Minimal residual disease is an independent predictor for 10-year survival in CLL

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

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

This is an open access paper.-- et al.The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.The meetings were sponsored by the European Myeloma.Peer Reviewe

Results of the randomized phase IIB ADMIRE trial of FCR with or without mitoxantrone in previously untreated CLL

ADMIRE was a multi-center, randomized-controlled, open, phase IIB superiority trial in previously untreated Chronic Lymphocytic Leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). Initial evidence from non-randomized Phase II trials suggested that the addition of mitoxantrone to FCR (FCM-R) improved remission rates. 215 patients were recruited to assess the primary endpoint of complete remission (CR) rates according to IWCLL criteria. Secondary endpoints were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity and safety. At final analysis, CR rates were 69.8% FCR vs 69.3% FCM-R [adjusted odds ratio (OR): 0.97; 95%CI: (0.53-1.79), P=0.932]. MRD-negativity rates were 59.3% FCR vs 50.5% FCM-R [adjusted OR: 0.70; 95% CI: (0.39-1.26), P=0.231]. During treatment, 60.0% (n=129) of participants received G-CSF as secondary prophylaxis for neutropenia, a lower proportion on FCR compared with FCM-R (56.1 vs 63.9%). The toxicity of both regimens was acceptable. There are no significant differences between the treatment groups for PFS and OS. The trial demonstrated that the addition of mitoxantrone to FCR did not increase the depth of response. Oral FCR was well tolerated and resulted in impressive responses in terms of CR rates and MRD negativity compared to historical series with intravenous chemotherapy

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

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

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

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background:Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.Methodology/Principal Findings:For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLloclones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhiand advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+clonal B-cells.Conclusions/Significance:These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLloclones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution

Repeat cycles of rituximab on clinical relapse in ANCA-associated vasculitis: identifying B cell biomarkers for relapse to guide retreatment decisions

OBJECTIVE: To assess clinical and B cell biomarkers to predict relapse after rituximab in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) using retreatment on clinical relapse strategy. METHODS: 35 patients with AAV received treatment with 2×1000 mg rituximab, repeated on clinical relapse (up to 5 cycles). Disease activity was assessed by Birmingham Vasculitis Activity Score (BVAS) and peripheral B cell subsets using highly sensitive flow cytometry (HSFC) as previously described; both performed at baseline and every 3 months. RESULTS: Response rates were high: >83%, with median time-to-relapse of 82 weeks for cycle 1 (C1) and >54 weeks for all cycles. Prior to rituximab, AAV was characterised by naïve B-lymphopenia compared to healthy controls. This dysregulation was more marked in patients with raised C-reactive protein (CRP) (p<0.05). In C1, no clinical feature predicted relapse. However, repopulation of naïve B cell at 6 months was associated with a reduced risk of relapse (HR: 0.326, 95% 0.114 to 0.930, p=0.036). Relapse rates at 12 and 18 months were 0% and 14% with naïve repopulation at 6 months, and 31% and 54% without naïve repopulation. CONCLUSIONS: Responses to B cell depletion therapy are long-lasting and relapse post-treatment may be predicted by absence of naïve B cell repopulation at 6 months. Naïve B-lymphopenia may be a biomarker of disease activity in AAV

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. METHODOLOGY/PRINCIPAL FINDINGS: The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis