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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., Competing Interests: Declaration of interests Prof Bruce has received research grants from GSK, consulting fees from GSK, UCB, Eli Lilly & Co, BMS, Aurinia, IL-TOO and AstraZeneca and speaker fees from AstraZeneca, GSK and UCB within the last 3 years. Dr Vital has received honoraria and consulting fees from Roche within the last 3 years. All other authors declare no competing interest related to the work described in this manuscript., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: SD has received honoraria from Roche and GSK. SS has received honoraria from Novartis, Swedish Orphan Biovitrum (SOBI), and Sire and grant support from Novartis, Swedish Orphan Biovitrum, Octapharma, and CSL Behring. EV has received honoraria and research grant support from Roche, GSK, and AstraZeneca. PE has received consultant fees from BMS, Abbott, Pfizer, MSD, Novartis, Roche, and UCB. He has received research grants paid to his employer from Abbott, BMS, Pfizer, MSD, and Roche. MYMY has received consultancy fees from Aurinia Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arnold, Vital, Dass, Aslam, Rawstron, Savic, Emery and Md Yusof.)

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., (© 2020 by The American Society of Hematology.)

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., (© 2019, American College of Rheumatology.)

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.

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.

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., (© 2017 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.)

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., Competing Interests: Competing interests: EMV is an NIHR Clinician Scientist. He has received honoraria and research grant support from Roche, GSK and AstraZeneca. PE has received consultant fees from BMS, Abbott, Pfizer, MSD, Novartis, Roche and UCB. He has received research grants paid to his employer from AstraZeneca, Abbott, BMS, Pfizer, MSD and Roche., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

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.

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.

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., (Copyright © 2017 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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/m 2 in cycle 1 and 500 mg/m 2 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.

With immunochemotherapy, remission duration and survival in patients with chronic lymphocytic leukaemia is dependent on the level of minimal residual disease (MRD) after treatment. This phase II trial assessed alemtuzumab consolidation post-chemotherapy in patients who responded with persistent low levels of detectable disease. Blood was screened for MRD using multi-parameter flow cytometry, 6-24 months post-chemotherapy. MRD-positive participants received alemtuzumab 30 mg subcutaneously 3 times weekly for 6 weeks. Following a marrow assessment, MRD-negative participants or non-responders stopped therapy and MRD-positive participants with 1 + log reduction had 6 more weeks of alemtuzumab. Alemtuzumab consolidation was received by 47 participants. One death and 19 of 22 serious adverse events reported from 17 (36%) participants were alemtuzumab-related. MRD eradication from blood and bone marrow was achieved in 39 (83%) participants at the end of consolidation, with 18 (38%) remaining MRD-negative in the blood 6 months later. Of the 18 participants who were MRD-negative at 6 months, the median time to MRD relapse was 46 months, which was similar to patients who were MRD-negative at baseline and were followed up. The 5-year progression-free survival (PFS) and overall survival (OS) of participants who were MRD-negative at 6 months was significantly better than MRD-positive participants [PFS: 78% vs. 39% (P = 0·010), OS: 89% vs. 64% (P = 0·029)]., (© 2016 John Wiley & Sons Ltd.)

Importance: Numerous studies have evaluated the prognostic value of minimal residual disease (MRD) in patients with multiple myeloma (MM). Most studies were small and varied in terms of patient population, treatment, and MRD assessment methods., Objective: To evaluate the utility of MRD detection in patients with newly diagnosed MM., Data Sources: A Medline search was conducted for articles published in English between January 1990 and January 2016., Study Selection: Eligible studies reported MRD status and progression-free survival (PFS) or overall survival (OS) in 20 or more patients following treatment. Among 405 articles identified, 21 met the initial eligibility criteria and were included in the analysis., Data Extraction and Synthesis: Information on patient characteristics, treatment, MRD assessment, and outcomes were extracted using a standard form., Main Outcomes and Measures: The impact of MRD status on PFS and OS was assessed by pooling data from relevant trials. Data were adjusted to allow for different proportions of patients with MRD in different studies, and analyzed using the Peto method. Forest plots were created based on Cox model analysis. Other prespecified research questions were addressed qualitatively., Results: Fourteen studies (n = 1273) provided data on the impact of MRD on PFS, and 12 studies (n = 1100) on OS. Results were reported specifically in patients who had achieved conventional complete response (CR) in 5 studies for PFS (n = 574) and 6 studies for OS (n = 616). An MRD-negative status was associated with significantly better PFS overall (hazard ratio [HR], 0.41; 95% CI, 0.36-0.48; P < .001) and in studies specifically looking at CR patients (HR, 0.44; 95% CI, 0.34-0.56; P < .001). Overall survival was also favorable in MRD-negative patients overall (HR, 0.57; 95% CI, 0.46-0.71; P < .001) and in CR patients (HR, 0.47; 95% CI, 0.33-0.67; P < .001). Tests of heterogeneity found no significant differences among the studies for PFS and OS., Conclusions and Relevance: Minimal residual disease-negative status after treatment for newly diagnosed MM is associated with long-term survival. These findings provide quantitative evidence to support the integration of MRD assessment as an end point in clinical trials of MM.

Minimal residual disease (MRD) negativity, defined as <1 chronic lymphocytic leukemia (CLL) cell detectable per 10 000 leukocytes, has been shown to independently predict for clinical outcome in patients receiving combination chemoimmunotherapy in the frontline setting. However, the long-term prognostic value of MRD status in other therapeutic settings remains unclear. Here, we retrospectively analyzed, with up to 18 years follow-up, all patients at our institution who achieved at least a partial response (PR) with various therapies between 1996 and 2007, and received a bone marrow MRD assessment at the end of treatment according to the international harmonized approach. MRD negativity correlated with both progression-free survival (PFS) and overall survival (OS) independent of the type and line of treatment, as well as known prognostic factors including adverse cytogenetics. The greatest impact of achieving MRD negativity was seen in patients receiving frontline treatment, with 10-year PFS of 65% vs 10% and 10-year OS of 70% vs 30% for MRD-negative vs MRD-positive patients, respectively. Our results demonstrate the long-term benefit of achieving MRD negativity, regardless of the therapeutic setting and treatment modality, and support its use as a prognostic marker for long-term PFS and as a potential therapeutic goal in CLL., (© 2016 by The American Society of Hematology.)

Background: Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia. Combination immunochemotherapy such as fludarabine, cyclophosphamide and rituximab is the standard first line therapy in fit patients, but there is limited evidence regarding the optimal treatment of patients after relapse. Ofatumumab as monotherapy has been proven to be effective in the treatment of relapsed, refractory CLL, and as it is not myelotoxic, it is an ideal drug to combine with chemotherapy. However, the optimal dose of ofatumumab in this setting is not known. The Chemotherapy plus Ofatumumab at Standard or Mega dose in relapsed CLL (COSMIC) trial will assess the efficacy and safety of standard and high (mega) doses of ofatumumab combined with bendamustine or a combination of fludarabine and cyclophosphamide to determine which, if either, schedule should progress to a phase III trial., Methods/design: COSMIC is a phase II, multi-centre, randomised, open, parallel group trial for patients with relapsed CLL who are not refractory to fludarabine-based chemotherapy. Participants will be randomised to receive either standard dose or mega dose ofatumumab. Both doses will be given in combination with either bendamustine or fludarabine and cyclophosphamide chemotherapy backbone. The primary objective is to assess the proportion of participants achieving a complete remission following therapy with the two treatment arms (mega versus standard), as assessed at 3 months post treatment. The treatment groups will be assessed independently to determine whether the level of response is acceptable in relation to pre-specified criteria. If both treatment groups show an acceptable level of response, selection criteria will be used to determine which to take forward to a confirmatory phase III trial. A key secondary objective is to assess the dynamics of minimal residual disease (MRD) levels in relapsed disease. Eighty-two participants are planned to be recruited from 18 research centres in the UK., Discussion: Currently there is limited evidence regarding the optimal treatment of patients with relapsed or refractory CLL, and so suitable therapies are urgently needed. The COSMIC trial will identify whether ofatumumab given in combination with chemotherapy is safe and effective in this population, and will identify the optimal doses for further investigation., Trial Registration: ISRCTN51382468 . Registered on 21 September 2011.

Background: Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing., Methods: A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM., Results/conclusion: Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively., (© 2015 International Clinical Cytometry Society.)

