Peripheral B-cell Counts Research Articles

Poor Hematopoietic Reserve at the Time of CD19 CAR T-Cell Infusion Is Associated with Long Term B-Cell Aplasia

Background: On-target off-tumor toxicities of anti-CD19 CAR T-cell therapy include B-cell aplasia and hypogammaglobulinemia. Long term B-cell aplasia is hypothesized to represent functional CAR T-cell engraftment (Melenhorst, Nature 2022), while peripheral blood B-cell recovery represents the loss of functional CAR-T cells against target over time. Patients with large B-cell lymphoma (LBCL) who are clinical responders have a 50-70% chance of B-cell recovery in the first year, with the remainder experiencing longer term B-cell aplasia (Logue, Haematologica 2021). Here, we sought to identify factors associated with long term B-cell aplasia using a cohort of responding patients who received CAR T-cell therapy for LBCL. Methods:This retrospective cohort study included 57 patients with LBCL treated with CD19-targeted CAR T-cell therapy between June 2016 and August 2020 at Moffitt Cancer Center who exhibited complete or partial response at six months after CAR T-cell infusion. Patients were treated with with axicabtagene ciloleucel (axi-cel; n=50) or tisagenlecleucel (tisa-cel; n=7), either as standard-of-care therapy (n=47) or as part of a previously published clinical trial (n=10; NCT02348216, NCT03391466, NCT03153462). Peripheral blood B-cells were measured by flow cytometry using CD19 expression as part of routine clinical care at baseline and periodically after infusion. Prolonged B-cell aplasia was defined as a CD19+ B cell count comprising less than 1% of peripheral blood mononuclear cells during a minimum of two separate time points beyond 6 months of follow-up. Patients were defined as having B cell recovery if >1% B-cells were observed in peripheral blood at any time after CAR T-cell infusion. Results: At a median follow up of 33 months (95% CI: 28.7, 37.3 months), 29 (51%) patients had persistent B-cell aplasia and 28 (49%) had recovery of peripheral B-cell counts. Median B-cell count at 12 months in the recovery group was 84/µL (IQR 2.5-197) compared to 0/µL (IQR 0-1) in the aplasia group. Patients with long-term B-cell aplasia had lower CD4 T-cell counts prior to CAR T-cell infusion [median 200/µL (IQR 136-278) vs. 330/µL (IQR 224-451); P= 0.008] and over time compared to patients with B-cell recovery (Fig. A). Patients with long-term B cell aplasia also had lower pre-infusion absolute neutrophil counts (2.3K/µL (IQR 1.4-4.15) vs. 3.5K/µL (IQR 2.8-4.2); P<0.001) and higher baseline CAR-HEMATOTOX scores [2 (IQR 1-3.25) vs 1 (IQR 0-1)], consistent with poor baseline hematopoietic reserve (Rejeski, Blood 2021). While disease-related mortality did not significantly differ between groups, non-relapse mortality was markedly higher in patients with long-term B-cell aplasia due to infection: all 8 patient deaths in the aplasia group were due to infections occurring beyond day 30 post-infusion, with 6 of the 8 deaths due to COVID-19. However, no patients in the B-cell recovery group died of late infection (Fig. B). Conclusions: Long term B-cell aplasia is hypothesized to represent ongoing CAR T-cell activity against normal B-cells. However, our results demonstrate that B-cell aplasia is associated with low baseline hematopoietic reserve and subsequent poor immune reconstitution of multiple lineages including B-cells, T-cells, and myeloid cells. Further studies are warranted to assess whether poor immune reconstitution and B-cell aplasia are related to long term CAR T-cell activity or other processes. These patients are at higher risk for non-relapse mortality from infection, and mitigation strategies are needed.

Rituximab as Initial Therapy in Adult Patients With Minimal Change Disease

Rituximab as Initial Therapy in Adult Patients With Minimal Change Disease

Low circulating B cells in immunocompromised individuals are linked to poorer antibody responses to vaccines and a predisposition to viral infections.

B cells play an important role in protection against viral infections, not only through the production of antibodies but also through their ability to act as antigen-presenting cells and produce cytokines. To assess whether there is a link between low circulating B-cell counts and a predisposition to viral infections in immunocompromised individuals, we performed a retrospective cohort analysis at 2 National Health Service Clinical Immunology sites in England. Eligible patients were adults who were either diagnosed with or under investigation for an immunodeficiency and had recorded circulating B-cell counts. Information on viral infections was collected by using the departmental, hospital, and laboratory electronic information systems. Ageneralized linear model was used to analyze the relationship between B-cell counts and relevant indices of viral infection while controlling for patient age, diagnosis group, and T-cell and natural killer cell counts. A total of 376 eligible patients were identified, 134 of whom had B-cell counts that were below the laboratory-defined refence range (<0.11 ×109/L). Patients with low numbers of circulating B cells had lower pretreatment immunoglobulin levels and poorer antibody responses to vaccines (Streptococcus pneumonia, Clostridium tetani, and Haemophilus influenzae type B). An increased number of chronic or recurrent (P= .001), severe or unusual (P= .001), and PCR-confirmed viral infections (P= .04) were recorded in these patients versus in those with normal numbers of circulating B cells. Overall, there was a statistically significant association between low circulating B-cell counts and the incidence of clinically important viral infections in this patient cohort, even when controlling for relevant covariates. Clinicians caring for patients with immunodeficiency should be vigilant for these types of infections, particularly in patients with low peripheral B-cell counts. Aprospective study will be required to confirm these findings.

Low Peripheral B-Cell Counts in Patients With Systemic Rheumatic Diseases Due to Treatment With Belimumab and/or Rituximab Are Associated With Low Antibody Responses to Primary COVID-19 Vaccination.

Background: Immunosuppressive agents inhibit COVID-19 vaccine antibody (Ab) responses in patients with systemic rheumatic diseases. Rituximab may fully block Ab responses when B cells become undetected. The effect of detected but low number of B cells due to treatment with a B-cell agent (belimumab and/or rituximab) has not been established. Purpose: We sought to examine whether there is an association between a low number of B cells due to treatment with belimumab and/or rituximab and impaired primary COVID-19 vaccination spike Ab responses in patients with systemic rheumatic diseases. Methods: We retrospectively examined Ab responses to COVID-19 vaccinations, especially in relation to B-cell counts after treatment with belimumab and/or rituximab, in 58 patients with systemic rheumatic diseases: 22 on and 36 not on B-cell agents. We used Kruskal-Wallis and Mann-Whitney U tests for comparison of Ab values between the groups and Fisher exact test for relative risk calculations. Results: Median (interquartile range) postvaccination Ab responses were lower in patients on versus those not on B-cell agents: 3.91 (0.77-20.00) versus 20.00 (14.32-20.00), respectively. Among patients on belimumab and/or rituximab, Ab responses of less than 25% of the assay's upper limit were exclusively observed in those with B-cell counts lower than 40/µL. Patients with B-cell counts lower than 40/µL exhibit a relative risk of 6.092 (95% CI: 2.75-14.24) for Ab responses of less than 25% of the upper limit compared with patients not on B-cell agents. This relative risk remained significant, even after excluding patients with undetected B cells. Conclusion: This retrospective study found an association between low B-cell counts (less than 40/µL) and decreased Ab responses to primary COVID-19 vaccination in patients with systemic rheumatic diseases treated with belimumab and/or rituximab. Despite the small number of patients studied, these findings add to the accumulating evidence on the importance of B-cell count in predicting spike Ab responses to COVID-19 vaccination.

Characteristics of COVID-19 Patients With SARS-CoV-2 Positivity in Feces.

BackgroundSARS-CoV-2 is highly contagious and poses a great threat to epidemic control and prevention. The possibility of fecal-oral transmission has attracted increasing concern. However, viral shedding in feces has not been completely investigated.MethodsThis study retrospectively reviewed 97 confirmed coronavirus disease 2019 (COVID-19) patients hospitalized at the First Affiliated Hospital, School of Medicine, Zhejiang University, from January 19 to February 17, 2020. SARS-CoV-2 RNA in samples of sputum, nasopharyngeal or throat swabs, bronchoalveolar lavage and feces was detected by real-time reverse transcription polymerase chain reaction (RT–PCR). Clinical characteristics and parameters were compared between groups to determine whether fecal RNA was positive.ResultsThirty-four (35.1%) of the patients showed detectable SARS-CoV-2 RNA in feces, and 63 (64.9%) had negative detection results. The median time of viral shedding in feces was approximately 25 days, with the maximum time reaching 33 days. Prolonged fecal-shedding patients showed longer hospital stays. Those patients for whom fecal viral positivity persisted longer than 3 weeks also had lower plasma B-cell counts than those patients in the non-prolonged group [70.5 (47.3-121.5) per μL vs. 186.5 (129.3-376.0) per μL, P = 0.023]. Correlation analysis found that the duration of fecal shedding was positively related to the duration of respiratory viral shedding (R = 0.70, P < 0.001) and negatively related to peripheral B-cell counts (R = -0.44, P < 0.05).ConclusionsCOVID-19 patients who shed SARS-CoV-2 RNA in feces presented similar clinical characteristics and outcomes as those who did not shed SARS-CoV-2 RNA in feces. The prolonged presence of SARS-CoV-2 nucleic acids in feces was highly correlated with the prolonged shedding of SARS-CoV-2 RNA in the respiratory tract and with lower plasma B-cell counts.

Humoral responses to SARS-CoV-2 vaccination in rituximab-treated patients depend on peripheral B cell re-population rather than the timings of the dosing

Introduction: Previous exposure to Rituximab affects the immunogenicity of vaccination including that against coronavirus disease (COVID-19). However, dynamics of the effect of rituximab on vaccination are not well understood. This study aims to assess the role of timing of vaccine dosing and B-cell repopulation on vaccine seroconversion.Methods: Autoimmune rheumatic disease (AIRD) patients treated with rituximab who had completed two doses of COVID-19 vaccination were enrolled. Peripheral B-cell counts were estimated along with the titer of immunoglobulin G antibody-directed against the receptor-binding domain of spike1 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody titers of participants were compared against B-cell re-population.Results: Out of the 33 participants with available prevaccination B-cell counts, 11 did not have any detectable peripheral B cells before vaccination, and out of these patients, only 1 developed antibodies postvaccination, whereas, of the remaining 22, 18 (81.8%) had a positive seroconversion. Although all patients who had received the last dose of Rituximab at least 1 year before vaccination had antibodies, there was no direct correlation between time from the last dose and antibody positivity. B-cell re-population was strongly associated with seroconversion (P = 0.0001).Conclusion: In rituximab-treated patients, humoral responses to SARS-CoV-2 vaccination depend on peripheral B-cell re-population rather than the timing of the vaccination postrituximab infusion.

