Results of the randomized phase IIB ARCTIC trial of low-dose rituximab in previously untreated CLL.

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

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

ADMIRE was a multicenter, randomized-controlled, open, phase IIB superiority trial in previously untreated chronic lymphocytic leukemia. Conventional front-line 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. Two hundred and fifteen patients were recruited to assess the primary end point of complete remission (CR) rates according to International Workshop on Chronic Lymphocytic Leukemia criteria. Secondary end points 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% confidence interval (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 granulocyte colony-stimulating factor 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 with historical series with intravenous chemotherapy.

An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases.

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL.

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

Heterogeneity of histological transformation events in Waldenström's macroglobulinemia (WM) and related disorders.

Histological transformation, typically to diffuse large B-cell lymphoma (DLBCL) is reported to occur in 5%-10% of patients with WM and recent studies have highlighted a possible aetiological role for the nucleoside analogues. It is however becoming increasingly clear that histological transformation is a complex phenomenon and may include clonally unrelated disorders. In order to highlight this pathological heterogeneity we describe 5 patients with diverse histological progression events. These included EBV-associated events namely DLBCL, peripheral T-cell lymphoma and spontaneously resolving mucocutaneous ulcer. A further 2 patients demonstrated a localised plasma cell rich lesion simulating plasmacytoma and a de novo DLBCL arising in an unrelated B-cell clone. It is clear therefore that detailed pathological assessments are required in all suspected cases of transformation and that the pathological heterogeneity demonstrated by this study needs to be taken into account when potential aetiological factors are being assessed.

Human peripheral blood B-cell compartments: a crossroad in B-cell traffic.

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) naïve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL).

Anti-CD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma.

Antibody-drug conjugates (ADCs) are potent cytotoxic drugs linked to antibodies through chemical linkers, and allow specific targeting of drugs to neoplastic cells. The expression of CD22 is limited to B-cells, and we show that CD22 is expressed on the vast majority of non-Hodgkin's lymphomas (NHLs). An ideal target for an ADC for the treatment of NHL would have limited expression outside the B-cell compartment and be highly effective against NHL. We generated an ADC consisting of a humanized anti-CD22 antibody conjugated to the anti-mitotic agent maytansine with a stable linker (anti-CD22-MCC-DM1). Anti-CD22-MCC-DM1 was broadly effective in in vitro killing assays on NHL B-cell lines. We did not find a strong correlation between in vitro potency and CD22 surface expression, internalization of ADC or sensitivity to free drug. We show that anti-CD22-MCC-DM1 was capable of inducing complete tumor regression in NHL xenograft mouse models. Further, anti-CD22-MCC-DM1 was well tolerated in cynomolgus monkeys and substantially decreased circulating B-cells as well as follicle size and germinal center formation in lymphoid organs. These results suggest that anti-CD22-MCC-DM1 has an efficacy, safety and pharmacodynamic profile that support its use as a treatment for NHL.

Management of nonresponse to rituximab in rheumatoid arthritis: predictors and outcome of re-treatment.

OBJECTIVE: A proportion of patients with rheumatoid arthritis (RA) have disease that fails to respond to an initial cycle of rituximab. Using highly sensitive flow cytometry (HSFC), it has been shown that most patients who do not exhibit a response, as measured using the European League Against Rheumatism (EULAR) criteria, have persistent circulating B cell levels at week 2 after initial treatment with rituximab. This study was undertaken to examine whether an additional cycle of rituximab would improve B cell depletion and clinical response in patients whose disease did not respond to the initial cycle. METHODS: Patients with RA (n = 158) were treated with a first cycle of rituximab (2 infusions of 1 gm each). Clinical responses were assessed using EULAR criteria, and patients were categorized as either first-cycle responders or first-cycle nonresponders. Baseline characteristics of first-cycle nonresponders (n = 38) and first-cycle responders (n = 65) with complete data were compared. First-cycle nonresponders (n = 25) were treated with a second cycle of rituximab at least 6 months after the first cycle. HSFC was performed at baseline, immediately prior to the second infusion (week 2), 1 month after the second infusion (week 6), and then every 3 months for each cycle of rituximab. Complete B cell depletion was defined as being <0.0001 x 10(9) cells/liter. RESULTS: At baseline, the number of preplasma cells was significantly higher in first-cycle nonresponders than in first-cycle responders (P = 0.003). Following the first infusion of the first cycle of rituximab, only 9% of first-cycle nonresponders (3 of 34) exhibited complete depletion of B-lineage cells, compared with 37% of first-cycle responders (22 of 59) (P = 0.007). Following the first infusion of the second cycle of rituximab, 38% of first-cycle nonresponders exhibited complete depletion. Twenty-six weeks after the second cycle, there was a significant improvement in the Disease Activity Score in 28 joints, with 72% of patients exhibiting a EULAR response. CONCLUSION: RA patients whose disease did not respond to an initial cycle of rituximab have higher circulating preplasma cell numbers at baseline and incomplete depletion. Our findings indicate that an additional cycle of rituximab administered prior to total B cell repopulation enhances B cell depletion and clinical responses.

Monoclonal B-cell lymphocytosis (MBL): biology, natural history and clinical management.

Chronic lymphocytic leukemia (CLL) and the other low-grade non-Hodgkin lymphomas are among the most common lymphoid malignancies. Recent studies suggest that more than 4% of the general population over age 40 harbor a population of clonal B cells with the phenotype of either CLL or another B-cell malignancy, a condition now designated monoclonal B-cell lymphocytosis (MBL). Although all cases of CLL appear to be preceded by MBL, the majority of individuals with MBL will not develop a hematologic malignancy. The biologic characteristics and clinical implications of MBL appear to differ based on whether it is identified during the diagnostic evaluation of lymphocytosis or incidentally discovered through screening of individuals with normal lymphocyte counts as part of research studies using highly sensitive detection methods. In this paper, we provide a state of the art review on the prevalence, nomenclature, biology, natural history and clinical management of MBL.

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

Flow cytometric protein expression profiling as a systematic approach for developing disease-specific assays: identification of a chronic lymphocytic leukaemia-specific assay for use in rituximab-containing regimens.

Depletion of disease below the levels detected by sensitive minimal residual disease (MRD) assays is associated with prolonged survival in chronic lymphocytic leukaemia (CLL). Flow cytometric MRD assays are now sufficiently sensitive and rapid to guide the duration of therapy in CLL, but generally rely on assessment of CD20 expression, which cannot be accurately measured during and after therapeutic approaches containing rituximab. The aim of this study was to use analytical software developed for microarray analysis to provide a systematic approach for MRD flow assay development. Samples from CLL patients (n=49), normal controls (n=21) and other B-lymphoproliferative disorders (n=12) were assessed with a panel of 66 antibodies. The DNA-Chip analysis program was used to identify discriminating antibodies, with hierarchical cluster analysis to identify complementary combinations. An iterative process was used: increasing numbers of patients were assessed with smaller, more targeted antibody panels until a highly specific combination (CD81/CD22/CD19/CD5) was identified. This combination was as sensitive and specific as previously reported assays and potentially applicable to blood and marrow samples from patients treated with current therapeutic approaches including rituximab. This approach to the identification of disease-specific antibody combinations for MRD analysis is readily applicable to a variety of haematological disorders.

Prevalence and characteristics of monoclonal B-cell lymphocytosis (MBL) in healthy individuals and the relationship with clinical disease.

Identifying and characterizing the earliest stages of disease is particularly important in disorders such as Chronic Lymphocytic Leukemia (CLL), where the initiating oncogenic events are unknown. Improvements in diagnostic technology have led to the detection of disease-associated markers in increasing numbers of otherwise healthy individuals. However, many of these approaches do not necessarily constitute evidence of a neoplastic process: the identification of a disease-precursor state requires evidence at several levels. The advent of high-sensitivity flow cytometry techniques, developed to monitor disease in CLL patients undergoing treatment, has allowed accurate enumeration of sub-clinical levels of monoclonal B-cells with a CLL phenotype in healthy individuals from the general population and CLL families. The age and gender distribution parallels that of clinical disease. Emerging evidence confirms the association between the aberrant cells in healthy individuals and those in CLL patients at the phenotypic, genotypic and clinical level, particularly in families with inherited susceptibility. These cells provide a tool for studying the events responsible for disease initiation.

Isotype class switching and the pathogenesis of multiple myeloma.

Translocations at the immunoglobulin heavy chain locus (14q32) are now considered the commonest karyotypic change in multiple myeloma. These translocations are thought to be intimately involved in the pathogenesis of this disease. The heavy chain locus is strongly transcriptionally active in B and plasma cells and transfer of a potential oncogene to 14q32 would result in its dysregulation. Molecular characterization suggests that the majority of these breakpoints cluster in switch regions within the heavy chain locus. Switch regions are normally involved in the regulated process of isotype switching so that in myeloma the rearrangements are believed to be a result of so-called illegitimate (aberrant) switch recombination and are likely to be an early event in myeloma development. A legitimate switch recombination event occurs between two switch regions producing a hybrid switch; this is necessary for class switching to proceed on a productive allele. In this review we describe the process of isotype switching and how illegitimate class switching may be related to the pathogenesis of multiple myeloma.

Analysis of CD34+ cell subsets in stem cell harvests can more reliably predict rapidity and durability of engraftment than total CD34+ cell dose, but steady state levels do not correlate with bone marrow reserve.

In peripheral blood stem cell transplantation (PBSCT), the number of CD34+ cells transplanted has been shown to correlate well with both rapidity and durability of engraftment. However, it is clear that engraftment does not necessarily correlate with total CD34+ cell numbers in some patients. Consequently, there is increasing interest in evaluating the role of CD34+ subsets in haemopoietic recovery as a more accurate marker of harvest quality. We analysed the numbers of CD34+ cell subsets, namely Thy-1+, L-Selectin+ and CD38-, and correlated this with engraftment in 86 patients undergoing PBSCT. Adequate engraftment was defined as being a platelet count greater than 50 x 10(9)/l and a neutrophil count greater than 1.0 x 10(9)/l. CD34+L-Selectin+ provided the best prediction of engraftment rapidity, although the improvement over total CD34+ cell dose was minor. Only the dose of CD34+Thy-1+ cells transplanted correlated with durable engraftment. The probability of adequate 3-month engraftment increased with the dose of CD34+ cells transplanted, but 10% of patients receiving > 5 x 10(6)/kg still showed poor engraftment at 3 months. However, all patients receiving > 2.5 x 10(5)/kg CD34+Thy-1+ showed adequate engraftment at this time point. We also demonstrated that CD34+Thy-1+ progenitors were restricted to the bone marrow under normal conditions and, during stem cell mobilization, their kinetics generally paralleled total CD34+ numbers.

Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.