People with myeloma who obtain a good response to treatment have a better survival if sensitive molecular or flow-cytometric techniques show no detectable minimal residual disease (MRD). The application of MRD techniques to clinical trials is now considered to be increasingly important because treatment approaches are sufficiently effective that using survival outcomes is slowing down the identification of the best new treatments. The articles in this issue consider the laboratory requirements for harmonization of MRD analysis by flow cytometry but there are practical considerations that are also important in implementing a myeloma MRD assay in the cytometry laboratory. In particular, it is important to consider when to request, and how best to utilize, a bone marrow aspirate sample because the procedure is invasive and the cells obtained are valuable for a number of different investigations. This brief article considers some experience obtained over two decades of implementing a service for MRD detection, initially as a scientific bolt-on to clinical trials through to a routine clinical diagnostic assay., (© 2015 International Clinical Cytometry Society.)

Treatment options for myeloma continue to develop at a rapid pace, and it is becoming increasingly challenging to determine the optimal therapeutic approaches because demonstrating a clear survival benefit now requires many years of follow-up. The detection of minimal residual disease (MRD) is recognized as a sensitive and rapid approach to evaluate treatment efficacy that predicts progression-free and overall survival independent of categorical response assessment and patients' biology. The benefit of MRD analysis is reflected in the many different techniques (multiparameter flow cytometry, quantitative polymerase chain reaction, and high-throughput sequencing) and collaborative groups (including EMN, ESCCA, ICCS, EuroFlow, and EuroMRD) that have performed collaborative projects to harmonize quantitative MRD detection. The time has come to adopt a consensus approach, and this report reviews the benefits and disadvantages of different strategies for MRD detection in myeloma and highlights the requirements for a sensitive, reproducible, and clinically meaningful cellular analytical approach., (© 2015 International Clinical Cytometry Society.)

Background: Flow cytometric (FC) detection of minimal residual disease (MRD) in multiple myeloma (MM) is prognostic and predictive of response to therapy. Therefore, standardization of FC MM MRD testing is vital to ensure better and uniform assessment of response to therapy and clinical prognostication. The International Clinical Cytometry Society and European Society for Clinical Cell Analysis, recognizing the need for standardized FC approaches, organized a working group to develop consensus guidelines on good clinical practice in FC MM MRD. Consensus guidelines are presented for specimen quality, staining process, reagent combinations, and the data acquisition process, all key factors in achieving high quality FC MM MRD testing., Methods: A group of eight flow cytometrists currently performing FC testing in MM evaluated available literature on FC MM MRD testing. A document presenting best practice was developed and reviewed in successive rounds until consensus was reached., Results/conclusion: The consensus on best practice for detection of MRD in MM is that CD38, CD138, and CD45 are analyzed in combination with CD19, CD56, CD27, CD81, and CD117. Consensus guidelines on acceptable specimen quality, staining procedures, panel design, and data acquisition were developed., (© 2015 International Clinical Cytometry Society.)

The detection of minimal residual disease (MRD) in myeloma using a 0.01% threshold (10(-4)) after treatment is an independent predictor of progression-free survival (PFS), but not always of overall survival (OS). However, MRD level is a continuous variable, and the predictive value of the depth of tumor depletion was evaluated in 397 patients treated intensively in the Medical Research Council Myeloma IX study. There was a significant improvement in OS for each log depletion in MRD level (median OS was 1 year for ≥10%, 4 years for 1% to <10%, 5.9 years for 0.1% to <1%, 6.8 years for 0.01% to <0.1%, and more than 7.5 years for <0.01% MRD). MRD level as a continuous variable determined by flow cytometry independently predicts both PFS and OS, with approximately 1 year median OS benefit per log depletion. The trial was registered at www.isrctn.com as #68454111., (© 2015 by The American Society of Hematology.)

The purpose of this study was to use multiparameter flow cytometry to detect occult marrow disease (OMD) in patients with solitary plasmacytoma of bone and assess its value in predicting outcome. Aberrant phenotype plasma cells were demonstrable in 34 of 50 (68%) patients and comprised a median of 0.52% of bone marrow leukocytes. With a median follow-up of 3.7 years, 28 of 50 patients have progressed with a median time to progression (TTP) of 18 months. Progression was documented in 72% of patients with OMD vs 12.5% without (median TTP, 26 months vs not reached; P = .003). Monoclonal urinary light chains (ULC) were similarly predictive of outcome because progression was documented in 91% vs 44% without (median TTP, 16 vs 82 months; P < .001). By using both parameters, it was possible to define patients with an excellent outcome (lacking both OMD and ULC, 7.7% progression) and high-risk patients (OMD and/or ULC, 75% progression; P = .001). Trials of systemic therapy are warranted in high-risk patients., (© 2014 by The American Society of Hematology.)

Purpose: To investigate the prognostic value of minimal residual disease (MRD) assessment in patients with multiple myeloma treated in the MRC (Medical Research Council) Myeloma IX trial., Patients and Methods: Multiparameter flow cytometry (MFC) was used to assess MRD after induction therapy (n = 378) and at day 100 after autologous stem-cell transplantation (ASCT; n = 397) in intensive-pathway patients and at the end of induction therapy in non-intensive-pathway patients (n = 245)., Results: In intensive-pathway patients, absence of MRD at day 100 after ASCT was highly predictive of a favorable outcome (PFS, P < .001; OS, P = .0183). This outcome advantage was demonstrable in patients with favorable and adverse cytogenetics (PFS, P = .014 and P < .001, respectively) and in patients achieving immunofixation-negative complete response (CR; PFS, P = .0068). The effect of maintenance thalidomide was assessed, with the shortest PFS demonstrable in those MRD-positive patients who did not receive maintenance and longest in those who were MRD negative and did receive thalidomide (P < .001). Further analysis demonstrated that 28% of MRD-positive patients who received maintenance thalidomide became MRD negative. MRD assessment after induction therapy in the non-intensive-pathway patients did not seem to be predictive of outcome (PFS, P = .1)., Conclusion: MRD assessment by MFC was predictive of overall outcome in patients with myeloma undergoing ASCT. This predictive value was seen in patients achieving conventional CR as well as patients with favorable and adverse cytogenetics. The effects of maintenance strategies can also be evaluated, and our data suggest that maintenance thalidomide can eradicate MRD in some patients.

Serum immunoglobulin (Ig) M levels vary considerably among patients with Waldenström macroglobulinemia, and previous studies have failed to demonstrate a correlation with overall bone marrow disease burden. In this study, bone marrow B cells and plasma cells were enumerated by flow cytometry and correlated with serum IgM concentrations. Monotypic B cells comprised a median of 6% of bone marrow leukocytes but did not correlate with IgM levels (r = 0.071, P = .5). Plasma cells, although typically present in lower numbers (median, 0.52%) did show a correlation with IgM (r = 0.452, P = .01). IgM levels in Waldenström macroglobulinemia, at least in part, correlate with the degree of plasma cell differentiation seen within the tumor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Monoclonal B cell Lymphocytosis (MBL) or similar terms have been used for decades to describe the presence of light-chain restricted B lymphocytes with uncertain clinical significance, usually having a phenotype consistent with chronic lymphocytic leukemia (CLL). As diagnostic technology improved, ever smaller monoclonal B cell populations were identifiable in the population, and approximately half of people over 90 years old have a minimal (<1 cell/μL) circulating CLL-like B cell population. These minimal CLL-like B cell populations share some molecular characteristics with CLL, but have no clinical significance. In contrast, CLL-like MBL cases detected through hospital investigations are biologically indistinguishable from early stage CLL, but the neoplastic B cell levels are usually stable over time and the risk of progressive disease requiring treatment is much lower than for early stage CLL. However, there is usually partial or complete depletion of normal B cells, with an increased relative risk of severe infection, comparable to early stage CLL, which may impair overall survival.

This report represents a further update of the consensus panel criteria for the assessment of clinical response in patients with Waldenström macroglobulinaemia (WM). These criteria have been updated in light of further data demonstrating an improvement in categorical responses with new drug regimens as well as acknowledgement of the fact that such responses are predictive of overall outcome. A number of key changes are proposed but challenges do however remain and these include the variability in kinetics of immunoglobulin M (IgM) reduction with different treatment modalities and the apparent discrepancy between IgM and bone marrow/tissue response noted with some regimens. Planned sequential bone marrow assessments are encouraged in clinical trials., (© 2012 Blackwell Publishing Ltd.)

