Outcomes and mutational analysis of patients with lower-risk non-del5q myelodysplastic syndrome treated with antithymocyte globulin with or without ciclosporine A.

Immunosuppressive treatment is a disease-modifying therapy for lower-risk myelodysplastic syndromes (MDS). However, IST is relatively rarely used and long-term outcomes of patients are seldom reported. We retrospectively studied outcomes of 20 patients with lower-risk non del 5q MDS with transfusion dependency, with horse or rabbit antithymocyte globulin ±â€¯ciclosporine A, and frontline eltrombopag in two of them. IPSS-R was low, intermediate and high in 30%, 55% and 10% of the patients, respectively. Fifty-five percent of the patients had hypocellular bone marrow (BM). Baseline mutations were detected in 31.5% of the patients and were more frequent in patients with normo/hypercellular MDS than in patients with hypocellular MDS. Transfusion independence rate for both red blood cells (RBC) and platelets was achieved in 45% of patients. RBC transfusion duration ≤6 months, B-cell counts >0.2 G/L and, marginally, BM blasts ≤2% were associated with higher transfusion independence rate. Age and cellularity did not influence the response rate. Median transfusion independence duration was 53 months. Cumulative incidence of progression to a more aggressive myeloid disease was 0 in patients without baseline mutations and 33% in patients with baseline mutations (P = .008). Median progression-free and overall survival after treatment onset and median overall survival after loss of transfusion independence were 45.5 months, 68 months and not reached, respectively. In conclusion, antithymocyte globulin ±â€¯ciclosporine A results in durable responses in MDS, irrespective of age, in patients with lower-risk disease without B-cell lymphopenia and treated early in the course of the disease.

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.

Routine diagnostic procedures of myelodysplastic syndromes: value of a structural blood cell parameter (NEUT-X) determined by the Sysmex XE-2100™.

INTRODUCTION: Diagnostic features of myelodysplastic syndromes (MDS) are often polymorphic and nonspecific including anemia in most cases. Standard parameters provided by an automated analyzer seldom bring any argument for this diagnosis. The aim of this study was to investigate whether some structural parameters, not routinely provided by Sysmex™ XE 2100 analyzer, could help diagnose MDS in a simple way, adapted to routine practice. METHODS: Blood samples from 184 MDS fully annotated cases and 3545 normal blood count controls were performed with XE 2100 Sysmex™ analyzer. Quantitative and structural parameters were considered. RESULTS: We found that the structural neutrophil parameter, NEUT-X, converted into a semi-quantitative parameter, the granularity index (GI), could be used as a flag for MDS in front of anemia. Negative GI and anemia were able to make otherwise unrecognized MDS stand out in routine practice, increasing the number of slides addressed to review from 67% to 96%, without leading to a large excess of unfounded slide review among non-MDS. CONCLUSION: Including the GI index in the routine parameters provided by the Sysmex analyzer could be of major help for nonspecialized routine laboratories in detecting MDS.

[Expression of proliferation marker Ki 67 in chronic lymphocytic leukemia]. / Expression du marqueur de proliferation Ki 67 dans les leucemies lymphoides chroniques.

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of low proliferating mature B and T lymphocytes in the bone marrow and peripheral blood. The nuclear antigen Ki 67 is a protein detected in G1, S, G2 and M phases of the cell cycle, but not in G0, and thus, is a widely accepted proliferation marker of Human tumors. The aim of this study was to evaluate Ki 67 monoclonal antibody in CLL. We studied 48 patients diagnosed as CLL on the presence of clinical signs, over 4.109/l circulating lymphoid cells and immunophenotyping by flow cytometry using CD19, CD5, CD22, CD23, FMC7 and immunoglobulin light chains monoclonal antibodies. Ki 67 immunostaining was determined by Avidin Biotin Complex method. Our results allows to characterize between CLL: one group which proliferation rate (percentage of Ki 67 positive cells) was equal or less than 2%, represented by 14 cases (29,2%) with morphological aspect of typical CLL, one group which proliferation rate was between 3% and 9% represented by 32 cases (66,6%) with morphological aspect of polymorph CLL or prolymphocytic leukemia, and a last group with proliferation rate equal or up to 10% and corresponding to two cases (4,2%) of transformation of CLL to high grade Non Hodgkin lymphoma. There were no correlation between Matutes immunological score and proliferation rate, as this rate was 2.9% in score < 3 and 2.7% in score > 3. This study confirm the Ki 67 usefulness in studying cellular proliferation, and underline that CLL with polymorphic cytology are more proliferate than typical CLL. These data reinforce the notion that CLL is a disease with heterogeneity in clinical behavior, immunophenotype, cytogenetic, molecular aspects, and thus, prognostic.

