Comprehensive genetic and functional analyses of Fc gamma receptors influence on response to rituximab therapy for autoimmunity.

BACKGROUND: Rituximab is widely used to treat autoimmunity but clinical response varies. Efficacy is determined by the efficiency of B-cell depletion, which may depend on various Fc gamma receptor (FcγR)-dependent mechanisms. Study of FcγR is challenging due to the complexity of the FCGR genetic locus. We sought to assess the effect of FCGR variants on clinical response, B-cell depletion and NK-cell-mediated killing in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: A longitudinal cohort study was conducted in 835 patients [RA = 573; SLE = 262]. Clinical outcome measures were two-component disease activity score in 28-joints (2C-DAS28CRP) for RA and British Isles Lupus Assessment Group (BILAG)-2004 major clinical response (MCR) for SLE at 6 months. B-cells were evaluated by highly-sensitive flow cytometry. Single nucleotide polymorphism and copy number variation for genes encoding five FcγRs were measured using multiplex ligation-dependent probe amplification. Ex vivo studies assessed NK-cell antibody-dependent cellular cytotoxicity (ADCC) and FcγR expression. FINDINGS: In RA, carriage of FCGR3A-158V and increased FCGR3A-158V copies were associated with greater 2C-DAS28CRP response (adjusted for baseline 2C-DAS28CRP). In SLE, MCR was associated with increased FCGR3A-158V, OR 1.64 (95% CI 1.12-2.41) and FCGR2C-ORF OR 1.93 (95% CI 1.09-3.40) copies. 236/413 (57%) patients with B-cell data achieved complete depletion. Homozygosity for FCGR3A-158V and increased FCGR3A-158V copies were associated with complete depletion in combined analyses. FCGR3A genotype was associated with rituximab-induced ADCC, and increased NK-cell FcγRIIIa expression was associated with improved clinical response and depletion in vivo. Furthermore, disease status and concomitant therapies impacted both NK-cell FcγRIIIa expression and ADCC. INTERPRETATION: FcγRIIIa is the major low affinity FcγR associated with rituximab response. Increased copies of the FCGR3A-158V allele (higher affinity for IgG1), influences clinical and biological responses to rituximab in autoimmunity. Enhancing FcγR-effector functions could improve the next generation of CD20-depleting therapies and genotyping may stratify patients for optimal treatment protocols. FUNDING: Medical Research Council, National Institute for Health and Care Research, Versus Arthritis.

Obinutuzumab as consolidation after chemo-immunotherapy: Results of the UK National Cancer Research Institute phase II/III GALACTIC trial.

The GA101 (obinutuzumab) monocLonal Antibody as Consolidation Therapy In chronic lymphocytic leukaemia (CLL) (GALACTIC) was a seamless phase II/III trial designed to test whether consolidation with obinutuzumab is safe and eradicates minimal residual disease (MRD) and, subsequently, whether this leads to prolonged progression-free survival (PFS) in patients with CLL who have recently responded to chemo-immunotherapy. Patients with a response 3-24 months after chemotherapy were assessed for MRD. MRD-positive patients were randomised to receive consolidation therapy with obinutuzumab or no consolidation. The trial closed after the phase II part due to slow recruitment. In all, 48 patients enrolled of whom 19 were MRD negative and were monitored. Of the 29 MRD-positive patients, 14 were randomised to receive consolidation and 15 to no consolidation. At 6 months after randomisation, 10 and 13 consolidated patients achieved MRD negativity by flow cytometry (sensitivity 10-4 ) in bone marrow and peripheral blood respectively. PFS was significantly better in consolidated patients compared to non-consolidated patients (p = 0.001). No difference was observed in PFS, overall survival or duration of MRD negativity when comparing the 10 MRD-negative patients after consolidation with the 19 MRD-negative patients in the monitoring group. Common adverse events in the consolidation arm were thrombocytopenia, infection, and cough. Only 1% of events were infusion-related reactions. This observation provides further evidence that consolidation to achieve MRD negativity improves outcomes in CLL and that obinutuzumab is well tolerated in patients with low levels of disease.