A multi-center, open-label, randomized, parallel-controlled phase II study comparing pharmacokinetic, pharmacodynamics and safety of ripertamab (SCT400) to rituximab (MabThera®) in patients with CD20-positive B-cell non-Hodgkin lymphoma.

This multi-center, open-label, randomized, parallel-controlled phase II study aimed to compare the pharmacokinetics (PK), pharmacodynamics (PD) and safety profile of ripertamab (SCT400), a recombinant anti-CD20 monoclonal antibody, to rituximab (MabThera®) in patients with CD20-positive B-cell non-Hodgkin lymphoma (NHL). Patients with CD20-positive B-cell NHL who achieved complete remission or unconfirmed complete remission after standard treatment were randomly assigned at a 1:1 ratio to receive a single dose of ripertamab (375 mg/m2) or rituximab (MabThera®, 375 mg/m2). PK was evaluated using area under the concentration-time curve (AUC) from time 0 to d 85 (AUC0-85 d), AUC from time 0 to week 1 (AUC0-1 w), AUC from time 0 to week 2 (AUC0-2 w), AUC from time 0 to week 3 (AUC0-3 w), AUC from time 0 to week 8 (AUC0-8 w), maximum serum concentration (Cmax), terminal half-life (T1/2), time to maximum serum concentration (Tmax) and clearance (CL). Bioequivalence was confirmed if the 90% confidence interval (90% CI) of the geometric mean ratio of ripertamab/rituximab was within the pre-defined bioequivalence range of 80.0%-125.0%. PD, immunogenicity, and safety were also evaluated. From December 30, 2014 to November 24, 2015, a total of 84 patients were randomized (ripertamab, n=42; rituximab, n=42) and the PK analysis was performed on 76 patients (ripertamab, n=38; rituximab, n=38). The geometric mean ratios of ripertamab/rituximab for AUC0-85 d, AUC0-inf, and Cmax were 96.1% (90% CI: 87.6%-105.5%), 95.9% (90% CI: 86.5%-106.4%) and 97.4% (90% CI: 91.6%-103.6%), respectively. All PK parameters met the pre-defined bioequivalence range of 80.0%-125.0%. For PD and safety evaluation, there was no statistical difference in peripheral CD19-positive B-cell counts and CD20-positive B-cell counts at each visit, and no difference in the incidence of anti-drug antibodies was observed between the two groups. The incidences of treatment-emergent adverse events and treatment-related adverse events were also comparable between the two groups. In this study, the PK, PD, immunogenicity, and safety profile of ripertamab (SCT400) were similar to rituximab (MabThera®) in Chinese patients with CD20-positive B-cell NHL.

ALL Maintenance Treatment for Early Loss of B-Cell Aplasia after Tisagenlecleucel Therapy

Chimeric antigen receptor (CAR) T-cell therapy is a new, effective treatment for patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukaemia (ALL). Tisagenlecleucel achieved a complete remission (CR) rate and minimal residual disease (MRD) negativity of 81% at 3 months in the pivotal study; overall survival (OS) was 76% at 12 months (Maude et al, 2018). Real world data confirmed similar outcomes, with 1-year OS of 77% and event free survival (EFS) of 52% (Pasquini et al, 2020). Relapse can occur in the form of CD19 negative or CD19 positive ALL. The latter is associated with lack of persistence of the CAR T product. B-cell aplasia (BCA) is an indirect measure of CAR T presence. Early (<6 months from infusion) loss of BCA is associated with high relapse risk (Pillai et al, 2019); therefore, allogeneic stem cell transplantation (SCT) is often considered. However, SCT is associated with therapy-related morbidity and mortality and not all patients will find a suitable donor. Therefore, the optimal management of patients with loss of BCA is yet to be defined.In our centre, we administered maintenance therapy to a cohort of children with early loss of BCA. When compared to UK patients undergoing SCT for the same indication, we noted promising early outcomes. We report the findings here.We collected data on children with r/r ALL treated with tisagenlecleucel at Great Ormond Street Hospital (GOSH) from January 2018 to January 2021 who presented loss of BCA without evidence of disease (negative molecular or flow cytometry MRD) within 12 months from infusion. Loss of BCA was defined as peripheral B-cell count ≥0.10 x 10^9/L or bone marrow (BM) CD19+ events ≥0.1%. We compared outcomes of children who received maintenance as per UKALL 2011 protocol at GOSH to those who received SCT for the same indication from all UK paediatric centres.Fourteen patients from GOSH met the inclusion criteria. Four had loss of BCA after 6 months from CAR T infusion, none of them received additional therapy and they are all alive and in CR at a median of 535 days after CAR T infusion (Figure 1, A and B). Ten patients recovered B cells at <6 months: 3 proceeded to SCT, 6 started on maintenance therapy, 1 received other treatment. In 2 cases, maintenance was commenced after a second CAR T infusion. Two patients from the UK cohort met the inclusion criteria for the SCT group. Analysis was performed on 6 children who received maintenance and 5 who had SCT. Baseline characteristics of the 2 groups were similar (male/female ratio, median age at infusion, cytogenetics). Time from infusion to loss of BCA did not differ: the median was 80 days (range 28-168) in children who had maintenance vs 93 days (range 28-150) in those who had SCT. At a median follow up of 511 days (range 222-812), 3/6 children who received maintenance relapsed at median 210 days after infusion and proceeded to further treatment, no patient relapsed post SCT. One child died of disease in the maintenance group 237 days after infusion, 2 children died of transplant related mortality in the SCT group at 222 and 422 days post infusion. OS and EFS did not differ statistically between the 2 groups, as shown in Figure 1 (C and D). We observed that outcome for patients who presented loss of BCA within or at 2 months from infusion was poor regardless of the intervention (maintenance or SCT).Management of patients who experience early loss of BCA after CAR T is challenging and there are little data to support optimal treatment. In our experience, maintenance therapy compared favourably with SCT with similar rate of OS and EFS. Of note, 2/5 patients died of TRM in the SCT group highlighting the toxicity of this approach in such heavily pre-treated patients. On the other hand, maintenance is a well tolerated, low-cost treatment which can be easily delivered on an out-patient basis. Our preliminary data support investigation of this strategy in larger, prospectively-recruited cohorts of patients. Moreover, our preliminary data suggest that the time of loss of BCA is a crucial clinical parameter, as children who developed it within 2 months from infusion had the worst outcome, possibly reflecting prior therapy intensity and its impact on autologous T cells. [Display omitted] DisclosuresAmrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Ghorashian: UCLB: Patents & Royalties: CARPALL; Novartis: Honoraria.