Combination fludarabine (F), cyclophosphamide (C) and rituximab (R) is the standard front-line therapy in chronic lymphocytic leukaemia (CLL), but appropriate treatment of relapsed/refractory CLL is less clear. Combined FC and mitoxantrone (M) has been reported to be effective in a single arm study, and rituximab when added to chemotherapy in CLL is synergistic. A randomized, two-stage, Phase II trial of FCM and FCM-R was conducted in relapsed CLL. The primary endpoint was response rate 2 months after therapy, assessed according to the 2008 International Workshop CLL criteria. In addition, minimal residual disease (MRD) in the marrow was studied 2 months after therapy, with MRD negativity defined as <0·01% CLL cells. Fifty-two patients were entered, 26 in each arm. The overall response rates to FCM and FCM-R were 58% and 65% respectively. Combined complete response (CR) and CR with incomplete marrow recovery [CR(i)] was 15% (95% confidence interval [CI]:4-35%) for FCM and 42% (95%CI:23-63%) for FCM-R, with eight patients achieving MRD negativity (3 FCM; 5 FCM-R). The toxicity of both regimens was acceptable. In conclusion, the addition of rituximab to FCM improves the response rates in relapsed CLL, resulting in more complete remissions and without additional safety concerns. Efficacy and safety should be fully tested in a randomized Phase III trial., (© 2011 Blackwell Publishing Ltd.)

The term monoclonal B-cell lymphocytosis (MBL) was recently introduced to identify individuals with a population of monoclonal B cells in the absence of other features that are diagnostic of a B-cell lymphoproliferative disorder. MBL is often identified through hospital investigation of a mild lymphocytosis, and approximately 1% of such individuals develop progressive disease requiring treatment per year. However, in population studies using high-sensitivity flow cytometry, MBL may be detectable in more than 10% of adults aged over 60 years, and clinical progression is rare. The majority of MBL cases have features that are characteristic of chronic lymphocytic leukaemia, but an increasing amount of information is becoming available about MBL with the features of other B-cell lymphoproliferative disorders. In addition to flow cytometry findings, the incidental detection of an occult B-cell lymphoproliferative disorder is also occurring in a significant proportion of tissue biopsy samples. In this review, the clinical and biological relationship between MBL and B-cell lymphoproliferative disorders will be discussed, with a focus on identifying the differences between low levels of peripheral blood or bone marrow involvement with lymphoma and the monoclonal B-cell populations that commonly occur in elderly adults., (© 2011 Blackwell Publishing Limited.)

Monoclonal B-cell lymphocytosis (MBL) is detectable in > 3% of the general population. Recent data are compatible, at least in a proportion of cases, with MBL being a progenitor lesion for chronic lymphocytic leukemia (CLL) and a surrogate for inherited predisposition. Common single nucleotide polymorphisms (SNPs) at 2q13 (rs17483466), 2q37.1 (rs13397985), 2q37.3 (rs757978), 6p25.3 (rs872071), 8q24.21 (rs2456449), 11q24.1 (rs735665), 15q21.3 (rs7169431), 15q23 (rs7176508), 16q24.1 (rs305061), and 19q13.32 (rs11083846) have been shown to confer a modest but significant increase in CLL risk. To examine the impact of these 10 SNPs on MBL, we analyzed 3 case-control series totaling 419 cases and 1753 controls. An association between genotype and MBL risk was seen for 9 SNPs, 6 of which were statistically significant: rs17483466 (odds ratio [OR] =1.27; P = .02), rs13397985 (OR = 1.40; P = 1.72 × 10(-3)), rs757978 (OR = 1.38; P = .02), rs872071 (OR = 1.27; P = 7.75 × 10(-3)), rs2456449 (OR = 1.31; P = 3.14 × 10(-3)), and rs735665 (OR = 1.63; P = 6.86 × 10(-6)). Collectively, these data provide support for genetic variation influencing CLL risk through predisposition to MBL.

Monoclonal B-lymphocytosis (MBL) is defined as the presence of a population of monoclonal B-cells, usually with a chronic lymphocytic leukaemia (CLL) phenotype, which comprise fewer than 5000 cells per microl with no evidence of tissue involvement. Over the past few years, MBL has been clearly defined and differentiated from CLL so that individuals with MBL are no longer inappropriately labelled as suffering from leukaemia. In this review, we will describe the entity of MBL and summarise the evidence that underlies the current theory on the pathophysiology of the disorder, the relationship with CLL and the probability of developing progressive disease requiring treatment. In addition, we will evaluate the importance of further clinical investigations, in particular, the relevance of screening for MBL and undertaking bone marrow investigations according to the clinical setting and B-cell phenotype., (Copyright 2010. Published by Elsevier Ltd.)

Even though chronic lymphocytic leukemia (CLL) is the most prevalent leukemia of the Western world, the development of treatment approaches for CLL has lagged behind the development of approaches to various other hematologic malignancies for a variety of reasons. In recent years, the treatment approach to patients with CLL has evolved rapidly, with the addition of several new prognostic markers, highly effective immunochemotherapy combinations, and attainment of remission up to the point of the eradication of minimal residual disease (MRD). Highly sensitive methods now available to detect MRD can detect a single CLL cell in 10(4) leukocytes using either allele-specific oligonucleotide polymerase chain reaction or four-color or six-color flow cytometry. Over the past decade, several studies have examined the possible advantage of MRD eradication in CLL. This article reviews our current understanding of MRD eradication and analyzes whether it is a desirable goal in the routine clinical treatment of CLL, which will optimize the management of individual patients.

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be., (Published 2010 Wiley-Liss, Inc.)

The biological and clinical relationship between Chronic Lymphocytic Leukaemia (CLL) and Monoclonal B-cell Lymphocytosis (MBL) has now been reported in some detail. This review investigates associations between biology and disease activity as they relate to the absolute numbers of abnormal cells. The clonal B-cells in CLL-type MBL are indistinguishable from CLL with respect to surface phenotype and the presence of chromosomal abnormalities. However, the majority of CLL-type MBL cases in the general population have very low numbers of clonal B-cells, typically in the range 0.1-10 per μL, and such cases use different IGHV genes than higher-count CLL-type MBL cases and often show intraclonal heterogeneity. Cases with higher counts are biologically similar to CLL although there is a relationship between the CLL cell count at presentation and the likelihood of further clonal expansion. Individuals presenting with CLL cell counts above 2,000 per μL are more likely to have gradually increasing B-cell counts over time and although the risk of requiring treatment for progressive CLL remains low there may be impaired normal B-cell activity., (© 2010 International Clinical Cytometry Society.)

Background: Individuals with monoclonal B-cell lymphocytosis (MBL) have been identified in clinic outpatients, in unaffected relatives of patients with chronic lymphocytic leukemia (CLL), and in general populations. MBL and its relationship with CLL have been actively investigated over the last decade. This report systematically reviews the prevalence of MBL in the context of the populations studied and the evolution of laboratory methods used to define MBL., Methods: To identify published studies that have assessed the prevalence of MBL, we systematically searched the MEDLINE databases and consulted with members of the International MBL Study Group. We reviewed the 10 articles that were identified by this process. We abstracted information on study populations, laboratory tests, criteria for designating MBL, and the reported frequencies., Results: Three of the ten studies were published in 2009, three between 2007 and 2008, and four between 2002 and 2004. Reported prevalences varied widely, ranging from 0.12 to 18.2%. This variability was clearly associated with both the laboratory methods and the populations studied. MBL was more common among older individuals and kindred of persons with CLL. The most common MBL subtype was CLL-like MBL., Conclusions: Large population-based studies of MBL that employ standardized laboratory methods with a consensus case definition are needed to assess prevalence and establish risk factors. These studies should include prospective follow-up of MBL cases to determine the relationship between MBL and CLL. Data from original studies should be reported in sufficient detail to allow future synthesis of information from multiple studies, such as meta-analysis., (Published 2010 Wiley-Liss, Inc.)

Introduction: The aim of this study was to screen for cell surface markers that could discriminate CLL-type MBL from CLL or identify CLL cases likely to have stable disease., Methods: Six color flow cytometry was performed on CLL-type MBL (n = 94) and CLL (n = 387) at diagnosis or relapse; 39 cases had poor-risk chromosomal abnormalities (17p and/or 11q deletion). Expression of 30 markers was analysed: CCR6, CD10, CD103, CD11c, CD138, CD200, CD22, CD23, CD24, CD25, CD27, CD31, CD38, CD39, CD43, CD49d, CD5, CD52, CD62L, CD63, CD79b, CD81, CD86, CD95, CXCR5, HLADR, IgD, IgG, IgM, LAIR1., Results: There was no difference in expression between CLL-type MBL and CLL for the majority of markers. Differential expression was observed for several markers, mainly between MBL and CLL cases with adverse-risk chromosomal abnormalities. These differences included lower expression of CD38 (9.4-fold lower, P = 0.007) and CD49d (3.2-fold lower, P = 0.008) and higher expression of LAIR-1 (3.7-fold higher, P = 0.003), CXCR5 (1.25-fold higher, P = 0.002), and CCR6 (1.9-fold higher P < 0.001) on CLL-type MBL compared to CLL with adverse chromosomal abnormalities. CD62L (L-selectin) which mediates lymphocyte adhesion to endothelial venules of lymphoid tissue, was expressed at a significantly different level between CLL-type MBL and both CLL sub-groups, with 1.3-fold lower (P = 0.04) expression levels on the MBL cases. However, there was broad overlap in expression levels., Conclusions: CLL-type MBL is phenotypically identical to CLL for a very broad range of markers. Differential expression is predominantly related to known prognostic markers and proteins involved in homing to lymphoid tissue., (© 2010 International Clinical Cytometry Society.)

In this study we used bone marrow flow cytometry and immunohistochemistry to evaluate response to fludarabine therapy in patients with Waldenström's macroglobulinemia (WM)/lymphoplasmacytic lymphoma. Responses in serum M protein were typically delayed with a median time to maximum response of 6 months following the completion of therapy (range, 0-18 months). In contrast, bone marrow responses occurred promptly in responding patients such that there were no detectable clonal B cells at the end of therapy in 55% of patients assessed. Persistent monoclonal plasma cells were, however, readily identified by CD138 immunohistochemistry, explaining the persistence of serum M protein in these patients. This simple observation has significant implications for the assessment of responses in WM as well as the design of future therapeutic strategies.

The incidence and presenting features of chronic lymphocytic leukemia (CLL) have changed significantly over the last century. Routine diagnostic techniques can now detect very low levels of CLL phenotype cells. Monoclonal B-cell lymphocytosis (MBL) is a relatively recent diagnostic category encapsulating individuals with an abnormal B-cell population but not meeting the diagnostic criteria for a B-cell malignancy. This review focuses on CLL-type MBL, which represents the majority of MBL cases identified in diagnostic laboratories. CLL-type MBL has a phenotype identical to CLL and shares the same chromosomal abnormalities even at the lowest levels detectable. Recent evidence suggests that the immunoglobulin gene usage plays a key role in whether the abnormal cells will develop in significant numbers. In most cases, CLL-type MBL is a stable condition with only 1% per year among those presenting for clinical attention developing progressive disease requiring treatment, although suppressed immune function may have a more significant impact on outcome.

Objective: In rheumatoid arthritis (RA), B cell depletion occurs in all patients treated with rituximab, but the clinical responses to rituximab are variable. A highly sensitive assay was used to test the hypothesis that B cell depletion is variable, and that incomplete depletion leads to a poorer outcome., Methods: Sixty patients with active RA unresponsive to anti-tumor necrosis factor agents received two 1-gram infusions of rituximab. B cell numbers were measured by highly sensitive flow cytometry before and after each infusion and at 3-month intervals thereafter. A reduction in B cell levels below 0.0001x10(9)/liter was defined as complete depletion (compared with 0.05x10(9)/liter by conventional cytometry). Clinical responses were measured using the European League Against Rheumatism (EULAR) criteria., Results: At 6 months, 92% of patients had a moderate-to-good clinical response according to the EULAR criteria. B cells were detected in 63% of patients after the first infusion of rituximab (median level 0.0009x10(9)/liter [range<0.0001-0.0015x10(9)/liter), and these patients had poorer clinical outcomes than patients with complete depletion. At 9 months, 82% of patients with complete depletion had a moderate-to- good EULAR response, compared with 43% of those with partial depletion (P=0.01). At 12 months, 59% of complete responders had a moderate-to-good EULAR response, compared with 21% of those with partial depletion (P=0.01). Patients in whom B cells were depleted only after the second infusion did no better than those in whom depletion was never complete and had poorer clinical outcomes than those in whom depletion was initially complete., Conclusion: This study is the first to show, using a highly sensitive analysis, that rituximab therapy is associated with variable diminution in B cell numbers. A lack of complete depletion of B cells after 1 infusion was associated with a poorer outcome.