Prognostic impact of p27KIP1 expression in cyclin D1 positive lymphoproliferative disorders.

Nodal mantle cell lymphoma (MCL) is a well-defined entity, but non-nodal leukemic cyclin D1 positive lymphoproliferative disorders have been reported and their relationship with MCL remains controversial and their prognosis heterogeneous. We prospectively studied the expression of cyclin D1 in CD5 positive leukemic B lymphoproliferative disorders at diagnosis and identified 65 cases overexpressing cyclin D1. We did not distinguish any clinical or biological criteria allowing one to identify a non-MCL group. Multivariate analysis identified age, anemia and p27kip1 expression as independent prognostic factors of survival. By univariate analysis, p27kip1 high expression proved to be the strongest predictor of prolonged survival. The median survival of p27 low expressors was 30 months, while it was not reached for p27 high expressors. A high level of p27 expression was often found associated with the absence of nodal involvement and the presence of somatic mutations, but neither of them was restricted to the p27 high expression group. In conclusion, we hypothesize that MCL and these cyclin D1 positive leukemic lymphoproliferative disorders represent a continuous spectrum of diseases. Determination of p27 expression level appears as a routine applicable test allowing identification of a subset of patients who could be considered for different therapeutic approaches.

[Bone marrow aspiration for diagnostic purposes]. / La ponction aspiration médullaire à visée diagnostique.

Examination of bone marrow aspiration is an important tool in the diagnosis of haematological diseases. First attempts of bone marrow sampling took place at the beginning of the twentieth century. Thereafter, numerous methods were proposed and different materials were described. The commonly accepted sites for sampling are sternum and the iliac crest. We describe here a sampling procedure for each site. Bone marrow aspiration is a safely investigation, but not recommended for patients with impaired haemostasis. The physician must be aware of its side effects and complications which could occur. The consequence of the complications varies according to the type of iatrogenic injury. Prevention and rapid diagnosis are a crucial point in the management of bone marrow aspiration accidents. To avoid malpractice, the procedure should be taught by senior physicians including theoretical as well as practical learning. The purpose of the learning is a high quality of care to ensure patients the best comfort in subsequent bone marrow examinations, this point being particularly important in paediatrics.

Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.

The glycoprotein (Gp) IIb/IIIa integrin, also called CD41, is the platelet receptor for fibrinogen and several other extracellular matrix molecules. Recent evidence suggests that its expression is much wider in the hematopoietic system than was previously thought. To investigate the precise expression of the CD41 antigen during megakaryocyte (MK) differentiation, CD34(+) cells from cord blood and mobilized blood cells from adults were grown for 6 days in the presence of stem cell factor and thrombopoietin. Two different pathways of differentiation were observed: one in the adult and one in the neonate cells. In the neonate samples, early MK differentiation proceeded from CD34(+)CD41(-) through a CD34(-)CD41(+)CD42(-) stage of differentiation to more mature cells. In contrast, in the adult samples, CD41 and CD42 were co-expressed on a CD34(+) cell. The rare CD34(+)CD41(+)CD42(-) cell subset in neonates was not committed to MK differentiation but contained cells with all myeloid and lymphoid potentialities along with long-term culture initiating cells (LTC-ICs) and nonobese diabetic/severe combined immune-deficient repopulating cells. In the adult samples, the CD34(+)CD41(+)CD42(-) subset was enriched in MK progenitors, but also contained erythroid progenitors, rare myeloid progenitors, and some LTC-ICs. All together, these results demonstrate that the CD41 antigen is expressed at a low level on primitive hematopoietic cells with a myeloid and lymphoid potential and that its expression is ontogenically regulated, leading to marked differences in the surface antigenic properties of differentiating megakaryocytic cells from neonates and adults. (Blood. 2001;97:2023-2030)

Thromboxane synthase has the same pattern of expression as platelet specific glycoproteins during human megakaryocyte differentiation.

Regulation of the platelet formation process is poorly understood. It has been shown that p45NF-E2 deficient mice have a profound defect in platelet formation and recently the first platelet/megakaryocytic gene regulated by NF-E2, thromboxane synthase (TXS), has been identified. In this study, we investigated TXS expression as a model of a gene regulated by NF-E2 during MK differentiation. Megakaryocytic cells derived from blood CD34+ cells were purified according to their stage of maturation on the basis of expression of CD34, CD41a and CD42a, permitting to define different stages in MK differentiation. By means of real-time quantitative RT-PCR, we could determine that the level of TXS increased during differentiation in parallel with the expression of c-mpl and GPIIb (CD41). However, amounts of TXS transcripts increased about 1.6-fold more than that of GPIIb or c-mpl transcripts during maturation. Expression of TXS and MK specific proteins such as CD41a, CD42a and vWF was also correlated in maturing MKs. In addition, staining by anti-TXS antibody of proplatelet bearing MKs was not increased in comparison to that observed in mature MK, suggesting that TXS is not upregulated during platelet formation. In addition, we investigated whether TXS and cyclooxygenase could be involved in platelet formation by adding aspirin into the cultures. No significant decrease of platelet production was observed. In conclusion, this study shows that TXS is coordinately expressed with the other platelet proteins during MK differentiation but is not directly involved in platelet formation.

Existence of a differentiation blockage at the stage of a megakaryocyte precursor in the thrombocytopenia and absent radii (TAR) syndrome.

The thrombocytopenia and absent radii (TAR) syndrome is a rare disease associating bilateral radial agenesis and congenital thrombocytopenia. Here, we investigated in vitro megakaryocyte (MK) differentiation and expression of c-mpl in 6 patients. Using blood or marrow CD34(+) cells, the colony-forming unit (CFU)-MK number was markedly reduced. CD34(+) cells were also cultured in liquid medium in the presence of a combination of 3 cytokines (stem cell factor, interleukin-3, and interleukin-6) or megakaryocyte growth and development factor (PEG-rHuMGDF) with or without SCF. In the presence of PEG-rHuMGDF, the majority of mature megakaryocytes (CD41 high, CD42 high) underwent apoptosis. This phenomenon was also observed in cultures stimulated by three cytokines. However, this last combination of cytokines allowed a more complete terminal MK differentiation. Surprisingly, a homogeneous population of CD34(-)CD41(+)CD42(-) cells accumulated during the cultures. This population was unable to differentiate along the myeloid pathways. This result suggests that a fraction of MK cells is unable to differentiate in the TAR syndrome. We subsequently investigated whether this could be related to an abnormality in c-mpl. No mutation or rearrangement in the c-mpl gene was found by Southern blots or by sequencing of the c-mpl coding region and its promoter in any of the patients. Using Western blot analysis, a decreased level of Mpl was found in patient platelets. A decreased level of c-mpl messenger RNA in TAR platelets was also detected with a lower c-mpl-P to c-mpl-K ratio in comparison to adult platelets. Altogether, these results demonstrate that the thrombocytopenia of the TAR syndrome is associated with a dysmegakaryocytopoiesis characterized by cells blocked at an early stage of differentiation. (Blood. 2000;95:1633-1641)

Intracellular trafficking of the alphaIIbbeta3 receptor antagonist, abciximab, in normal and Glanzmann's disease megakaryocytes.

The alphaIIbbeta3 platelet receptor antagonist abciximab (c7E3Fab, ReoPro(R)) has proved to be effective in preventing arterial thrombosis. However, its binding capacity to the platelet precursors, megakaryocytes (MKs), which also express alphaIIbbeta3, is not known. The purpose of this study was to establish whether abciximab is able to react with alphaIIbbeta3 located on human MKs, and to follow its subsequent intracellular trafficking. MKs were grown from CD34+ progenitors from normal subjects and from a patient with type I Glanzmann's thrombasthenia, and abciximab was added at day 10 of culture (4 microgram/ml). Cells were fixed at day 12, cryosectioned, and immunolabelled for abciximab. Labelling was prominent on the MK plasma membrane; it also lined the demarcation membration system. Interestingly, alpha-granule membranes were labelled showing that the antibody was internalized and further stored into MK secretory granules. Abciximab was also strongly detected on and in newly-formed platelets. Glanzmann's disease MKs (which completely lacked alphaIIbbeta3) were consistently negative, confirming that the antibody fragment was specifically interacting with alphaIIbbeta3. In conclusion, this study demonstrated that abciximab: (i) binds MK plasma membrane and demarcation membranes, (ii) trafficks into alpha-granules, and (iii) is expressed on and in nascent platelets. These findings could be taken in account when monitoring anti-alphaIIbbeta3 receptor therapy.

Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.