Minimal Residual Disease After Autologous Stem-Cell Transplant for Patients With Myeloma: Prognostic Significance and the Impact of Lenalidomide Maintenance and Molecular Risk.

PURPOSE: Minimal residual disease (MRD) can predict outcomes in patients with multiple myeloma, but limited data are available on the prognostic impact of MRD when assessed at serial time points in the context of maintenance therapy after autologous stem-cell transplant (ASCT) and the interaction between MRD and molecular risk. METHODS: Data from a large phase III trial (Myeloma XI) were examined to determine the relationship between MRD status, progression-free survival (PFS), and overall survival (OS) in post-ASCT patients randomly assigned to lenalidomide maintenance or no maintenance at 3 months after ASCT. MRD status was assessed by flow cytometry (median sensitivity 0.004%) before maintenance random assignment (ASCT + 3) and 6 months later (ASCT + 9). RESULTS: At ASCT + 3, 475 of 750 (63.3%) patients were MRD-negative and 275 (36.7%) were MRD-positive. MRD-negative status was associated with improved PFS (hazard ratio [HR] = 0.47; 95% CI, 0.37 to 0.58 P < .001) and OS (HR = 0.59; 95% CI, 0.40 to 0.85; P = .0046). At ASCT + 9, 214 of 326 (65.6%) were MRD-negative and 112 (34.4%) were MRD-positive. MRD-negative status was associated with improved PFS (HR = 0.20; 95% CI, 0.13 to 0.31; P < .0001) and OS (HR = 0.33; 95% CI, 0.15 to 0.75; P = .0077). The findings were very similar when restricted to patients with complete response/near complete response. Sustained MRD negativity from ASCT + 3 to ASCT + 9 or the conversion to MRD negativity by ASCT + 9 was associated with the longest PFS/OS. Patients randomly assigned to lenalidomide maintenance were more likely to convert from being MRD-positive before maintenance random assignment to MRD-negative 6 months later (lenalidomide 30%, observation 17%). High-risk molecular features had an adverse effect on PFS and OS even for those patients achieving MRD-negative status. On multivariable analysis of MRD status, maintenance therapy and molecular risk maintained prognostic impact at both ASCT + 3 and ASCT + 9. CONCLUSION: In patients with multiple myeloma, MRD status at both ASCT + 3 and ASCT + 9 is a powerful predictor of PFS and OS.

A Personalized Rituximab Retreatment Approach Based on Clinical and B-Cell Biomarkers in ANCA-Associated Vasculitis.

Background: Time to relapse after rituximab for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is variable, and optimal retreatment strategy has remained unclear. In AAV following rituximab induction, the study objective was to evaluate clinical and B-cell predictors of relapse in order to develop a retreatment algorithm. Methods: A retrospective observational study was conducted in 70 rituximab-treated ANCA-associated vasculitis patients followed up for over 10 years. Complete response (CR) was defined as Birmingham Vasculitis Activity Score v3.0 = 0. Retreatment was given on clinical relapse, defined as new features or worsening of persistent disease (not by biomarker status). Peripheral B-cell subsets were measured using highly sensitive flow cytometry. Predictors were tested using multivariable Cox regression. Results: Median time to retreatment for cycles 1-5 were 84, 73, 67, 60, and 73 weeks. Over 467 patient-years follow-up, 158 relapses occurred in 60 patients; 16 (in 15 patients) were major (renal = 7, neurological = 4, ENT = 3, and respiratory = 2). The major-relapse rate was 3.4/100 patient-years. In multivariable analysis, concomitant immunosuppressant [HR, 0.48 (95% CI, 0.24-0.94)], achieving CR [0.24 (0.12-0.50)], and naïve B-cell repopulation at 6 months [0.43 (0.22-0.84)] were associated with longer time to relapse. Personalized retreatment using these three predictors in this cohort would have avoided an unnecessary fixed retreatment in 24% of patients. Area under the receiver operating characteristic for prediction of time to relapse was greater if guided by naïve B-cell repopulation than if previously evaluated ANCA and/or CD19+ cells return at 6 months had been used, 0.82 and 0.53, respectively. Conclusion: Our findings suggest that all patients should be coprescribed oral immunosuppressant. Those with incomplete response or with absent naïve B cells should be retreated at 6 months. Patients with complete response and naïve repopulation should not receive fixed retreatment. This algorithm could reduce unnecessary retreatment and warrant investigation in clinical trials.

Integrative analysis of spontaneous CLL regression highlights genetic and microenvironmental interdependency in CLL.

Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.

Ibrutinib Plus Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia: The CLARITY Study.

PURPOSE: The treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by targeted therapies that either inhibit proliferation (ibrutinib) or reactivate apoptosis (venetoclax). Both significantly improve survival in CLL and replace chemoimmunotherapy for many patients. However, individually, they rarely lead to eradication of measurable residual disease (MRD) and usually are taken indefinitely or until progression. We present the CLARITY trial that combined ibrutinib with venetoclax to eradicate detectable CLL with the intention of stopping therapy. PATIENTS AND METHODS: CLARITY is a phase II trial that combined ibrutinib with venetoclax in patients with relapsed or refractory CLL. The primary end point was eradication of MRD after 12 months of combined therapy. Key secondary end points were response by International Workshop on CLL criteria, safety, and progression-free and overall survival. RESULTS: In 53 patients after 12 months of ibrutinib plus venetoclax, MRD negativity (fewer than one CLL cell in 10,000 leukocytes) was achieved in the blood of 28 (53%) and the marrow of 19 (36%). Forty-seven patients (89%) responded, and 27 (51%) achieved a complete remission. After a median follow-up of 21.1 months, one patient progressed, and all patients were alive. A single case of biochemical tumor lysis syndrome was observed. Other adverse effects were mild and/or manageable and most commonly were neutropenia or GI events. CONCLUSION: The combination of ibrutinib plus venetoclax was well tolerated in patients with relapsed or refractory CLL. There was a high rate of MRD eradication that led to the cessation of therapy in some patients. The progression-free and overall survival rates are encouraging for relapsed and refractory CLL.

Ibrutinib induces chromatin reorganisation of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries. It has recently been shown that the homogeneity of the chromatin landscape between CLL cells contrasts with the important observed genetic heterogeneity of the disease. To gain further insight into the consequences of disease evolution on the epigenome's plasticity, we monitored changes in chromatin structure occurring in vivo in CLL cells from patients receiving continuous Ibrutinib treatment. Ibrutinib, an oral inhibitor of the Bruton's tyrosine kinase (BTK) has proved to be remarkably efficient against treatment naïve (TN), heavily pre-treated and high-risk chronic lymphocytic leukaemia (CLL), with limited adverse events. We established that the chromatin landscape is significantly and globally affected in response to Ibrutinib. However, we observed that prior to treatment, CLL cells show qualitative and quantitative variations in chromatin structure correlated with both EZH2 protein level and cellular response to external stimuli. Then, under prolonged exposure to Ibrutinib, a loss of the two marks associated with lysine 27 (acetylation and trimethylation) was observed. Altogether, these data indicate that the epigenome of CLL cells from the peripheral blood change dynamically in response to stimuli and suggest that these cells might adapt to the Ibrutinib "hit" in a process leading toward a possible reduced sensitivity to treatment.

Predicting Severe Infection and Effects of Hypogammaglobulinemia During Therapy With Rituximab in Rheumatic and Musculoskeletal Diseases.