Background: A diagnosis of chronic lymphocytic leukemia (CLL) requires a count of over 5000 circulating CLL-phenotype cells per cubic millimeter. Asymptomatic persons with fewer CLL-phenotype cells have monoclonal B-cell lymphocytosis (MBL). The goal of this study was to investigate the relation between MBL and CLL., Methods: We investigated 1520 subjects who were 62 to 80 years of age with a normal blood count and 2228 subjects with lymphocytosis (>4000 lymphocytes per cubic millimeter) for the presence of MBL, using flow cytometry. Monoclonal B cells were further characterized by means of cytogenetic and molecular analyses. A representative cohort of 185 subjects with CLL-phenotype MBL and lymphocytosis were monitored for a median of 6.7 years (range, 0.2 to 11.8)., Results: Monoclonal CLL-phenotype B cells were detected in 5.1% of subjects (78 of 1520) with a normal blood count and 13.9% (309 of 2228) with lymphocytosis. CLL-phenotype MBL had a frequency of 13q14 deletion and trisomy 12 similar to that of CLL and showed a skewed repertoire of the immunoglobulin heavy variable group (IGHV) genes. Among 185 subjects presenting with lymphocytosis, progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The absolute B-cell count was the only independent prognostic factor associated with progressive lymphocytosis. During follow-up over a median of 6.7 years, 34% of subjects (62 of 185) died, but only 4 of these deaths were due to CLL. Age above 68 years and hemoglobin level below 12.5 g per deciliter were the only independent prognostic factors for death., Conclusions: The CLL-phenotype cells found in the general population and in subjects with lymphocytosis have features in common with CLL cells. CLL requiring treatment develops in subjects with CLL-phenotype MBL and with lymphocytosis at the rate of 1.1% per year., (2008 Massachusetts Medical Society)

IgM myeloma is a very rare and poorly defined entity. In a detailed assessment of 10 cases, it was demonstrated that 70% had an aberrant phenotype based on the expression of CD19, CD45, CD27 and Cyclin D1 but all cases lacked CD56 and CD117. Interphase fluorescence in situ hybridization demonstrated deletion 13 in 50% while 5/8 cases assessed had a t(11;14). Despite the high incidence of the t(11;14), CD20 was only expressed in one of nine cases. We conclude that IgM myeloma is a distinctive subset characterized by a CD20-CD56-CD117- phenotype and the t(11;14).

New treatment approaches have substantially improved response rates in chronic lymphocytic leukemia. Accurate assessment of effective combination chemoimmunotherapy requires more sensitive measures of response, and a variety of techniques to measure minimal residual disease (MRD) have been developed. Because many studies demonstrate that MRD eradication is associated with prolonged treatment-free survival, detection of MRD is becoming a standard component of clinical trials. Quantitative approaches using polymerase chain reaction or multiparameter flow cytometry are preferable because they allow comparison of efficacy between different studies. In most clinical settings, the levels of chronic lymphocytic leukemia always increase from the first detection of MRD; the exception is allogeneic transplantation, in which there may be stable MRD levels or delayed MRD eradication. MRD analysis in the peripheral blood also may be used during therapy to predict eventual response and potentially to guide therapy to achieve the optimal outcome.

A CD5(+)23(+) monoclonal B-cell population is detectable in approximately 3% of the general adult population. The phenotype of the monoclonal CD5(+)23(+) B cells is identical to chronic lymphocytic leukaemia (CLL) with respect to a large number of proteins in addition to the standard diagnostic markers used to identify CLL. Studies in CLL families and direct assessment of genetic features indicate a close biological association between indolent CLL and the CLL-phenotype cells detected in individuals with a normal blood count. Patients with a CLL-phenotype monoclonal B-cell lymphocytosis (MBL) often have increasing CLL cell counts with time and some progress to a stage requiring treatment. Analysis of intraclonal variation in the immunoglobulin heavy chain gene suggests a process of clonal diversification rather than clonal selection in the early stages of disease progression. CLL-phenotype MBL is detectable in approximately 10% of cases referred for investigation of a lymphocytosis and future studies should be directed towards the detection of factors which identify MBL patients at risk of disease progression.