl in the concurrent core biopsy with one exception. This patient had zero B-cells on day 180 but no evidence of concurrent BM-GvHD while on 12 mg/d of prednisone for appetite stimulation.Conclusions: Using a carefully selected cohort of fully chimeric allo-SCT recipients with delayed B lymphopoiesis, on no or minimal amounts of steroids, and with minimal or no systemic GvHD, we demonstrated a high frequency of concurrent increased marrow T-cell infiltration. These results support the recently introduced concept of BM-GvHD and highlight its negative effect on B lymphopoiesis. We show that bone marrow damage by allogeneic T-cells can occur even in the absence of systemic GvHD. Given the difficulties in quantification of marrow T-cells on the core, a more reproducible definition for BM-GvHD is needed. DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetic and Pharmacodynamic Properties of a Biosimilar Rituximab (Reditux®) Are Identical to the Innovator Brand MabThera®– Experience from a Tertiary Cancer Centre in Western India

Background: Rituximab is a chimeric IgG1 monoclonal antibody targeting the CD20 surface antigen on normal and neoplastic B cells. Its use in combination with chemotherapy in CD20 positive B cell lymphomas has translated into better progression free and disease free survival making its incorporation, a standard of care in treatment of these lymphomas. However, the benefits of this important molecule may not have been available to a majority of patient in the developing countries but for the emergence of biosimilars in the market. The lack of defined regulatory norms in approving biosimilars for patient care has left some doubts regarding their true efficacy in comparison to the innovator MabThera® (Roche). Although our clinical experience suggests comparable efficacy and safety for the biosimilar, we embarked on a formal pharmacokinetic study of the biosimilar, Reditux®, used in combination with standard CHOP or CHOP like regimen in patients with diffuse large B cell lymphoma planned for chemo-immunotherapy. We present our data on twenty-one prospectively treated patients and compared it with the published data on MabThera®.Methods: This was a prospective, single centre pharmacokinetic study conducted between Nov 2011 and June 2013. Patients diagnosed with DLBCL, and treated with Reditux® in combination with chemotherapy, were enrolled into the study. All patients received six cycles of R-CHOP, wherein they received 375mg/m2 Reditux as a graded infusion of 50mg, 100mg and maximum 200mg during the first, second and third hour respectively on day 1 of first cycle. The chemotherapy consisted of standard CHOP including cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2 and vincristine 1.4 mg/m2 (for a maximum single dose of 2 mg), administered intravenously on day 1 after Rituximab infusion, along with 100 mg oral prednisone for 5 days from first day. Serial blood sampling was done during the first cycle of R-CHOP at pre-dose, immediately post infusion and thereafter at 24, 48, 144 hours post infusion with a last sample collected on day 21. The plasma level of rituximab was determined by ELISA (Life Technologies, USA) with a sensitivity of 50 ng/mL. Rituximab AUC and other pharmacokinetic parameters were calculated from the observed concentrations at each time point using WinNonlin® version 6.3. The peripheral blood B-cell count was done on day 3 and day 21 of chemoimmunotherapy. The absolute B cell percentage was calculated by flow-cytometry using CD45, CD19 and CD20. Influence of covariates on AUC of Reditux was analyzed by linear regression.Results: A total of 21 patients were enrolled into the study including 18 males and 3 females. The median age was 50 years in the Reditux® group (range 24-70). Four patients were above 60. Majority (n=12) had Stage 4 disease. The pharmacokinetic parameters described in the table were comparable with the pharmacokinetic profile of MabThera® from published literature. High inter patient variability in pharmacokinetics was observed (CV=88%). Age and gender did not influence pharmacokinetics of the biosimilar though the numbers are few to derive firm conclusions. The median B cell count on day 3 and day 21 of first cycle was 1.75 ± 0.27 and 5.56 ± 1.24 cells/µl respectively. Of the 21 patients, four were lost to follow up while 2 patients had progressive disease and died. At a median follow of 19 months 70.3% of patients from the Reditux® group were progression free. There was no untoward immediate or late toxicity attributable to Reditux® on follow up.Conclusions: Reditux® has similar pharmacokinetic profile as MabThera®. The B-cell kinetics following Reditux® administration are similar to that reported for MabThera®. This study gives further credence to Reditux® as a suitable alternative to MabThera®in the treatment of CD20 positive B cell lymphomas.Table:Pharmacokinetic parameters of Reditux® and Mabthera®PK parameter (mean ± SD)Reditux®(N=21)MabThera® (published literature)AUC (0-t) hr* µg / mL54,236 ± 47,555-C max (µg / mL )555.74 ± 141.46408Vd ( L)1.3 ± 0.642.7CL (l/day)0.15 ± 0.160.14Half life (days )10.9 ± 8.622MRT last (days)2.78 ± 3.08-Vd=Volume of distribution, CL=Clearance, MRT=Mean Residence Time DisclosuresNo relevant conflicts of interest to declare.

Novel Use of Rituximab for Steroid-Dependent Nephrotic Syndrome in Children

Background: Though rituximab (RTX) is effective for childhood steroid-dependent nephrotic syndrome (SDNS), an established regimen does not exist. The relapses tend to occur when the peripheral blood B-cell count re-arises at 3 months upon single RTX infusion. This study was conducted to clarify whether the long-term remission of SDNS can be obtained by repeated RTX administrations. Methods: RTX was administered 4 times at 3-month intervals at 375 mg/m²/time to 5 children with SDNS. The changes in the clinical indicators were analyzed. Results: The median (range) observation period was 6.3 (0.9-8.4) years before RTX and 3.2 (1.9-3.8) years following the commencement of RTX. The changes in the clinical indicators were as follows (median and range): (1) annual number of relapses: before administration 1.4 (1.1-3.5) times/year, after administration 0.0 (0.0-0.0) times/year, and (2) median steroid dosage: before administration 0.80 (0.23-0.96) mg/kg/day, after administration 0.00 (0.00-0.00) mg/kg/day. All changes were significant at p < 0.05. Relapse occurred 3 times following the start of RTX (the period to relapse was 2.2, 1.9, and 2.3 years, respectively). No serious side effects were seen. Conclusions: Repeated RTX against SDNS in children may be a useful therapeutic option.

AB0024 Systemic inflammation and b-cells in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is heterogeneous in clinical symptoms, clinical parameters, pathogenesis and gene expression levels. Previously, we demonstrated variation in B-cell related gene expression between RA patients.ObjectivesThe aim was to...

Determination of Rituximab Dose According to Immunologic Risk in ABO-Incompatible Kidney Transplantation

The adequate rituximab (RTX) dosage in ABO-incompatible transplantation (ABO-IKT) remains undetermined. We used two kinds of RTX dosage groups [low RTX (100 mg/m2) and typical RTX (375 mg/m2) dosage groups] according to immunologic risks and investigated the change of B-cell, anti-ABO antibodies, and the clinical outcome in ABO-IKT according to the RTX dose. Fifteen patients with high immunologic risk [panel reactive antibody (PRA) > 50%, retransplant, AB to O transplant] were assigned to typical RTX group and 17 patients without risk were assigned to low RTX group. We compared the changes of B-cell, anti-ABO antibody titer, required number of plasmapheresis (PP), and the clinical outcome after transplantation between the two groups. After infusion of RTX, peripheral blood B-cell counts were successfully depleted to <1% in both groups. Before kidney transplantation (KT), the minimal number of PP to achieve the target titer (1:16) (2.6 ± 2.7 vs. 2.2 ± 2.5; p = 0.66) and the titer reduction rate of anti-ABO antibodies did not differ between the two groups (low RTX: 1.52 ± 1.21 vs. typical RTX: 1.53 ± 1.20, p = 0.94). After KT, anti-ABO antibody titer was suppressed less than 1:32 in both groups up to posttransplant 1 year. The allograft function and infectious complication did not differ between the two groups as well. In ABO-IKT, low RTX is comparable with typical RTX dosing with respect to B-cell depletion, antibody rebound suppression, the effect on clinical outcome in patients with low immunologic risk.

B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy

AbstractCharacterization of lymphocytes including B cells during early versus chronic HIV infection is important for understanding the impact of chronic viremia on immune cell function. In this setting, we investigated B cells before and after reduction of HIV plasma viremia by antiretroviral therapy (ART). At baseline, peripheral blood B-cell counts were significantly lower in both early and chronic HIV-infected individuals compared with uninfected controls. Similar to CD4+ but not CD8+ T cells, B-cell numbers in both groups increased significantly after ART. At baseline, B cells of early HIV-infected individuals were composed of a higher percentage of plasmablasts and resting memory B cells compared with chronic HIV-infected individuals whose B cells were composed of a higher percentage of immature/transitional and exhausted B cells compared with their early infection counterparts. At 1 year after ART, the percentage of resting memory B cells remained higher in early compared with chronic HIV-infected individuals. This difference translated into a better functional profile in that memory B-cell responses to HIV and non-HIV antigens were superior in early- compared with chronic-treated HIV infected individuals. These findings provide new insights on B cells in HIV infection and how early initiation of ART may prevent irreversible immune system damage.