OBJECTIVE: To evaluate predictors of serious infection events (SIEs) during rituximab (RTX) therapy and effects of hypogammaglobulinemia on SIE rates, and humoral response and its persistence after discontinuation of RTX in the treatment of rheumatic and musculoskeletal diseases (RMDs). METHODS: A retrospective longitudinal study of 700 RMD patients treated with RTX in a single center was conducted. Immunoglobulin levels were measured at baseline and at 4-6 months after each treatment cycle. Baseline predictors of SIEs were assessed using multivariable logistic regression; for RTX cycles 2-4, a mixed-effects logistic regression model was used. RESULTS: A total of 507 patients (72%) had rheumatoid arthritis, 94 (13%) had systemic lupus erythematosus, 49 (7%) had antineutrophil cytoplasmic antibody-associated vasculitis, and 50 (7%) had other RMDs. The number of SIEs recorded was 281 in 176 patients (9.8 per 100 person-years). Predictors of SIEs included non-RTX-specific comorbidities (previous history of SIE, cancer, chronic lung disease, diabetes mellitus, and heart failure), higher corticosteroid dose, and RTX-specific factors, including low IgG (<6 gm/liter) both at baseline and during treatment, RTX-associated neutropenia, higher IgM, and longer time to RTX re-treatment, but not B cell count or depletion status. Of 110 patients with low IgG, SIE rates were higher in those with low IgG at baseline (16.4 per 100 person-years) and in those who acquired low IgG during or after RTX treatment (21.3 per 100 person-years) versus those with normal IgG (9.7 per 100 person-years). Five of 8 patients (63%) had impaired humoral response to pneumococcus and hemophilus following vaccination challenge, and only 4 of 11 patients (36%) had IgG normalized after switching biologic disease-modifying antirheumatic drugs. CONCLUSION: Immunoglobulin levels should be monitored at baseline and before each RTX cycle to identify patients at risk of SIEs. Individualized risk-benefit assessment should be undertaken in those with lower IgG as this is a consistent SIE predictor and may increase infection profiles when RTX is switched to different therapies.

Minimal residual disease analysis in chronic lymphocytic leukemia: a way for achieving more personalized treatments.

Therapeutic approaches for chronic lymphocytic leukemia (CLL) have dramatically changed over the recent past. In parallel, quantification of minimal residual disease (MRD) proved to be an independent prognostic factor for progression-free and overall survival. The European Research Initiative on CLL (ERIC) in collaboration with American and Australasian partners developed harmonised assays that could be applied reproducibly to compare the efficacy of different treatments. The potential for MRD analysis to identify the most effective treatments prior to reaching survival endpoints was recognised by regulatory agencies and approved as an intermediate endpoint for licensure in randomized studies, in Europe. More recently treatment approaches have evolved, in particular with BCL2-pathway inhibitors, so that MRD analysis may be informative for most patients and clinical trials, potentially becoming a tool for managing CLL patients in clinical practice. In the recent past the importance of the type of MRD assay used, the most appropriate timing and compartment to assess for different treatment types have been learnt as we move towards eradicating residual disease beyond the guideline threshold of one cell in ten thousand. Nowadays, MRD assessment in CLL has quickly become an indispensable tool for clinical research and development that promise to change the way we manage our patients in the future.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project.

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society.

Assessment of ibrutinib plus rituximab in front-line CLL (FLAIR trial): study protocol for a phase III randomised controlled trial.

BACKGROUND: Treatment of chronic lymphocytic leukaemia (CLL) has seen a substantial improvement over the last few years. Combination immunochemotherapy, such as fludarabine, cyclophosphamide and rituximab (FCR), is now standard first-line therapy. However, the majority of patients relapse and require further therapy, and so new, effective, targeted therapies that improve remission rates, reduce relapses, and have fewer side effects, are required. The FLAIR trial will assess whether ibrutinib plus rituximab (IR) is superior to FCR in terms of progression-free survival (PFS). METHODS/DESIGN: FLAIR is a phase III, multicentre, randomised, controlled, open, parallel-group trial in patients with previously untreated CLL. A total of 754 participants will be randomised on a 1:1 basis to receive standard therapy with FCR or IR. Participants randomised to FCR will receive a maximum of six 28-day treatment cycles. Participants randomised to IR will receive six 28-day cycles of rituximab, and ibrutinib taken daily for 6 years until minimal residual disease (MRD) negativity has been recorded for the same amount of time as it took to become MRD negative, or until disease progression. The primary endpoint is PFS according to the International Workshop on CLL (IWCLL) criteria. Secondary endpoints include: overall survival; proportion of participants with undetectable MRD; response to therapy by IWCLL criteria; safety and toxicity; health-related quality of life (QoL); and cost-effectiveness. DISCUSSION: The trial aims to provide evidence for the future first-line treatment of CLL patients by assessing whether IR is superior to FCR in terms of PFS, and whether toxicity rates are favourable. TRIAL REGISTRATION: ISRCTN01844152 . Registered on 8 August 2014, EudraCT number 2013-001944-76 . Registered on 26 April 2013.

GA101 (obinutuzumab) monocLonal Antibody as Consolidation Therapy In CLL (GALACTIC) trial: study protocol for a phase II/III randomised controlled trial.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia. Achieving minimal residual disease (MRD) negativity in CLL is an independent predictor of survival even with a variety of different treatment approaches and regardless of the line of therapy. METHODS/DESIGN: GA101 (obinutuzumab) monocLonal Antibody as Consolidation Therapy In CLL (GALACTIC) is a seamless phase II/III, multi-centre, randomised, controlled, open, parallel-group trial for patients with CLL who have recently responded to chemotherapy. Participants will be randomised to receive either obinutuzumab (GA-101) consolidation or no treatment (as is standard). The phase II trial will assess safety and short-term efficacy in order to advise on continuation to a phase III trial. The primary objective for phase III is to assess the effect of consolidation therapy on progression-free survival (PFS). One hundred eighty-eight participants are planned to be recruited from forty research centres in the United Kingdom. DISCUSSION: There is evidence that achieving MRD eradication with alemtuzumab consolidation is associated with improvements in survival and time to progression. This trial will assess whether obinutuzumab is safe in a consolidation setting and effective at eradicating MRD and improving PFS. TRIAL REGISTRATION: ISRCTN, 64035629 . Registered on 12 January 2015. EudraCT, 2014-000880-42 . Registered on 12 November 2014.

Monoclonal B-cell lymphocytosis in a hospital-based UK population and a rural Ugandan population: a cross-sectional study.

BACKGROUND: Reported incidence of B-cell malignancies shows substantial geographical variation, being more common in the Americas and Europe than in Africa. This variation might reflect differences in diagnostic capability, inherited susceptibility, and infectious exposures. Monoclonal B-cell lymphocytosis (MBL) is a precursor lesion that can be screened for in apparently healthy people, allowing comparison of prevalence across different populations independently of health-care provision. We aimed to compare the prevalence and phenotypic characteristics of MBL in age-and-sex-matched populations from rural Uganda and the UK. METHODS: In this cross-sectional study, we recruited volunteers aged at least 45 years who were seronegative for HIV-1 from the established Ugandan General Population Cohort and obtained their whole-blood samples. We also obtained blood samples from anonymised waste material of age-and-sex-matched individuals (aged >45 years, with a normal blood count and no history of cancer) in the UK. We used flow cytometry to determine the presence of MBL, defined according to standard diagnostic criteria, in the samples and compared differences in the proportion of cases with chronic lymphocytic leukaemia (CLL)-phenotype MBL and CD5-negative MBL, as well as differences in absolute monoclonal B-cell count between the two cohorts. FINDINGS: Between Jan 15 and Dec 18, 2012, we obtained samples from 302 Ugandan volunteers and 302 UK individuals who were matched by age and sex to the Ugandan population. Overall MBL prevalence was higher in the Ugandan participants (42 [14%] individuals) than in the UK cohort (25 [8%]; p=0·038). CLL-phenotype MBL was detected in three (1%) Ugandan participants and 21 (7%) UK participants (p=0·00021); all three Ugandan participants had absolute monoclonal B-cell count below one cell per µL, whereas the 21 UK participants had a median absolute number of circulating neoplastic cells of 4·6 (IQR 2-12) cells per µL. The prevalence of CD5-negative MBL was higher in the Ugandan cohort (41 [14%], of whom two [5%] also had CLL-phenotype MBL) than in the UK cohort (six [2%], of whom two [33%] also had CLL-phenotype MBL; p<0·0001), but the median absolute B-cell count was similar (227 [IQR 152-345] cells per µL in the Ugandan cohort vs 135 [105-177] cells per µL in the UK cohort; p=0·13). INTERPRETATION: MBL is common in both Uganda and the UK, but the substantial phenotypic differences might reflect fundamental differences in the pathogenesis of B-cell lymphoproliferative disorders. FUNDING: UK Medical Research Council and UK Department for International Development.