Percentage of residual B cells after 2weeks of rituximab treatment predicts the improvement of systemic sclerosis-associated interstitial lung disease.

The benefit of rituximab (RTX) for systemic sclerosis-associated interstitial lung disease (SSc-ILD) has been shown in previous clinical trials. However, predictors of RTX efficacy have not been clarified. We investigated whether B-cell responsiveness to RTX is related to therapeutic effect. Ten SSc-ILD patients treated with RTX in an independent clinical trial (Japan Registry of Clinical Trials, jRCTs031180373) were included in this analysis. Peripheral B-cell counts were examined retrospectively before RTX administration (baseline) and at 2, 4, 12, and 24weeks after the first RTX administration, along with percent-predicted forced vital capacity (%FVC) before and 24weeks after RTX treatment. Relative to baseline, the percentage of residual peripheral blood B cells at 2weeks after RTX was negatively correlated with the %FVC improvement at the 24-week assessment (r=-0.41, p=0.04). In the subgroup with less than 5% B-cell persistence at week 2, %FVC at the 24-week assessment was significantly improved compared to baseline (p=0.02). In another subgroup with more than 5% residual B cells, %FVC was not significantly different after 24weeks compared to baseline (p=0.41). In conclusion, the removal rate of B cells after 2weeks of RTX treatment may be a useful surrogate marker of subsequent SSc-ILD improvement.

Lung Transplant Patients Show a Dissimilar Peripheral B-Cell Subset Ratio Compared With Healthy Controls.

Lung transplant is a last treatment option for patients with end-stage pulmonary disease. Chronic lung allograft dysfunction, which generally manifests as bronchiolitis obliterans syndrome, is a major long-term survival limitation. Bronchiolitis obliterans syndrome is diagnosed when forced expiratory volume in 1 second declines > 20% in the absence of known causes. B cells can either contribute or restrain the development of bronchiolitis obliterans syndrome (eg, via induction of alloimmune antibodies, regulation of cellular immunity, and induction of tolerance). Here, we explored how peripheral B-cell subsets were altered in lung transplant recipients with bronchiolitis obliterans syndrome. Fresh whole blood samples were analyzed from 42 lung transplant recipients, including 17 with bronchiolitis obliterans syndrome; samples from these groups were compared with 10 age-matched healthy control samples. B-cell subsets were analyzed using flow cytometry, and relative distributions of subsets were compared. Changes in forced expiratory volume in 1 second were also determined. Absolute B-cell count was significantly increased in transplant recipients with bronchiolitis obliterans syndrome. Transitional (CD24+CD38+) and naïve (CD27-IgD+) B cells were decreased in lung transplant patients, with transitional B cells almost absent in those with bronchiolitis obliterans syndrome. Double-negative (CD27-IgD-) memory B cells were significantly increased (P < .001). No differences were found for plasmablasts (CD38+CD24-) and switched (CD27+IgD-) and non-switched (CD27+IgD+) memory B cells. Correlation analyses showed positive correlations between lung function and naïve B cells in transplant recipients (P = .0245; r = -0.458). Peripheral B-cell count and subset distribution were altered in lung transplant recipients with and without bronchiolitis obliterans syndrome compared with healthy controls. Transitional and naïve B-cell decreases may be caused by differentiation toward double-negative B-cells, which were increased. The correlation between forced expiratory volume and naïve B cells during follow-up care may be clinically interesting to investigate.

