Correction: NKp30 expression is a prognostic immune biomarker for stratification of patients with intermediate-risk acute myeloid leukemia.

[This corrects the article DOI: 10.18632/oncotarget.17747.].

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.

Prognostic value of multicenter flow cytometry harmonized assessment of minimal residual disease in acute myeloblastic leukemia.

The assessment of minimal residual disease (MRD) in acute myeloblastic leukemia is of growing interest as a prognostic marker of patients' outcome. Multiparameter flow cytometry (MFC), tracking leukemia-associated immunophenotypic patterns, has been shown in several studies to be a useful tool to investigate MRD. Here, we report a multicenter prospective study which allowed to define a harmonized analysis strategy, as well as the efficacy of MFC MRD to predict outcome. This study included 276 patients, in 10 different MFC centers, of whom 268 had at least 1 MRD check point. The combination of a CD45, CD34, and CD33 backbone, with the addition of CD117, CD13, CD7, and CD15 in 2 five-color tubes allowed to define each patient's multiparameter immunophenotypic characteristics at diagnosis, according to a Boolean combination of gates. The same individual diagnosis gating strategy was then applied at each MRD time point for each patient. MRD levels were stratified according to log by log thresholds, from 5 × 10-2 (the classical morphological threshold to define remission) down to <5 × 10-5 . MRD was found to be constantly negative (<5 × 10-5 ) for 148 patients. Survival analyses significantly associated MRD negativity with a good prognosis and any positive value with poorer outcome. All P values were <0.0001 both for disease-free and overall survival at the earliest time point (post-induction, MRD1) as well as when considering all time points together. Finally, MRD levels were independent of cytogenetics and allowed in fact to further stratify all cytogenetics risk groups. In summary, this multicenter study demonstrates that a simple combination of immunophenotypic markers successfully allows for the detection of MRD in acute myeloblastic leukemia patients, with a strong correlation to outcome.

NKp46 expression on NK cells as a prognostic and predictive biomarker for response to allo-SCT in patients with AML.

NKp46 is a major determinant of natural killer (NK) cell function and it is implicated in tumor immune surveillance in acute myeloid leukemia (AML). The purpose of this study was to investigate the prognostic significance of NKp46 expression in an independent cohort of patients with AML, and to investigate the impact of NKp46 on clinical outcome after allogeneic stem cell transplantation (allo-SCT). NKp46 expression was assessed at diagnosis on NK cells by flow cytometry (N = 180 patients). Clinical outcome was evaluated with regard to NKp46 expression. Patients with NKp46high phenotype at diagnosis had better progression-free survival (PFS) and overall survival (OS) than patients with NKp46low phenotype (74.3% vs. 46.6%, p = 0.014; 82.6% vs. 57.1%, p = 0.010, respectively). In multivariate analysis, high NKp46 was an independent factor for improved OS (HR = 0.409, p = 0.010) and PFS (HR = 0.335, p = 0.011). Subgroup analysis revealed that allo-SCT had a favorable impact on PFS in patients with NKp46high phenotype (p = 0.025). By contrast, allo-SCT did not impact PFS in patients with low NKp46 expression (p = 0.303). In conclusion, we validate the prognostic value of NKp46 expression at diagnosis in AML. However, the prognostic value of NKp46 expression is limited to patients treated with allo-SCT, thus suggesting that NKp46 status may be predictive for allo-SCT responsiveness.

Natural Killer Defective Maturation Is Associated with Adverse Clinical Outcome in Patients with Acute Myeloid Leukemia.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15, P = 0.004 and HR = 8.23, P = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.

NKp30 expression is a prognostic immune biomarker for stratification of patients with intermediate-risk acute myeloid leukemia.