Predicting and managing primary and secondary non-response to rituximab using B-cell biomarkers in systemic lupus erythematosus.

OBJECTIVE: To assess factors associated with primary and secondary non-response to rituximab in systemic lupus erythematosus (SLE) and evaluate management of secondary non-depletion non-response (2NDNR). METHODS: 125 patients with SLE treated with rituximab over 12 years were studied prospectively. A major clinical response was defined as improvement of all active British Isles Lupus Assessment Group (BILAG)-2004 domains to grade C/better and no A/B flare. Partial responders were defined by one persistent BILAG B. B-cell subsets were measured using highly sensitive flow cytometry. Patients with 2NDNR, defined by infusion reaction and defective depletion, were treated with ocrelizumab or ofatumumab. RESULTS: 117 patients had evaluable data. In cycle 1 (C1), 96/117 (82%) achieved BILAG response (major=50%, partial=32%). In multivariable analysis, younger age (OR 0.97, 95% CI 0.94 to 1.00) and B-cell depletion at 6 weeks (OR 3.22, 95% CI 1.24 to 8.33) increased the odds of major response. Complete depletion was predicted by normal complement and lower pre-rituximab plasmablasts and was not associated with increased serious infection post-rituximab. Seventy-seven (with data on 72) C1 responders were retreated on clinical relapse. Of these, 61/72 (85%) responded in cycle 2 (C2). Of the 11 C2 non-responders, nine met 2NDNR criteria (incidence=12%) and tested positive for anti-rituximab antibodies. Lack of concomitant immunosuppressant and higher pre-rituximab plasmablasts predicted 2NDNR. Five were switched to ocrelizumab/ofatumumab, and all depleted and responded. CONCLUSION: Treatment with anti-CD20 agents can be guided by B-cell monitoring and should aim to achieve complete depletion. 2NDNR is associated with anti-rituximab antibodies, and switching to humanised agents restores depletion and response. In SLE, alternative anti-CD20 antibodies may be more consistently effective.

Clinical effectiveness and cost-effectiveness results from the randomised, Phase IIB trial in previously untreated patients with chronic lymphocytic leukaemia to compare fludarabine, cyclophosphamide and rituximab with fludarabine, cyclophosphamide, mitoxantrone and low-dose rituximab: the Attenuated dose Rituximab with ChemoTherapy In Chronic lymphocytic leukaemia (ARCTIC) trial.

BACKGROUND: The conventional frontline therapy for fit patients with chronic lymphocytic leukaemia (CLL) is fludarabine, cyclophosphamide and rituximab (FCR). Rituximab (Mabthera®, Roche Products Ltd) targets the CD20 antigen, which is expressed at low levels in CLL. The standard dose of rituximab in CLL (375 mg/m2 in cycle 1 and 500 mg/m2 in cycles 2-6) was selected based on toxicity data only. Small doses of rituximab (as low as 20 mg) have biological activity in CLL, with an immediate reduction in circulating CLL cells and down-regulation of CD20. Phase II trials had suggested improved efficacy with the addition of mitoxantrone to FCR. The key assumption for the Attenuated dose Rituximab with ChemoTherapy In CLL (ARCTIC) trial was that the addition of mitoxantrone to fludarabine, cyclophosphamide and low-dose rituximab would be more effective than conventional FCR. OBJECTIVES: To assess whether fludarabine, cyclophosphamide, mitoxantrone and low-dose rituximab (FCM-miniR) (100 mg of rituximab per cycle) was non-inferior to FCR in frontline CLL. Complete response (CR) rate was the primary end point, with the secondary end points being progression-free survival (PFS), overall survival (OS), overall response rate, eradication of minimal residual disease (MRD), safety and cost-effectiveness. DESIGN: ARCTIC was a UK multicentre, randomised, controlled, open, Phase IIB non-inferiority trial in previously untreated CLL. A total of 206 patients with previously untreated CLL who required treatment, according to the International Workshop on Chronic Lymphocytic Leukaemia criteria, were to be randomised to FCR or FCM-miniR. There was an independent Data Monitoring and Ethics Committee (DMEC) with a pre-planned interim efficacy assessment on 103 participants. RESULTS: The DMEC's interim analysis led to early trial closure. Although the response rates in both arms were higher than anticipated, FCM-miniR had a lower CR rate than FCR. This was partly attributable to the higher toxicity associated with mitoxantrone. A total of 100 participants completed FCR, 79 completed FCM-miniR and 21 commenced FCM-miniR but switched to FCR following DMEC recommendations. The CR rate for participants receiving FCR was 76%, compared with 55% for FCM-miniR (adjusted odds ratio 0.37; 95% confidence interval 0.19 to 0.73). Key secondary end points also showed that FCR was superior, with more participants achieving MRD negativity (57% for FCR vs. 46% for FCM-miniR). More participants experienced a serious adverse reaction with FCM-miniR compared with FCR (50% vs. 41%). At a median of 37.3 months' follow-up, the PFS and OS rates are good compared with previous studies, with no significant difference between the treatment arms. The economic analysis indicates that because FCM-miniR is less effective than FCR, FCM-miniR is not expected to be cost-effective over a lifetime horizon, producing a mean cost-saving of -£7723, a quality-adjusted life-year loss of -0.73 and a resulting incremental net monetary loss of -£6780. CONCLUSIONS: FCM-miniR is less well tolerated, with poorer response rates, than FCR, partly owing to the additional toxicity associated with mitoxantrone. In view of this, FCM-miniR will not be taken forward into a larger definitive Phase III trial. The trial demonstrated that oral FCR yields extremely high response rates compared with historical series with intravenous chemotherapy. FUTURE WORK: We shall compare the results of ARCTIC with those of the ADMIRE (Does the ADdition of Mitoxantrone Improve Response to FCR chemotherapy in patients with CLL?) trial, which compared FCR with FCM-R to assess the efficacy of low- versus standard-dose rituximab, allowing for the toxicity associated with mitoxantrone. TRIAL REGISTRATION: Current Controlled Trials ISRCTN16544962. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 28. See the NIHR Journals Library website for further project information.

Early Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell Transition.

Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19neg human PCs. However, whether human CD19neg PCs are necessarily aged cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19neg plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19pos, CD19low, and CD19neg ASCs secrete vaccine-specific Abs and show linked IGHV repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19neg state can be established at the plasmablast to PC transition, that CD19neg PCs increase as a percentage of surviving PCs in vitro, and that CD19neg and CD19pos PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19neg state to be established.