Immune Profiling of Relapsed/Refractory Multiple Myeloma Patients Treated with Venetoclax in Combination with Dexamethasone (VenDex), Daratumumab and Dexamethasone (VenDd), and Daratumumab, Bortezomib and Dexamethasone (VenDVd)

Background: The BCL-2 family of anti-apoptotic proteins (e.g., BCL-2, BCL-XL, MCL-1) are important regulators of lymphocyte development and are therapeutic targets for hematologic malignancies, including multiple myeloma (MM). Venetoclax (Ven) is a highly selective, potent, oral BCL-2 inhibitor that induces apoptosis in MM cells and has shown synergistic activity with bortezomib (Velcade; V) and dexamethasone (Dex or d). Combination of the CD38 monoclonal antibody (mAb) daratumumab (D) with Ven is hypothesized to further increase anti-myeloma activity based upon dual mechanisms of pro-apoptotic effects on tumor cells as well as enhanced immune stimulation. Pre-clinically and in healthy human subjects, Ven treatment leads to enrichment of CD8+ T effector memory cells, while resulting in loss of CD4+ and CD8+ naïve T-cells, however the potential immunomodulatory effect of Ven in MM is unknown. Results presented herein describe the pharmacodynamic changes observed in immune cell subsets in relapsed/refractory (R/R) MM patients (pts) treated with VenDex, VenDd, and VenDVd. Methods: Peripheral blood samples were collected at day 1 of cycles 1-5 to characterize pharmacodynamic changes in B- and T-cell sub-populations by multicolor flow cytometry in Ven MM clinical trials M13-367 (NCT01794520) and M15-654 (NCT03314181). M13-367 is a phase 1/2 study of VenDex in t(11;14) R/R MM, and M15-654 is a phase 1/2 study of VenDd in t(11;14) R/R MM (Part 1) and VenDVd in R/R MM (Part 2). As of 12 June 2019, 15 out of 31 pts treated with VenDex in phase 2 of M13-367, 19 out of 24 pts treated in Part 1 (VenDd), and 19 out of 24 pts treated in Part 2 (VenDVd) of M15-654 had baseline and post-treatment specimens available for analysis. Results: Consistent with previous findings that B-cells are highly dependent upon BCL-2 for cell survival, each Ven-containing regimen (VenDex, VenDd, and VenDVd) resulted in rapid and sustained reduction (~90% decrease from baseline by end of cycle 1) in peripheral B-cell (CD19+/CD5-) counts (Figure). In subgroup analyses, naïve B-cells (CD27-/IgM+) were significantly reduced in pts treated with each regimen, however regulatory B-cells (CD27+/CD24+) remained largely unaffected. In addition, a decrease in plasmablasts (CD27+CD38+CD20-) were only observed in pts treated with D-containing regimens (VenDd and VenDVd), which is consistent with expression of CD38 in this subgroup. In contrast to B-cells, CD3+ T-cells were minimally reduced (~20-30% reduction from baseline through end of cycle 4) with VenDex and VenDd treatment. A more prominent reduction was observed in pts treated with VenDVd (~50-60% reduction from baseline through end of cycle 4). CD4+ T-cells were more sensitive than CD8+ T-cells to VenDex and VenDd, while both CD4+ and CD8+ T-cells were reduced in VenDVd treated pts (Figure). T-cell subgroup analyses were also performed, including naïve T-cells (CD45RA+/CD197+), effector (CD45RA+/CD197-), effector memory (CD45RA-/CD197-), and central memory (CD45RA-/CD197+). Overall, there were no significant changes in the composition of the T-cell pool (Tnaive, TCM, TEM, TEMRA) or in Th1 (CD4+/CD183+/CD196-), Th2 (CD4+/CD183-/CD196-), and Th17 (CD4+/CD183-/CD196+) cells in pts treated with VenDex, VenDd, or VenDVd. Finally, a decrease in Tregs (CD4+/CD25+/CD127low/CD194+) was observed in VenDd and VenDVd treated pts, but not in VenDex treated pts (Figure). While no reduction in overall Tregs was observed with VenDex, a decrease in the relative proportion of activated Tregs (HLA-DR+/CD45RO+) to naïve (HLA-DR-/CD45RO-) and memory (HLA-DR-/CD45RO+) Tregs was observed. Conclusions: Comprehensive immunophenotyping studies were conducted to characterize the potential effects of Ven in B- and T-cell subsets when used in combination with standard of care anti-myeloma agents of various mechanisms of action (i.e. glucocorticoid, CD38 mAb and/or proteasome inhibitor). While each Ven combination regimen resulted in significant reductions in circulating B-cells, T-cell effects were largely dependent upon the combination regimen. Minimal reduction in total T-cell counts were observed with VenDex and VenDd, which were primarily due to reduction in CD4+ T-cells. However, the extent of T-cell depletion (CD4+ and CD8+ T-cells) was greatest in pts treated with the bortezomib-containing regimen VenDVd. Correlative studies between immune profiles and pt outcomes are ongoing. Disclosures Vishwamitra: AbbVie: Employment, Other: stock or other options. Mantis:AbbVie Inc: Employment, Other: Stock/stock options. Haribhai:AbbVie: Employment, Other: stock or other options. Uziel:AbbVie: Employment, Other: stock or other options. Leverson:AbbVie Inc: Employment, Other: Stock or options. Pauff:AbbVie Inc: Employment, Other: may own stock or stock options. Bueno:AbbVie: Employment, Other: Stock/stock options. Maciag:AbbVie: Employment, Other: Stock/stock options. Ross:AbbVie: Employment, Other: Stock/stock options.

Addition of Obinutuzumab to Ibrutinib Enhances Depletion of CLL Cells in the Peripheral Blood and Bone Marrow after 1 Month of Combination Therapy: Initial Results from the Bloodwise TAP Iciclle Extension Study

Background: A major aim of treatment in CLL is to eradicate detectable minimal residual disease (MRD). Ibrutinib is a major step forward in the treatment of CLL but results in an immediate lymphocytosis that persists in most patients for at least several months. Obinutuzumab is a second generation anti-CD20 monoclonal antibody which appears to be highly effective in CLL resulting in a rapid eradication of peripheral blood lymphocytosis and the eradication of MRD in a proportion of patients. The IcICLLe Extension Study expands on the IcICLLe trial (ISRCTN 12695354) to examine the efficacy and safety of the combination treatment of obinutuzumab and ibrutinib.Patients: The IcICLLetrial recruited 40 participants with CLL requiring treatment (20 treatment-naïve, 20 relapsed/refractory disease) receive continuous ibrutinib therapy until achievement of <0.01% residual disease in bone marrow or disease progression. The IcICLLe Extension Study involves 40 participants with relapsed/refractory CLL requiring treatment who will all receive continuous oral therapy with ibrutinib and 6 cycles of obinutuzumab. Of the 40 participants, >20 will have had no prior Ibrutinib treatment (ibrutinib naive), and ≤20 will have received at least 12 months of Ibrutinib monotherapy as part of the IcICLLe trial before enrolling in the Extension Study . Adverse Events (AEs) are collected from registration until 30 days after treatment cessation and reported at 1, 3 and 6 months, and 6-monthly thereafter using the Common Terminology Criteria for Adverse Events v4.0.Results: 17 participants (12 ibrutinib naïve and 5 IcICLLe monotherapy patients) are evaluable for response assessment after one month of combination treatment. In the 12 Ibrutinib-naïve cases, the peripheral CLL counts remained at or below baseline levels in most (9/12) cases from week 1 onwards. After one month of combination therapy the peripheral B-cell count was a median 35% of baseline levels (range <1% to 174%) compared to a median 215% (range 29% to 3570%) for relapsed/refractory patients on Ibrutinib monotherapy. The percentage of CLL cells in the bone marrow aspirate for patients after 1 month of combination therapy showed a median 20% reduction (range 1% increase to 55% decrease, paired t-test P<0.001, baseline median 74% CLL cells, range 23-94%) while there was no change for relapsed/refractory patients on ibrutinib monotherapy (median 1% increase, range 39% increase to 24% decrease, paired t-test P=0.35, baseline median 85% CLL cells, range 11-96%). The reduction in bone marrow aspirate CLL percentage was also evident by morphological assessment of the trephine biopsy with all 10 evaluable patients showing improvements in the cellularity and/or extent of infiltration.Bone marrow assessment was not mandated for the 5 patients previously on ≥12 months (range 14-20 months) continuous ibrutinib monotherapy because they had already undergone 4 biopsies in the prior year, however all five patients showed a reduction in peripheral CLL counts (median 1.3 log reduction, range 0.3 to 2.4 log) which was significantly greater than the reduction seen in the prior 6 months of ibrutinib monotherapy (median 0.5 log reduction, range -1.0 to 0.8, paired t-test P=0.043)Of the 17 participants recruited to the IcICLLe Extension Study there have been no reports of Tumour Lysis Syndrome within the first month of combination treatment. There was a single report of a grade 2 Infusion Related Reaction (IRR) for one participant following the first infusion (100mg) of obinutuzumab. No further IRRs have been reported.Conclusions: The data indicate that the addition of obinutuzumab to ibrutinib results in a substantial improvement in the depletion of CLL cells from the peripheral blood and bone marrow after only one month of combination therapy.Date of Data Freeze: 19-Jul-2016 DisclosuresRawstron:Gilead: Consultancy, Honoraria, Research Funding; BD Biosciences: Other: Remuneration; Celegene: Honoraria; Roche: Honoraria; Janssen: Research Funding; GlaxoSmithKline: Honoraria; AbbVie: Honoraria; Genzyme: Honoraria. Munir:Alexion pharmaceuticals: Honoraria. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Bloor:Janssen: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Consultancy, Speakers Bureau; Gilead: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees. Pettitt:Roche: Research Funding, Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Infinity: Research Funding. Fox:Roche: Consultancy; Janssen: Honoraria, Other: Travel support. Fegan:Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria. Devereux:Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy; Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

Progressive multifocal leukoencephalopathy and black fungus in a patient with rheumatoid arthritis without severe lymphocytopenia

Introduction:Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating brain infection caused by JC polyomavirus (JCV), primarily seen in patients with severely compromised cellular immunity. Clinical presentation varies depending on the affected white matter. PML prognosis is variable and effective treatments are lacking.Case presentation:A 75-year-old Chinese woman with type 2 diabetes mellitus, chronic kidney disease and rheumatoid arthritis, treated with low-dose methotrexate and prednisolone for 2.5 years, developed a Pleurostomophora richardsiae infection of her left arm. After 6 months of treating this rare black fungus infection with voriconazole, surgery and immunosuppression discontinuation, she presented with progressive afebrile encephalopathy with right-sided hemiparesis. There were no signs of inflammation or metabolic abnormalities. Brain magnetic resonance imaging revealed diffuse frontal white matter lesions and a cerebrospinal fluid PCR confirmed PML due to JC virus. Severe lymphopenia was never present, and at PML diagnosis, CD4 and CD8 T-cell counts were 454 mm−3 and 277 mm−3. CD8 T-cells were able to respond to JCV VP1 peptide stimulation with TNFα secretion. Peripheral B-cell count was only 8 mm−3. Mirtazapine and Maraviroc were started, but unfortunately, she rapidly deteriorated and died 5 weeks after PML diagnosis.Conclusion:Although peripheral lymphocyte counts were never low and CD4 T-cell count was close to normal, the persistent black fungus infection was a hallmark of severely compromised cellular immunity. The unexpected extremely low absolute B-cell count might suggest a protective role for B-cells. The paradoxical, clinical PML onset months after immunosuppressive discontinuation suggests that it was only discovered in the context of an immune reconstitution inflammatory syndrome.