Biologic activity and safety of belimumab, a neutralizing anti-B-lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus

IntroductionThis trial evaluated the safety, biologic activity, and pharmacokinetics of belimumab, a fully human monoclonal antibody that inhibits the biologic activity of the soluble form of the essential B-cell survival factor B-lymphocyte stimulator (BLyS) in patients with systemic lupus erythematosus (SLE).MethodsSeventy patients with mild-to-moderate SLE were enrolled in a phase I, double-blind, randomized study and treated with placebo (n = 13) or belimumab (n = 57) at four different doses (1.0, 4.0, 10, and 20 mg/kg) as a single infusion or two infusions 21 days apart. Patients were followed for 84 to 105 days to assess adverse events, pharmacokinetics, peripheral blood B-cell counts, serology, and SLE disease activity. Data from the study were summarized using descriptive statistics. χ2 type tests were used to analyze discrete variables. The Kruskal-Wallis test, the Wilcoxon test, and the analysis of covariance were used to analyze the continuous variables, as appropriate. The analysis was performed on all randomized patients who received study agent.ResultsThe incidences of adverse events and laboratory abnormalities were similar among the belimumab and placebo groups. Belimumab pharmacokinetics were linear across the 1.0 to 20 mg/kg dose range. Long terminal elimination half-life (8.5 to 14.1 days), slow clearance (7 ml/day per kg), and small volume of distribution (69 to 112 ml/kg) were consistent with a fully human antibody. Significant reductions in median percentages of CD20+ B cells were observed in patients treated with a single dose of belimumab versus placebo (day 42: P = 0.0042; and day 84: P = 0.0036) and in patients treated with two doses of belimumab versus placebo (day 105: P = 0.0305). SLE disease activity did not change after one or two doses of belimumab.ConclusionsBelimumab was well tolerated and reduced peripheral B-cell levels in SLE patients. These data support further studies of belimumab in autoimmune disorders.Trial RegistrationNCT00657007 [clinicaltrials.gov].

Monoclonal antibody therapy of chronic lymphocytic leukemia.

Chemotherapeutic approaches during the last decade have failed to result in major advances in the outcome of patients with chronic lymphocytic leukemia (CLL). The recent availability of an increasing number of active monoclonal antibodies, immunotoxins, and radioimmunoconjugates (RICs) has stimulated considerable interest in clinical research in CLL. Alemtuzumab was the first antibody approved for CLL on the basis of responses in one third of patients with advanced disease. However, infusion reactions and immunosuppression with opportunistic infections present a challenge that may be overcome with altered schedules and routes of administration. Rituximab has limited activity as a single agent in patients relapsed or refractory after prior chemotherapy; however, response rates seem to be higher in previously untreated patients. More importantly, combinations with chemotherapy drugs such as fludarabine are showing promise in early trials. Newer antibodies in development as single agents and in combinations include apolizumab (Hu1D10), a humanized antibody against an epitope of HLA-DR, and IDEC-152, a primatized anti-CD23 antibody. BL22, an immunotoxin with impressive activity in hairy cell leukemia, is in phase II trials in CLL as well. The safe use of RICs is complicated by the elevated peripheral blood B-cell count, and the extent of bone marrow involvement in CLL; studies will explore the use of agents to eliminate malignant cells from the bone marrow before RIC therapy. It is hoped that the rational development of combinations of the various promising antibodies with chemotherapy and each other will lead to more effective approaches for patients with CLL.

The effect of exposure of hospital employees to patients with tuberculosis on dermal reactivity to four new tuberculins

An early (6–8 h) erythematous response to Purified Protein Derivative and to sonicate antigens (new tuberculins) prepared from Mycobacterium tuberculosis M. vaccae, M. scrofulaceum, and M. leprae occurred much more frequently amongst hospital employees exposed to patients with tuberculosis than amongst factory workers. Biopsies taken from the skin test sites at 48 h revealed a more intense inflammatory cell infiltrate in response to PPD and the sonicate of M. tuberculosis, but not to the antigens of the other mycobacteria, amongst the hospital employees thus indicating a degree of specificity. The early response appears to be directed towards species specific antigens, but not, apparently, to the same as those that elicit the 48 h reactions. The hospital employees also had higher peripheral blood B-cell counts and total IgG levels, suggestive of an adjuvant effect. It is postulated that the early reaction results from repeated exposure to tubercle bacilli and the possible nature of the reaction is discussed.