Cytogenetics and European Leukemia Net (ELN) genetic classification predict patients at increased risk of relapse in acute myeloid leukemia (AML) except in the intermediate risk group for which further prognostic determinants are required. We have previously shown that Natural Killer (NK) cell defects in AML are predictors of poor overall survival (OS). This study aimins at validating NKp30, a receptor that mediates NK activation, as a prognostic biomarker for AML patients with intermediate prognosis.NKp30 expression was prospectively assessed at diagnosis on NK cells from peripheral blood by flow cytometry (N = 201 patients). Clinical outcome was evaluated with regard to NKp30 status.In patients with intermediate cytogenetic (N = 162), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.26; 95%CI = [0.14-0.50]; P < 0.0001) and relapse-free survival (RFS) (HR = 0.21; 95%CI = [0.08-0.52]; P = 0.0007). In patients with intermediate ELN (N = 116), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.33; 95%CI = [0.16-0.67]; P = 0.0019) and RFS (HR = 0.24; 95%CI = [0.08-0.67]; P = 0.0058). In multivariate analysis, high NKp30 expression independently predicted improved OS (HR = 0.56, P = 0.046) and RFS (HR = 0.37, P = 0.048). Consistently, cumulative incidence of relapse (CIR) was lower in patients with high NKp30 expression (HR = 0.37, P = 0.026).In conclusion, we propose NKp30 status as a simple and early prognostic biomarker that identifies intermediate-risk patients with poor prognosis who otherwise may not be identified with existing risk stratification systems.

Increased NK Cell Maturation in Patients with Acute Myeloid Leukemia.

Understanding immune alterations in cancer patients is a major challenge and requires precise phenotypic study of immune subsets. Improvement of knowledge regarding the biology of natural killer (NK) cells and technical advances leads to the generation of high dimensional dataset. High dimensional flow cytometry requires tools adapted to complex dataset analyses. This study presents an example of NK cell maturation analysis in Healthy Volunteers (HV) and patients with Acute Myeloid Leukemia (AML) with an automated procedure using the FLOCK algorithm. This procedure enabled to automatically identify NK cell subsets according to maturation profiles, with 2D mapping of a four-dimensional dataset. Differences were highlighted in AML patients compared to HV, with an overall increase of NK maturation. Among patients, a strong heterogeneity in NK cell maturation defined three distinct profiles. Overall, automatic gating with FLOCK algorithm is a recent procedure, which enables fast and reliable identification of cell populations from high-dimensional cytometry data. Such tools are necessary for immune subset characterization and standardization of data analyses. This tool is adapted to new immune cell subsets discovery, and may lead to a better knowledge of NK cell defects in cancer patients. Overall, 2D mapping of NK maturation profiles enabled fast and reliable identification of NK cell subsets.

Role of ASXL1 and TP53 mutations in the molecular classification and prognosis of acute myeloid leukemias with myelodysplasia-related changes.

Acute myeloid leukemias (AML) with myelodysplasia-related changes (AML-MRC) are defined by the presence of multilineage dysplasia (MLD), and/or myelodysplastic syndrome (MDS)-related cytogenetics, and/or previous MDS. The goal of this study was to identify distinct biological and prognostic subgroups based on mutations of ASXL1, RUNX1, DNMT3A, NPM1, FLT3 and TP53 in 125 AML-MRC patients according to the presence of MLD, cytogenetics and outcome. ASXL1 mutations (n=26, 21%) were associated with a higher proportion of marrow dysgranulopoiesis (mutant vs. wild-type: 75% vs. 55%, p=0.030) and were mostly found in intermediate cytogenetic AML (23/26) in which they predicted inferior 2-year overall survival (OS, mutant vs. wild-type: 14% vs. 37%, p=0.030). TP53 mutations (n=28, 22%) were mostly found in complex karyotype AML (26/28) and predicted poor outcome within unfavorable cytogenetic risk AML (mutant vs. wild-type: 9% vs. 40%, p=0.040). In multivariate analysis, the presence of either ASXL1 or TP53 mutation was the only independent factor associated with shorter OS (HR, 95%CI: 2.53, 1.40-4.60, p=0.002) while MLD, MDS-related cytogenetics and previous MDS history did not influence OS. We conclude that ASXL1 and TP53 mutations identify two molecular subgroups among AML-MRCs, with specific poor prognosis. This could be useful for future diagnostic and prognostic classifications.

Array comparative genomic hybridization and sequencing of 23 genes in 80 patients with myelofibrosis at chronic or acute phase.

Myelofibrosis is a myeloproliferative neoplasm that occurs de novo (primary myelofibrosis) or results from the progression of polycythemia vera or essential thrombocytemia (hereafter designated as secondary myelofibrosis or post-polycythemia vera/ essential thrombocythemia myelofibrosis). To progress in the understanding of myelofibrosis and to find molecular prognostic markers we studied 104 samples of primary and secondary myelofibrosis at chronic (n=68) and acute phases (n=12) from 80 patients, by using array-comparative genomic hybridization and sequencing of 23 genes (ASXL1, BMI1, CBL, DNMT3A, EZH2, IDH1/2, JAK2, K/NRAS, LNK, MPL, NF1, PPP1R16B, PTPN11, RCOR1, SF3B1, SOCS2, SRSF2, SUZ12, TET2, TP53, TRPS1). We found copy number aberrations in 54% of samples, often involving genes with a known or potential role in leukemogenesis. We show that cases carrying a del(20q), del(17) or del(12p) evolve in acute myeloid leukemia (P=0.03). We found that 88% of the cases were mutated, mainly in signaling pathway (JAK2 69%, NF1 6%) and epigenetic genes (ASXL1 26%, TET2 14%, EZH2 8%). Overall survival was poor in patients with more than one mutation (P=0.001) and in patients with JAK2/ASXL1 mutations (P=0.02). Our study highlights the heterogeneity of myelofibrosis, and points to several interesting copy number aberrations and genes with diagnostic and prognostic impact.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts.

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum.

Combination of cytarabine and topotecan in patients treated for acute myeloid leukemia with persistent disease after frontline induction.

After a first course of induction chemotherapy, 30-40% of patients with acute myeloid leukemia (AML) do not achieve a complete response (CR). A second course of an anthracycline and intermediate-dose cytarabine (IDAC) allows a significant number of patients with persistent AML at day 14 to finally achieve a CR. We hypothesized that use of a topotecan and cytarabine combination in this setting might improve tolerance and efficacy. Cytarabine (1000 mg/m(2)/12 h days 1-4) was combined with topotecan (TA, 1.25 mg/m(2)/day by continuous intravenous infusion [CIV] days 1-4) in 31 consecutive patients with ≥ 5% marrow blasts by day 14 of induction. The median follow-up was 36 months. The CR rate was 81%, and the 2-year probability of overall survival and cumulative incidence of relapse were 66% and 38%, respectively. No unexpected toxicity was observed. Comparison with historical controls treated with the combination of a similar schedule of cytarabine and an anthracycline showed a better CR rate (p = 0.054), overall survival (p = 0.03) and cumulative incidence of relapse (p = 0.03). These results were confirmed in a multivariate analysis model. This work shows that the substitution of an anthracycline by topotecan is feasible and associated with significant efficacy for patients with AML with persistent leukemia at day 14 after standard-dose anthracycline induction.

Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms.

Since the discovery of the JAK2V617F tyrosine kinase-activating mutation several genes have been found mutated in nonchronic myeloid leukemia (CML) myeloproliferative neoplasms (MPNs), which mainly comprise three subtypes of "classic" MPNs; polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We searched for mutations in ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 genes in 149 non-CML MPNs, including 127 "classic" MPNs cases. JAK2 was mutated in 100% PV, 66% ET and 68% MF. We found a high incidence of ASXL1 mutation in MF patients (20%) and a low incidence in PV (7%) and ET (4%) patients. Mutations in the other genes were rare (CBL, DNMT3A, IDH2, MPL, SF3B1, SUZ12, NF1) or absent (IDH1).

Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

Improved outcome of patients with low- and intermediate-risk cytogenetics acute myeloid leukemia (AML) in first relapse with gemtuzumab and cytarabine versus cytarabine: results of a retrospective comparative study.