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.

Abstract 1744: ACP-196: A second generation Btk inhibitor demonstrates biologic activity in a canine model of B-cell non-Hodgkin lymphoma

Abstract Recent recognition of B-cell receptor (BCR) signaling as a critical factor in the progression of B-cell malignancies, including non-Hodgkin lymphoma (NHL), has resulted in the development of numerous targeted therapeutics that inhibit this signaling pathway. Ibrutinib, a small molecule inhibitor of Bruton tyrosine kinase (Btk) a key signaling molecule in the BCR pathway, has demonstrated significant clinical activity in a broad range of B-cell cancers. ACP-196 is a second generation Btk inhibitor with increased target selectivity and enhanced in vivo potency compared with ibrutinib and, thus, may represent an improvement over its predecessor. With the following studies, we sought to evaluate ACP-196 in canine models of B-cell NHL with the ultimate goal of providing the preclinical data necessary to move ACP-196 into human clinical trials. Using two immunophenotypically confirmed canine B-cell lymphoma cell lines, CLBL-1 and 17-71, we demonstrate potent in vitro inhibition of activation of Btk and the downstream effectors ERK 1/2 and PLCγ2 following 1 hour of treatment with ACP-196 at concentrations as low as 10nM. In vivo studies were performed in companion dogs as part of an ongoing clinical trial. Twelve dogs with immunophenotypically confirmed, spontaneously occurring B-cell NHL were orally administered ACP-196 at dosages of 2.5mg/kg every 24 hours (6 dogs), 5mg/kg every 24 hours (5 dogs), or 10mg/kg every12 hours (1 dog). Btk occupancy in peripheral blood and lymphoma cells was assessed using a biotin-tagged probe derived from ACP-196. Using this assay we found that at 2.5mg/kg full Btk occupancy was achieved in peripheral B cells 3h after dosing for all dogs, except for a single dog with high peripheral B-cell count. At 24 hours after dosing, 83-99% Btk target occupancy was observed for all dogs. Partial response, as assessed by a modified RECIST scheme, was achieved in 2 dogs in the 2.5mg/kg group and the dog in the 10mg/kg group. Upon relapse, one of the responders in the 2.5mg/kg group was dose escalated to 10mg/kg q12 on day 42 and partial response from relapse was reestablished. Of the remaining 9 dogs, 3 achieved stable disease for &amp;gt; 28 days and 6 discontinued the study after developing progressive disease within 28 days of starting treatment. In total, to date, 3 dogs achieved a partial response, 3 dogs stable disease, and 6 dogs progressive disease. ACP-196 was well tolerated with only mild anorexia noted in some dogs. These data demonstrate that ACP-196 has single agent biologic activity in a spontaneous large animal model of human NHL. Studies in dogs with NHL are ongoing to define regimens prior to initiation of human phase I clinical trials. Additional cohorts are planned combining ACP-196 with a phosphatidylinositide 3-kinase (PI3K) delta-specific inhibitor. Citation Format: Heather L. Gardner*, Bonnie K. Harrington*, Raquel Izumi, Ahmed Hamdy, Allard Kaptein, Bart Van Lith, Cheryl A. London, John C. Byrd, Amy J. Johnson, William C. Kisseberth. ACP-196: A second generation Btk inhibitor demonstrates biologic activity in a canine model of B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1744. doi:10.1158/1538-7445.AM2014-1744

AB0434 Relation of Subclinical Ultrasound Detected Synovitis and Peripheral B-Cell Counts in Rheumatoid Arthritis Patients Treated with Rituximab

BackgroundThe clinical response in patients with Rheumatoid Arthritis (RA) to rituximab (RTX) has been shown to be related with the presence of B-cells in the synovi. To date this has...

Preemptive Rituximab Infusions Efficiently Prevent Relapses In Acquired Thrombotic Thrombocytopenic Purpura. Experience Of The French Thrombotic Microangiopathies Reference Center

