The presence of a chromosomal abnormality in cytopenia without dysplasia identifies a category of high-risk clonal cytopenia of unknown significance.

Myelodysplastic syndromes (MDS) are hematological malignancies classically defined by the presence of cytopenia(s) and dysmorphic myeloid cells. It is now known that MDS can be preceded by a pre-malignant condition called clonal cytopenia of unknown significance (CCUS), which associates a clonality marker with cytopenia in the absence of criteria of dysplasia. However, to date, it is not clear whether chromosomal abnormalities should be considered in the definition of CCUS or if they carry a prognostic impact in CCUS patients. In this study, we analyzed the clinico-biological features and outcomes of 34 patients who presented with one or more cytopenias, an absence of significant dysplasia, and a presence of a chromosomal abnormality (CA). We named this entity chromosomal abnormality with cytopenia of undetermined significance (CACtUS). We show that these patients are slightly older than MDS patients and that they more frequently presented with normocytic anemia. Most CACtUS patients exhibited only one unbalanced CA. The number and type of mutations were comparable between CACtUS patients and MDS patients. Regardless of the cytogenetic abnormality, the clinicobiological characteristics, overall survival, and risk of progression to high-risk (HR) MDS were similar between CACtUS patients and low-risk MDS patients. Thus, we suggest that CACtUS patients can be considered as HR-CCUS and should receive the follow-up regimen recommended for MDS patients.

Multicentric study underlining the interest of adding CD5, CD7 and CD56 expression assessment to the flow cytometric Ogata score in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative.

Very low levels of surface CD45 reflect CLL cell fragility, are inversely correlated with trisomy 12 and are associated with increased treatment-free survival.

It has recently been suggested that the percentage of smudge cells on blood smears from patients with chronic lymphocytic leukemia (CLL) could predict overall survival. However, smudge cells are a cytological artifact influenced by multiple physical factors not related to CLL. To identify simple parameters reflecting CLL cell fragility, we studied CD45 expression in a series of 66 patients with Binet stage A CLL. Decreased CD45 expression was specific for CLL cells when compared to 44 patients with a leukemic phase of B-cell non Hodgkin lymphoma and 42 control B-cells. CD45 expression was markedly decreased for all patients with CLL with high percentages of smudge cells. CLL cells with the lowest CD45 expression were the most sensitive to osmotic shock. Very low levels of CD45 expression were significantly associated with lack of CD38 expression, absence of trisomy 12, and with increased treatment free survival time. Altogether, these results demonstrate that low levels of CD45 expression are specific to CLL cells and reflect cell fragility, suggesting that this is an important intrinsic biological feature that determines disease course.

Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia.

Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.

Flow cytometry detection of CD138 expression continuum between monotypic B and plasma cells is associated with both high IgM peak levels and MYD88 mutation and contributes to diagnosis of Waldenström macroglobulinemia.

BACKGROUND: Differential diagnosis of Waldenström macroglobulinemia (WM) with other indolent B-cell malignancies is still a challenge. Here, we propose an original and simple analysis of routine flow cytometry (FCM) unraveling the characteristic ongoing plasma cell (PC) differentiation of WM tumor B-cells. METHODS: FCM analysis of both B-cells and PC was performed on a series of 77 patients with IgM peak. MYD88 and CXCR4 mutations were studied using an allele-specific PCR and by high throughput sequencing. RESULTS: Twenty seven (35%), 46 (58%) and 4 (5%) patients were classified as WM, IgM monoclonal gammopathy of undetermined significance (MGUS) or other B-NHL respectively. MYD88 mutation was found in 25/27 WM (93%) and in 29/46 MGUS (63%). Using FCM, monotypic B-cells were found in 27/27 WM (100%) and 34/46 MGUS (74%). Monotypic CD138pos/CD38pos PCs were detected in 23/27 WM (85%) and 25/46 MGUS (54%). Highlighting the ongoing PC differentiation of WM tumor B-cells by FCM, we evidenced a CD138 expression continuum between monotypic B-cells and PCs. This pattern remained absent in control samples and was significantly associated with higher IgM peaks (p = 6.10-5 ) and MYD88 mutations (p = 10-3 ) in both WM and MGUS cases. CONCLUSIONS: FCM exploration of both B-cells and PC led to identify a CD138 expression continuum as an objective marker of ongoing PC differentiation of WM tumor cells and was strongly associated with increased IgM peak levels and MYD88 mutations. This approach could contribute to place FCM at the forefront of WM diagnosis.

Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia.