BACKGROUND: Acute myeloid leukemia (AML) in first relapse is associated with a poor outcome even when treated with intermediate- to high-dose cytarabine (IHDAraC). Gemtuzumab ozogamycin (GO) used as a single agent has clinical activity in relapsed and refractory AML. Various combination regimens of GO have been developed, but few data are available regarding their efficacy compared with IHDAraC-based regimens. METHODS: The authors performed a retrospective analysis of response and survival in 90 AML patients in first relapse treated with either IHDAraC (n = 56) or IHDAraC + GO (n = 34). Patient characteristics of the two groups were comparable. RESULTS: Median follow-up was 37 months. Compared with IHDAraC, IHDAraC + GO induction was associated with a better response rate (68% vs 45%, P = .04), a better overall survival (median, 35 months vs 6 months, P = .001), and a better event-free survival (24 months vs 6 months, P = .002). This effect was limited to patients with low-risk and intermediate-risk cytogenetics. In multivariate analysis, age, cytogenetic risk, first complete remission duration, and the use of IHDAraC + GO were independently associated with better results. CONCLUSIONS: This study showed that the addition of GO to IHDAraC is associated with a better efficacy for patients in first relapse of AML with low- or intermediate-risk cytogenetics. Prospective controlled studies of GO in this population are warranted. Patients with high-risk cytogenetics should be offered investigational new drugs.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia.

Chronic myelomonocytic leukaemia (CMML) is a haematological disease currently classified in the category of myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) because of its dual clinical and biological presentation. The molecular biology of CMML is poorly characterized. We studied a series of 53 CMML samples including 31 cases of myeloproliferative form (MP-CMML) and 22 cases of myelodysplastic forms (MD-CMML) using array-comparative genomic hybridisation (aCGH) and sequencing of 13 candidate genes including ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2 and WT1. Mutations in ASXL1 and in the genes associated with proliferation (CBL, FLT3, PTPN11, NRAS) were mainly found in MP-CMML cases. Mutations of ASXL1 correlated with an evolution toward an acutely transformed state: all CMMLs that progressed to acute phase were mutated and none of the unmutated patients had evolved to acute leukaemia. The overall survival of ASXL1 mutated patients was lower than that of unmutated patients.

The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome.

The pseudo tyrosine kinase receptor 7 (PTK7) is an orphan tyrosine kinase receptor assigned to the planar cell polarity pathway. It plays a major role during embryogenesis and epithelial tissue organization. Here we found that PTK7 is also expressed in normal myeloid progenitors and CD34(+) CD38(-) bone marrow cells in humans. We performed an immunophenotyping screen on more than 300 patients treated for hematologic malignancies. We demonstrated that PTK7 is expressed in acute myeloid leukemia (AML) and is mostly assigned to granulocytic lineage differentiation. Patients with PTK7-positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced leukemia-free survival in a multivariate analysis model. In vitro, expression of PTK7 in cultured leukemia cells promotes cell migration, cell survival, and resistance to anthracycline-induced apoptosis. The intracellular region of PTK7 is required for these effects. Furthermore, we efficiently sensitized primary AML blasts to anthracycline-mediated cell death using a recombinant soluble PTK7-Fc protein. We conclude that PTK7 is a planar cell polarity component expressed in the myeloid progenitor compartment that conveys promigratory and antiapoptotic signals into the cell and that represents an independent prognosis factor of survival in patients treated with induction chemotherapy.

Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL.

BACKGROUND: The development of multiparameter flow cytometry (FCM) and increasingly sophisticated analysis software has considerably improved the exploration of hematological disorders. These tools have been widely applied in leukaemias, lymphomas, and myelodysplasias, yet with very heterogeneous approaches. Consequently, there is no extensive reference document reporting on the characteristics of normal human bone marrow (BM) in multiparameter FCM. Here, we report a reference analysis procedure using relevant antibody combinations in normal human BM. METHODS: A first panel of 23 antibodies, constructed after literature review, was tested in four-color combinations (including CD45 in each) on 30 samples of BM. After evaluation of the data, a second set of 22 antibodies was further applied to another 35 BM samples. All list-modes from the 65 bone marrow samples were reviewed collectively. A systematised protocol for data analysis was established including biparametric representations and color codes for the three major lineages and undifferentiated cells. RESULTS: This strategy has allowed to obtain a reference atlas of relevant patterns of differentiation antigens expression in normal human BM that is available within the European LeukemiaNet. This manuscript describes how this atlas was constructed. CONCLUSIONS: Both the strategy and atlas could prove very useful as a reference of normality, for the determination of leukemia-associated immunophenotypic patterns, analysis of myelodysplasia and, ultimately, investigation of minimal residual disease in the BM.