Context Acquired thrombotic thrombocytopenic purpura (TTP) results from a severe, antibody-mediated, deficiency in the von Willebrand factor-cleaving protease ADAMTS13. Rituximab is increasingly used in this indication in patients with a suboptimal response to plasma exchange. When severe acquired ADAMTS13 deficiency persists during remission, the estimated incidence rate is of 0.4/year. So far, it is still controversial whether preemptive rituximab efficiently prevents relapses in these patients. Patients and methods We defined two groups of patients with a history of acquired TTP who displayed a persistent severe ADAMTS13 deficiency during remission. Patients of group 1 were treated with preemptive infusions of rituximab. Patients of group 2 were managed in centers in which preemptive rituximab infusions were not the standard of care. The relapse incidence was evaluated and compared between both groups. Patients were treated according to National recommendations and enrolled from 12 French centers during a 12-year period. Patients were explored for ADAMTS13 activity and peripheral B-cell count every 3 months. Only patients with a &gt; 12-month follow-up after rituximab administration are reported here. Median (25th - 75th percentile) was determined for all continuous variables. Wilcoxon’s test was used to compare continuous variables and the chi-square test or Fisher’s exact test to compare binary data. Relapse-free survival was compared between both groups using the Kaplan-Meier estimator with the corresponding 95% confidence interval. Results Forty-eight patients (20.6%) with a history of acquired TTP displayed a persistent severe ADAMTS13 deficiency on remission or experienced a subsequent severe ADAMTS13 deficiency (24 cases each) after a median follow-up of 17 months (12-29 months). Anti-ADAMTS13 antibodies concentration was 44 U/mL (24-59 U/mL). Thirty patients received preemptive infusions of rituximab (group 1), whereas 18 others had no therapeutical intervention (group 2). In group 1, 16 patients experienced a past history of TTP with a median number of 2 (1-3) episodes, corresponding to a relapse incidence of 0.22 (0-0.57)/year. Rituximab infusions were performed 14.5 months (6.5-27.4 months) after the last TTP episode. A median number of 4 (1-4) rituximab infusions were performed. The median follow-up between the first preemptive infusion of rituximab and the last ADAMTS13 evaluation is of 36 months (24-65 months). After preemptive rituximab administration, only 3 patients experienced a clinical relapse (0 [0] episode/year), corresponding to a significant reduction in the relapse incidence (P &lt; .01). ADAMTS13 activity was 58.5% (30.5%-86.3%). Three months after the first rituximab infusion, ADAMTS13 activity was 46% (30-68); it further increased until the 12th month, and subsequently decreased. Accordingly, B-cell lymphocytes remained undetectable until the 6th month, and progressively increased at the 9th month to reach normal values at the 18th month. Nine patients (30%) required one (5 cases), two (2 cases), three (1 case) or ten (1 case) additional courses of rituximab for a further decrease or a persistent undetectable ADAMTS13 activity, which allowed to maintain a detectable ADAMTS13 activity in all but one patients. The time between two consecutive courses of rituximab was 26 months (5-59 months). At the end of follow-up, ADAMTS13 activity remained normal in 18 patients; 10 patients had a moderate ADAMTS13 deficiency, and 2 patients had a persistently undetectable ADAMTS13 activity. In four patients (13%), rituximab alone failed to increase durably ADAMTS13 activity, which required additional immunosuppressive drugs. In group 2, 14 patients relapsed after a 66-month follow-up (36-105 months), corresponding to a higher relapse incidence than in patients who received preemptive infusions of rituximab (0.23 [0.1-0.46] relapse/year, P&lt;.01). Moreover, 2 patients died of TTP in group 2, whereas no fatal outcome was recorded in group 1. Relapse free survival over time was significantly longer in group 1 (Log-rank test: P = .049). Five patients experienced adverse effects including benign infections in 2 cases. Conclusion Rituximab efficiently prevents TTP relapses in most patients with a persistent acquired ADAMTS13 deficiency, with acceptable side effects. Disclosures: Off Label Use: Rituximab Rituximab may prevent relapses in acquired thrombotic thrombocytopenic purpura.

Humoral Immunity after kidney transplantation: Impact of two randomized immunosuppressive protocols

Controlling alloimmune humoral response is a challenge in transplantation. Few studies have evaluated the impact of maintenance immunosuppression on blood humoral parameters. We performed a post-hoc analysis on 307 kidney transplant recipients included in a prospective randomized trial comparing tacrolimus/mycophenolate mofetil (Tac/MMF) vs. cyclosporine/azathioprine (CsA/AZA), both used with antithymocyte globulin induction and steroids. Humoral parameters were analyzed at D0, D15, and M12. IgG, IgA, and IgM levels decreased significantly as soon as D15 in both groups (–35%, –26%, and –35% respectively, vs. D0). At M12, although peripheral B-cell counts did not differ between the groups, Tac/MMF regimen was associated with lower IgG, IgA, and IgM levels than CsA/AZA (–5.9%, –14.6%, and –34%, respectively). Hypogammaglobulinemia at D15 was not associated with an increased risk of infections during the first year. The proportion of HLA-sensitized patients decreased in the Tac/MMF group (15.9% at D0 and 6.7% at M12, p=0.02) and remained stable in the CsA/AZA group (10.3% at D0 and 8.9% at M12, p=0.5). More patients sensitized at baseline became non-sensitized at M12 with Tac/MMF than with CsA/AZA. Our results suggest humoral immunosuppression is better with Tac/MMF than with CsA/AZA during the first year of kidney transplantation.

Rituximab Treatment for Adult Patients with Focal Segmental Glomerulosclerosis

We present two cases with steroid-resistant nephrotic syndrome (SRNS) and two cases with steroid-dependent nephrotic syndrome (SDNS) due to focal segmental glomerulonephritis (FSGS) who were treated with a single dose of rituximab (375 mg/m(2)). Although the two cases with SRNS showed no response, the two cases with SDNS achieved complete remission. The patients in whom the peripheral B-cell counts subsequently increased after the administration of rituximab demonstrated a relapse. Rituximab may be an effective treatment agent for SDNS with FSGS and the peripheral B-cell count may be a useful marker in such patients for preventing disease relapse.

Cutaneous Granulomatous Reaction After Herpes Zoster in Drug-Induced Hypersensitivity Syndrome

Cutaneous Granulomatous Reaction After Herpes Zoster in Drug-Induced Hypersensitivity Syndrome