Significance of Measurable Residual Disease in Adult Philadelphia Chromosome-Positive ALL: A GRAAPH-2014 Study.

PURPOSE: BCR::ABL1 quantification is widely regarded as the standard for monitoring measurable residual disease (MRD) in Philadelphia chromosome-positive (Ph+) ALL. However, recent evidence of BCR::ABL1 multilineage involvement questions the significance of BCR::ABL1 MRD. We aimed to define the prognostic role of MRD as assessed by BCR::ABL1 or lymphoid-specific immunoglobulin/T-cell receptor (IG/TR) gene markers. PATIENTS AND METHODS: We conducted BCR::ABL1 and IG/TR quantification after each treatment cycle in 264 patients treated in the GRAAPH-2014 trial, which used four cycles of reduced-intensity chemotherapy with nilotinib, followed by hematopoietic stem-cell transplantation (HSCT). RESULTS: Comparing BCR::ABL1 and IG/TR MRD revealed residual BCR::ABL1-positive non-ALL cells in 98 (43%) of 228 patients, defining multilineage Ph+ ALL. Despite poorer BCR::ABL1 responses, patients with multilineage Ph+ ALL had similar disease-free survival (DFS; hazard ratio [HR], 0.83 [95% CI, 0.49 to 1.41]; P = .50). Although BCR::ABL1 response failed to predict outcomes, IG/TR positivity (≥0.01%) was strongly associated with lower DFS (after cycle 2, HR, 2.49 [95% CI, 1.40 to 4.40]; P = .002; after cycle 4, HR, 4.13 [95% CI, 1.82 to 9.38]; P = .001). In multivariable analysis, both IG/TR positivity after cycle 2 and initial WBC count ≥30 × 109/L predicted poorer DFS, enabling to define a high-risk group having a 4-year DFS of 56.5% compared with 87.6% (HR, 3.72 [95% CI, 1.93 to 7.15]; P < .001). Moreover, allogeneic HSCT significantly improved DFS in the high-risk group (HR, 0.33 [95% CI, 0.18 to 0.60]; P < .001), whereas the standard-risk group had favorable outcomes regardless of allogeneic HSCT. CONCLUSION: Our findings challenge the significance of BCR::ABL1 monitoring in adult Ph+ ALL and demonstrate the prognostic role of IG/TR MRD. This study provides a framework for using MRD to guide treatment strategies in adults with Ph+ ALL.

Single-center, observational study of AML/MDS-EB with IDH1/2 mutations: genetic profile, immunophenotypes, mutational kinetics and outcomes.

OBJECTIVE: IDH1/2 mutations, intervening in epigenetic procedures, are frequently encountered in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Knowledge of the genetics, immunophenotypes, and mutational kinetics of IDH1/2-mutated AML can contribute to the understanding of AML clonal architecture and inform therapeutics and monitoring. METHODS: We retrospectively analyzed 50 IDH1/2-mutated AML/MDS-EB cases of our institution, to identify recurrent co-mutations, immunophenotypes, patterns of co-variance of IDH1/2 allele burdens with those of recurrent co-mutations, frequency of persistent IDH1/2 mutation as clonal hematopoiesis of indeterminate potential (CHIP) in remission and response to hypomethylating agents. RESULTS: Most frequently co-mutated genes were DNMT3A, SRSF2 and NPM1. Most cases with co-existent IDH1/2 and NPM1 mutations (11/13) showed an 'APL-like' immunophenotype (CD34-HLADR-). Allele burdens of mutated IDH1/2 were identical to mutated SRSF2 allele burdens at diagnosis and remission, but not always to mutated NPM1 allele burden in remission. We show persistence of significant mutIDH1/2 allele burden in approximately one-fourth of patients with deep remissions. IDH1/2 mutations were significantly more frequent among responders to first-line HMA-based regimens than among non-responders, in patients treated for myeloid neoplasms with excess blasts. CONCLUSIONS: IDH1/2 mutations are most frequently accompanied by DNMT3A, SRSF2 and NPM1 mutations. NPM1-IDH1/2 mutated AML has a mature phenotype possibly amenable to differentiation therapies. IDH1/2 and SRSF2 mutations probably arise at the same developmental stage of the disease, as their allele burdens covariate. IDH1/2 mutation represents CHIP in a substantial proportion of cases and is therefore no reliable residual disease marker. The preferential presence of IDH1/2 mutations among HMA-responders could inform therapeutic decisions if confirmed in larger series.

Daratumumab and venetoclax in combination with chemotherapy provide sustained molecular remission in relapsed/refractory CD19, CD20, and CD22 negative acute B lymphoblastic leukemia with KMT2A-AFF1 transcript.

Relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) has a very poor prognosis with a median overall survival of four to nine months. Achieving a complete molecular response is most often required to obtain a sustained leukemia-free survival after allogeneic hematopoietic stem cell transplantation. Immunotherapies targeting CD19, CD20, or CD22 are very efficient in achieving this goal. However, in the absence of the expression of these immunotherapeutic targets by lymphoblasts, treatment options are extremely scarce. We report the successful treatment of a 26-year-old man who suffered R/R, CD19, CD20, and CD22 negative B-ALL targeting Bcl-2 and CD38 by combining venetoclax and daratumumab with chemotherapy.

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.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry.

BACKGROUND: Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8-color tube for providing equivalent information, using flow cytometry. METHODS: 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies-CD3-FITC/CD10-PE/CD38-PerCP-Cy5.5/CD19-PECy7/CD36-APC/CD16-APC-H7/CD34-BV421/CD45-V500-was tested to identify the main cell subsets at different stages of maturation using a FACSCanto-II analyzer. RESULTS: We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B-lymphoid cells from hematogones to plasma-cells (5 subsets), T- and NK-cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible. CONCLUSION: A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.

An miRNA-DNMT1 Axis Is Involved in Azacitidine Resistance and Predicts Survival in Higher-Risk Myelodysplastic Syndrome and Low Blast Count Acute Myeloid Leukemia.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3' UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance.Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025-34. ©2016 AACR.

CD180 expression in B-cell lymphomas: A multicenter GEIL study.

CD180, a related member of the Toll-like receptor family, is lost or underexpressed at the plasma membrane in circulating cells of various B-cell lymphomas except marginal zone lymphomas (MZL). In order to confirm its clinical relevance in routine analysis, we evaluated prospectively the expression of CD180 in 236 patients from 5 French University Hospital laboratories on behalf of the GEIL. Highly comparable results were obtained in all centers using the EuroFlow standardization protocol. We observed that CD180 median fluorescence (MdFI) was significantly higher in MZL and hairy cell leukaemia (HCL) compared to all other B-cell proliferations (P < 0.05). CD180 intensity could distinguish lymphomas with numerous villous lymphocytes from other MZL. ROC curve analysis identified a CD180 MdFI threshold for which the diagnosis of MZL could be assessed with 77% sensitivity and 92% specificity. This study showed that CD180 can be considered as a single positive robust marker of MZL and should be therefore included in flow cytometry panels for the diagnosis of mature B-cell neoplasms. Harmonization process is of great interest in order to evaluate new markers in multicentric studies and to set up decisional thresholds. © 2015 International Clinical Cytometry Society.

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML).

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

In vivo and in vitro sensitivity of blastic plasmacytoid dendritic cell neoplasm to SL-401, an interleukin-3 receptor targeted biologic agent.

Blastic plasmacytoid dendritic cell neoplasm is an aggressive malignancy derived from plasmacytoid dendritic cells. There is currently no accepted standard of care for treating this neoplasm, and therapeutic strategies have never been prospectively evaluated. Since blastic plasmacytoid dendritic cell neoplasm cells express high levels of interleukin-3 receptor α chain (IL3-Rα or CD123), antitumor effects of the interleukin-3 receptor-targeted drug SL-401 against blastic plasmacytoid dendritic cell neoplasm were evaluated in vitro and in vivo. The cytotoxicity of SL-401 was assessed in patient-derived blastic plasmacytoid dendritic cell neoplasm cell lines (CAL-1 and GEN2.2) and in primary blastic plasmacytoid dendritic cell neoplasm cells isolated from 12 patients using flow cytometry and an in vitro cytotoxicity assay. The cytotoxic effects of SL-401 were compared to those of several relevant cytotoxic agents. SL-401 exhibited a robust cytotoxicity against blastic plasmacytoid dendritic cell neoplasm cells in a dose-dependent manner. Additionally, the cytotoxic effects of SL-401 were observed at substantially lower concentrations than those achieved in clinical trials to date. Survival of mice inoculated with a blastic plasmacytoid dendritic cell neoplasm cell line and treated with a single cycle of SL-401 was significantly longer than that of untreated controls (median survival, 58 versus 17 days, P<0.001). These findings indicate that blastic plasmacytoid dendritic cell neoplasm cells are highly sensitive to SL-401, and support further evaluation of SL-401 in patients suffering from blastic plasmacytoid dendritic cell neoplasm.

How mRNA is misspliced in acute myelogenous leukemia (AML)?

Approximately one-third of expressed genes are misspliced in AML, opening the possibility that additional factors than splicing factor mutations might cause RNA missplicing in these diseases. AML cells harbor a constellation of epigenetic modifications and regularly express large amounts of WT1 transcripts. Histone acetylation/methylation and DNA CpG methylation favor either exon skipping or inclusion, mainly through interfering with RNA Pol II-mediated elongation. This can result either from the binding of various factors on Pol II or alternatively from the recruitment of DNA binding factors that create roadblocks to Pol II-induced elongation. WT1 exhibits pleiotropic effects on mRNA splicing, which mainly result from the binding properties of WT1 via its zinc fingers domains to DNA, RNA, and proteins. Through the repression of the kinase SRPK1, WT1 modifies the splicing of VEGF, which plays important roles in hematopoiesis and angiogenesis. At the protein level, WT1 interacts with the splicing factors U2AF2, WTAP, and RPM4. Therefore, AML cells appear to have acquired numerous properties known to interfere with mRNA splicing. The challenge is now to elucidate these links in order to trigger mRNA splicing at the therapeutic level.

[CXCR4: a new therapeutic target of the leukaemic cell? Role of the SDF-1/CXCR4 axis in acute myeloid leukaemia]. / CXCR4 : nouvelle cible thérapeutique de la cellule leucémique? Rôle du couple SDF-1/CXCR4 dans la leucémie aiguë myéloïde.

CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. Recent flow cytometry analyses reported that CXCR4 expression levels have a major prognostic impact in acute myeloid leukaemia (AML). CXCR4 expression is associated with poor prognosis and can be useful to stratify patients, according to their phenotype, in order to establish risk-adapted strategies. Newly diagnosed AML are now routinely stratified according to molecular markers which guide prognosis and treatment. Many leukaemia are composed of multiples subclones with differential susceptibility to treatment and specific targeted therapies are missing. Despite therapeutic improvements for the treatment of AML, long term survival remains poor. Targeting CXCR4 is a novel promising approach of therapy. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies. This review summarises our current knowledge regarding the key role of CXCR4 in AML and discusses how targeting this pathway could provide an interesting approach to eradicate the LSC.

Comparable flow cytometry data can be obtained with two types of instruments, Canto II, and Navios. A GEIL study.

Flow cytometry (FC) instruments settings classically rely on local establishment of photomultipliers (PMT) voltages adapted to the measurements expected to be performed. In the era of multiparameter FC (MFC), it appears more and more desirable that comparable patterns of fluorescence are obtained in different settings. This relies on a harmonization of settings between instruments. Although this has been shown to be feasible within a given brand of flow cytometers, little information is available about broader comparisons in a given center or in a multicenter fashion. Here, we report a two-phase series of experiments first performed between a Canto II (BD Biosciences) and a Navios (Beckman Coulter) instruments in the same center. PMT values adjusted on the reference instrument (RI) Canto II were used to establish target values for PMT settings on the paired Navios practice instrument (PI). This allowed to show the good correlation of all but peaks 1 and 2 of Rainbow(®) beads between RI and PI. Using 4- or 8-color stained leukocytes, the similitude of the settings was further confirmed. A complex set of matrices was then established between five centers all equipped with both instruments. Using Bland & Altman difference comparisons for median fluorescence values, it was shown that using either Rainbow beads or CD16 stained polymorphonuclears to set-up target values on the RI CantoII, highly superimposable results could be obtained on all 9 PI. The latter were obtained using Rainbow beads or Compbeads(®) for comparisons. In summary, this two-phase study demonstrates the feasibility of different methods allowing for a robust harmonization of settings for MFC.

Heat Shock Protein 90 is overexpressed in high-risk myelodysplastic syndromes and associated with higher expression and activation of Focal Adhesion Kinase.

Myelodysplastic syndromes are characterized by a high risk of evolution into acute myeloid leukaemia which can involve activation of signalling pathways. As the chaperone heat shock protein 90 (HSP90) has a key role in signal transduction, we investigated its role in the pathogenesis and evolution of myelodysplastic syndromes. Expressions of HSP90 and signalling proteins clients (phosphorylated-AKT (pAKT), Focal Adhesion Kinase (FAK) and phosphorylated-FAK (pFAK)), were assessed in bone marrow mononuclear and CD34-positive (CD34+) cells from 177 patients with myelodysplasia. Effects of HSP90 inhibition were also evaluated in 39 samples. The levels of all proteins studied were significantly higher in patients with high grade disease, than those with low grade myelodysplastic syndrome or chronic myelomonocytic leukaemia. High levels of HSP90, FAK, pFAK and pAKT were associated with shorter survival and increased risk of progression into acute leukaemia. A down regulation of pFAK and pAKT and increased apoptosis was observed in mononuclear and CD34+ cells after 12 hours of incubation with 17-AAG. In conclusion, our data suggest the implication of HSP90 and FAK and AKT activation in the pathogenesis of myelodysplastic syndromes with excess of blasts and evolution to leukaemia. Moreover this signalling network could be a therapeutic target through HSP90 inhibition.

HSP90 inhibition results in apoptosis of Philadelphia acute lymphoblastic leukaemia cells: an attractive prospect of new targeted agents.

PURPOSE: HSP90 targeting is a promising therapeutic approach in cancer. 17-AAG is an HSP90 inhibitor with completed Phase I trials in patients with advanced cancer and recently published Phase II trials. The aim of this work was to study the expression of HSP 90 and apoptotic proteins, the effects in culture of 17-AAG on cell survival and apoptosis and to compare Philadelphia-positive (Ph+) ALL to common B cell ALL, in ALL cell lines and in patients' cells collected at ALL diagnosis. METHODS: We analysed 2 ALL cell lines and 63 leukaemic samples from patients treated in our institution (44 common B cell ALL and 19 Ph+ ALL). We performed flow cytometry analysis of bone marrow aspiration and cell lines with a combination of anti-HSP90, Bax, Bcl-2 and Bcl-xl antibodies. Apoptosis after cell culture (in presence or not of 17-AAG) was assessed using Annexin V and activated caspase-3 staining. RESULTS: Ph+ ALL cells appeared to be more sensitive to 17-AAG cytotoxicity with a 100 % mortality rate after exposure to 10 µM for 24 h (vs. 62 % for B-common ALL). A high percentage of HSP90-positive cells (in Ph+ ALL samples) was associated with high sensitivity to 17-AAG. 17-AAG induced apoptosis in a dose-dependent manner and was associated with down-regulation of Bcl-2 and Bcl-Xl expression and up-regulation of Bax expression. CONCLUSION: Considering that Bcr-Abl constitutes HSP 90 substrates, HSP 90 inhibition could be of particular interest for Ph+ ALL disease, even in patients harbouring resistance to tyrosine kinase inhibitor therapy.