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4(+) CD56(+) lineage(neg) CD45RA(+)/RO(neg) CD11c(neg) CD116(low) CD123(+) CD34(neg) CD36(+) HLA-DR(+). Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC - lymphoid or myeloid - acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4(+) CD56(+/-) MPO(neg) cCD3(neg) cCD79a(neg) CD11c(neg) profile and then the CD123(high), BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL.

How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients?

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)-like, acute lymphoid leukemia (ALL)-like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])-like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.

5-LOX, 12-LOX and 15-LOX in immature forms of human leukemic blasts.

Several reports have demonstrated an important role of leukotriene B(4) (LTB(4)) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB(4), 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M(0-2) and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB(4) in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A(2) suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB(4), 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB(4). However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB(4), 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB(4)-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Functional platelet-activating factor receptors in immature forms of leukemic blasts.

Platelet-activating factor (PAF) is a phospholipid mediator with potent immunoregulatory activities on mature leukocytes. PAF modulates leukocyte cytosolic Ca2+ concentration ([Ca2+]i) through a Gq mediated pathway. We highlight, for the first time, Gq transcripts, PAF receptor (PAF-R) transcripts and protein in blast cells of acute myeloid (AML) and lymphoid (ALL) leukemia patients. PAF stimulated [Ca2+]i in leukemic blast cells; PAF effects being prevented by a specific PAF-R antagonist. In conclusion, functional PAF-R are present in blast cells of patients with acute leukemia; a result that could be of physiologic importance regarding the important effect of PAF on leukocytes maturation and functions.

Harmonisation of full blood count reports, recommendations of the French-speaking cellular haematology group (GFHC).

AIMS: To propose recommendations related to the presentation, content and formulation of full blood count analysis reports. METHODS: Strong professional agreement among a group of experts from the French-Speaking Cellular Haematology Group (GFHC) was obtained. RESULTS: The following two proposals emerged from the consensus: (1) stratification of comments into three parts upon the discovery of an anomaly in blood cell analysis and (2) selection and/or redefinition of the terms recommended for designating the cell types found in normal and pathological peripheral blood. CONCLUSIONS: The recommendations can help biologists who are currently undergoing the process of accreditation.

PGE(2) receptor subtype functionality on immature forms of human leukemic blasts.

The ability of prostaglandin E2 (PGE2) to regulate the immune system is well documented. PGE2 effects are mediated through interactions with four distinct membrane EP receptors (EP(1-4)). We investigated, for the first time, the functionality of EP receptors on immature forms of blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. RT-PCR experiments documented the presence of the four EP receptor subtype transcripts in leukemic blasts of AML M0, AML M1, AML M2 and ALL patients. Western blot analysis only documented the presence of the EP2 receptor. Functional assays (cAMP production, calcium flux) confirmed Western blot results, i.e., the presence of functional EP2 receptors. Results of the present study suggest that the mechanism used by PGE2 to influence blast physiology is mediated through the EP2 receptor subtype, and subsequently through a cAMP-elevating effect. Results obtained with M0-2 subtypes have to be necessarily extended to more differentiated phenotype.

Platelet-activating factor and normal or leukaemic haematopoiesis.

Platelet-activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts directly during early human haematopoiesis by affecting the growth of haematopoietic progenitors and indirectly, by modulating cytokine synthesis by bone marrow stromal cells. At this time, its role during leukaemic diseases remains speculative. The lack of membrane PAF receptor (PAF-R) on leukaemic blasts suggest that this receptor represents a marker of mature cells and its membrane induction a consequence of cell maturation. While the couple PAF/PAF-R has been largely studied using B cell lines, few results are available using B cells of patients with haematopoietic malignancies casting some doubts concerning the potential role (if any) of this molecule during leukaemic diseases.

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B-cells due to ZAP70 overexpression in patient T-cells.

Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70.

Lipid mediators and human leukemic blasts.

Some of the most potent inflammatory mediators share a lipid origin. They regulate a wide spectrum of cellular processes including cell proliferation and apoptosis. However, the precise roles and ways (if any) in which these compounds impact the growth and apoptosis of leukemic blasts remain incompletely resolved. In spite of this, significant advances have been recently made. Here we briefly review the current knowledge about the production of lipid mediators (prostaglandins, leukotrienes, platelet-activating factor) by leukemic blasts, the enzymatic activities (phospholipase A(2), cyclooxygenases, lipoxygenases) involved in their productions and their effects (through specific membrane bound receptors) on the growth, and apoptosis of leukemic blasts.

"6 markers/5 colors" extended white blood cell differential by flow cytometry.

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE+CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes.