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Purpose: Acute myeloid leukemias (AML) are clonal diseases that develop from leukemic stem cells (LSC) that carry an independent prognostic impact on the initial response to induction chemotherapy, demonstrating the clinical relevance of LSC abundance in AML. In 2018, the European LeukemiaNet published recommendations for the detection of measurable residual disease (Bulk MRD) and suggested the exploration of LSC MRD and the use of multiparametric displays. Experimental Design: We evaluated the performance of unsupervised clustering for the post-induction assessment of bulk and LSC MRD in 155 patients with AML who received intensive conventional chemotherapy treatment. Results: The median overall survival (OS) for Bulk+ MRD patients was 16.7 months and was not reached for negative patients (HR, 3.82; P < 0.0001). The median OS of LSC+ MRD patients was 25.0 months and not reached for negative patients (HR, 2.84; P = 0.001). Interestingly, 1-year (y) and 3-y OS were 60% and 39% in Bulk+, 91% and 52% in Bulk-LSC+ and 92% and 88% in Bulk-LSC−. Conclusions: In this study, we confirm the prognostic impact of post-induction multiparametric flow cytometry Bulk MRD in patients with AML. Focusing on LSCs, we identified a group of patients with negative Bulk MRD but positive LSC MRD (25.8% of our cohort) with an intermediate prognosis, demonstrating the interest of MRD analysis focusing on leukemic chemoresistant subpopulations.

Sezary syndrome (SS) is a rare leukemic form of cutaneous T-cell lymphoma. Diagnosis mainly depends on flow cytometry, but results are not specific enough to be unequivocal. The difficulty in defining a single marker that could characterize Sezary cells may be the consequence of different pathological subtypes. In this study, we used multivariate flow cytometry analyses. We chose to investigate the expression of classical CD3, CD4, CD7, and CD26 and the new association of 2 markers CD158k and PD-1. We performed lymphocyte computational phenotypic analyses during diagnosis and follow-up of patients with SS to define new SS classes and improve the sensitivity of the diagnosis and the follow-up flow cytometry method. Three classes of SS, defined by different immunophenotypic profiles, CD158k+ SS, CD158k−PD-1+ SS, CD158k and PD-1 double-negative SS, showed different CD8+ and B-cell environments. Such a study could help to diagnose and define biological markers of susceptibility/resistance to treatment, including immunotherapy.

Classifications of acute myeloid leukemia (AML) patients rely on morphologic, cytogenetic, and molecular features. Here we have established a novel flow cytometry-based immunophenotypic stratification showing that AML blasts are blocked at specific stages of differentiation where features of normal myelopoiesis are preserved. Six stages of leukemia differentiation-arrest categories based on CD34, CD117, CD13, CD33, MPO, and HLA-DR expression were identified in two independent cohorts of 2087 and 1209 AML patients. Hematopoietic stem cell/multipotent progenitor-like AMLs display low proliferation rate, inv(3) or RUNX1 mutations, and high leukemic stem cell frequency as well as poor outcome, whereas granulocyte–monocyte progenitor-like AMLs have CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations, lower leukemic stem cell frequency, higher chemosensitivity, and better outcome. NPM1 mutations correlate with most mature stages of leukemia arrest together with TET2 or IDH mutations in granulocyte progenitors-like AML or with DNMT3A mutations in monocyte progenitors-like AML. Overall, we demonstrate that AML is arrested at specific stages of myeloid differentiation (SLA classification) that significantly correlate with AML genetic lesions, clinical presentation, stem cell properties, chemosensitivity, response to therapy, and outcome.

Postremission treatment is crucial to prevent relapse in acute myeloid leukemia (AML). High-dose cytarabine delivered every 12 hours on days 1, 3, and 5 (HDAC-135) is the standard of care for younger adult patients with AML. Although this standard has been unsuccessfully challenged by other treatment regimens, including multiagent chemotherapy, the timing of HDAC administration has attracted little attention. Here, we retrospectively compared the safety, efficacy, and health care resource consumption associated with HDAC-135 and another standard, condensed HDAC-123 regimen, as consolidation treatment in younger AML patients in first complete response. This study included 221 patients (median age, 46.6 years; range, 18-60 years). HDAC-123 and HDAC-135 were used in 92 and 129 patients, respectively. Both regimens were associated with similar rates of relapse-free survival, cumulative incidence of relapse, nonrelapse mortality, and overall survival, including in core binding factor AML subgroup in which levels of minimal residual disease reduction were similar in both schedules. Hematological recovery times regarding neutrophils and platelets were significantly shorter in patients receiving HDAC-123, with an average difference of 3 to 4 days for each consolidation cycle. The total duration of hospitalization for the whole postremission program was shorter with HDAC-123 (32 days; interquartile ratio [IQR], 22.0,36.5) compared with HDAC-135 (41 days; IQR, 30.5, 50.0) (P < .0001). In conclusion, the condensed HDAC-123 regimen induced faster hematological recovery and therefore significantly reduced the length of hospital stay without affecting treatment response or outcome in younger AML patients.

Erythroderma is challenging to diagnose. The aim of this single-centre retrospective study was to identify factors that can be used to improve the diagnosis of erythroderma. Among 91 patients with erythroderma, 21 were diagnosed with eczema, 17 with psoriasis, 20 with drug-induced erythroderma, 13 with erythrodermic mycosis fungoides and 20 with Sézary syndrome. Nail alterations, ear involvement, and severe scaling were significantly associated with psoriasis (p = 0.044). Fever and hypereosinophilia were associated with drug-induced erythroderma. Expression of programmed cell death protein 1 was observed in all skin biopsies. However, with Sézary syndrome, programmed cell death protein 1 expression was significantly higher than with other aetiologies. A programmed cell death protein 1 hormone receptor score (H-score) >50 was associated with Sézary syndrome (p

The Sezary syndrome has been defined by a triad combining erythrodermia, generalized lymphadenopathy, and the presence of circulating Sezary cells > 1 × 109/L characterized by a CD4+/CD8- phenotype with loss of one or more T antigens (mainly CD7 and/or CD26). We retrospectively reviewed the immunophenotypic profiles of 10 SS patients followed in our institution (University Hospital at Nancy, France). The application of the WHO criteria resulted in a diagnostic confirmation for 9 out of 10 cases. Since 2008, new diagnostic and staging criteria have been proposed, including the CD158k/KIR3DL2 receptor detection. The application of these new criteria to our cohort led us to notice a phenotypic heterogeneity of our cases but allowed to achieve a relevant diagnosis of Sezary syndrome in all cases, especially for patients with lymphopenia. The use of such a panel of monoclonal antibodies also optimized the follow-up of the patients.

TO THE EDITOR : Patients with chronic lymphocytic leukemia (CLL) exhibit a variety of skin lesions including mostly non-specific cutaneous manifestations (such as cutaneous infections or exaggerated reactions to insect bite) and secondary cutaneous malignancies, as patients are at high risk of developing basal cell carcinoma, squamous cell carcinoma, melanoma, and Merkel cell carcinoma. Specific cutaneous infiltration by neoplastic B lymphocytes with clinically identifiable skin lesions, also called leukemia cutis (LC), is more uncommon and has seldom been reported in chronic lymphocytic leukemia. [...]

Isolated trisomy 8 (+8) is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDS), but its characteristics are poorly reported. We performed a retrospective study of 138 MDS patients with isolated +8, classified or reclassified as MDS (excluding MDS/myeloproliferative neoplasm). Myeloproliferative (MP) features were defined by the repeated presence of one of the following: white blood cell count >10 × 109 /l, myelemia (presence of circulating immature granulocytes with a predominance of more mature forms) >2%, palpable splenomegaly. Fifty-four patients (39·1%) had MP features: 28 at diagnosis, 26 were acquired during evolution. MP forms had more EZH2 (33·3% vs. 12·0% in non-MP, P = 0·047), ASXL1 (66·7% vs. 42·3%, P = 0·048) and STAG2 mutations (77·8% vs. 21·7%, P = 0·006). Median event-free survival (EFS) and overall survival (OS) were 25 and 27 months for patients with MP features at diagnosis, versus 28 (P = 0·15) and 39 months (P = 0·085) for those without MP features, respectively. Among the 57 patients who received hypomethylating agent (HMA), OS was lower in MP cases (13 months vs. 23 months in non-MP cases, P = 0.02). In conclusion, MP features are frequent in MDS with isolated +8. MP forms had more EZH2, ASXL1 and STAG2 mutations, responded poorly to HMA, and tended to have poorer survival than non-MP forms.

Multiparameter flow cytometry (MFC) is a powerful and versatile tool to accurately analyze cell subsets, notably to explore normal and pathological hematopoiesis. Yet, mostly supervised subjective strategies are used to identify cell subsets in this complex tissue. In the past few years, the implementation of mass cytometry and the big data generated have led to a blossoming of new software solutions. Their application to classical MFC in hematology is however still seldom reported. Here, we show how one of these new tools, the FlowSOM R solution, can be applied, together with the Kaluza® software, to a new delineation of hematopoietic subsets in normal human bone marrow (BM). We thus combined the unsupervised discrimination of cell subsets provided by FlowSOM and their expert-driven node-by-node assignment to known or new hematopoietic subsets. We also show how this new tool could modify the MFC exploration of hematological malignancies both at diagnosis (Dg) and follow-up (FU). This can be achieved by direct comparison of merged listmodes of reference normal BM, Dg, and FU samples of a representative acute myeloblastic case tested with the same immunophenotyping panel. This provides an immediate unsupervised evaluation of minimal residual disease. © 2019 International Society for Advancement of Cytometry.

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.

The HematoFlow™ system is used in the hematology laboratory of the University Hospital of Bordeaux since July, 2011. The HematoFlow™ solution is the combination of a sample preparator (FP1000) and a 5 color flow cytometer (FC500) linked by a middleware (Remisol™). This system is used in second line when flags are activated by the hematology instrument and/or if the sample comes from the OncoHematology Department. Improvements in hematology disease diagnosis and follow-up were possible using this system. The laboratory has now entered in an accreditation procedure and needs to check this method in compliance with the COFRAC requirements.

Background: The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. Methods: We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. Results: We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. Conclusion: More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction. q 2006 International

The complete blood cell count and white blood cell differential are the first step in the biological diagnosis of hematological diseases. Both are currently performed by automated instruments which control data and produce alerts. If such flags are activated, the automated differential cannot be validated and the operator must activate a visual blood smear review. Microscopic examination is still today the reference method despite its lack of sensitivity and reproducibility. The HematoFlow™ (Beckman Coulter) system is the first flow cytometry commercialized method designed for the routine differential. Using six markers in five colors and an automated gating strategy, it provides differentials proven to be reliable for 17 leukocyte subpopulations detection. Relying first on a retrospective analysis of 6,462 blood samples processed by HematoFlow™, thresholds were determined to detect the presence of immature granulocytes and/or blast cells. All possible gating strategy misclassifications of leukocyte subpopulations were then summarized in a systematic nomenclature leading to the development of an original flag system based on the detection of aberrant localization of cell events in specific new bivariate histograms. Ultimately, more than 50% of the results could be automatically validated using the HematoFlow™ system, without any false negative, thereby dramatically contributing to an important decrease of technicians' workload. Moreover a noticeable help was given for smear review interpretation and new immunological flags led to the confirmation of blood disease after classical immunophenotyping. These results were confirmed in a second prospective study including 15,335 cases, where more than 50% of the results were automatically validated by this new flag system. MFC stands as being more and more essential for analyzing differentials in routine and this new flag system could greatly improve its implementation.

Cytogenetic abnormalities in acute myelocytic leukemia (AML) have been identified as one of the most important prognostic factors. The t(15;17) is associated with high rates of complete remission and event-free survival. Secondary chromosomal changes are also present in approximately one third of patients with newly diagnosed acute promyelocytic leukemia (APL). Indeed, the gain of whole chromosome 8 may be involved in the course of APL under C-MYC gene dosage effect theory. Complete or partial loss of the long arm of chromosome 7 region has been recognized in preleukemic myelodysplasia or unfavorable AML. We report here two original APL cases in which a new additional chromosomal abnormality, der(7)t(7;8)(q34;q21), is associated with the t(15;17)(q22;q21). This recurrent abnormality results in a partial loss of 7q associated with a partial 8q trisomy. As the 7q and 8q breakpoints were similar in both cases, the involvement of these critical regions in the pathogenesis and outcome of APL disease has to be determined.

A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MITO and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (10(-7) M) induced apoptosis at the G1-S boundary. Low concentrations of anthracyclines (40 nM DNR and20nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in G1 and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (IC50) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with G1 DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The IC50 of MITO was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the G1-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in vivo pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs.