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Abstract: We report here the results of the GRAAPH-2003 trial with long-term follow-up in 45 patients with de novo Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Imatinib-based strategy improved the 4-year overall survival (OS) up to 52% versus 20% in the pre-imatinib LALA-94 trial (P = .0001). Despite the selection in patients who actually underwent transplantation, these results suggest that allogeneic or autologous stem cell transplants (SCTs) still have a place in overcoming the poor prognosis of Ph+ ALL in the era of imatinib therapy. OS was 50% after allogeneic SCT (24 patients), 33% in patients without a transplantation (9 patients), and 80% after autologous SCT (10 patients without allogeneic donor or >55 years, including 7 patients in complete molecular response). [Copyright &y& Elsevier]

Abstract: Background: The median survival of patients with chronic myelogenous leukemia in myeloid blast crisis (MBC-CML) is poor even for patients treated with tyrosine kinase inhibitors (TKIs). Design and methods: We conducted a dose-escalating study of daunorubicin combined to fixed doses of IM (imatinib mesylate, 600mg/d) and cytarabine (200mg/d for 7 days), followed by hematopoietic stem cell transplantation or maintenance therapy with single-agent IM in patients with MBC-CML at onset or after failure of therapy excluding TKIs. Results: Thirty-six patients were evaluated. Median follow-up is 6.1 years. Daunorubicin was escaladed up to 45mg/m2/d 3 days. Twenty eight patients (77.7%) had hematologic response including 20 patients (55.5%) in complete hematologic response (CHR). Patients who received daunorubicin at 30–45mg/m2/d had higher CHR rates compared to other patients. Median overall survival was 16 months. Overall survival in patients with hematological response was 35.4 months. Better results were observed in patients diagnosed with MBC-CML at onset. Conclusions: The combination of IM with a standard “3+7” regiment was well tolerated and provided a high response rate. More than 55% of the patients achieved CHR and hematopoietic stem cell transplantation (SCT) was feasible in half of the cases. This trial was registered at www.clinicaltrials.gov as # NCT00219765. [Copyright &y& Elsevier]

The article presents a study on leukemic cell priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) which may modulate cell cycle kinetics and susceptible to phase-specific chemotherapeutic agents. In this study, cytogenetics, mutation detection and statistical analysis were used to 259 patients with acute myeloid leukemia (AML). The results show that priming of leukemia cells with GM-CSF may enhance complete remission (CR) rate and event-free survival (EFS).

• Our short series confirms the high response rate with CPX-351 in t-AML or MRC-AML. • The high rate of MRD negativity supports prior evidence that CPX-351 contributes to long-term remission. • MFC monitoring was confirmed as a reliable approach to assess response to treatment. [ABSTRACT FROM AUTHOR]

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNFα interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with β-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation. [Copyright &y& Elsevier]

Simple Summary: Acute myeloid leukemia refers to a large group of diseases that can be further defined by their genetic modifications. A specific alteration called KMT2A-PTD has been previously found in 5% of newly diagnosed acute myeloid leukemia cases. This work aimed to provide specific insight into outcomes of this specific disease and identify factors that may influence survival. Integrating KMT2A-PTD in ELN classification abrogates its predictive value on survival suggesting that this mutation may overcome other genomic marker effects. In patients receiving intensive chemotherapy, hematopoietic stem cell transplantation (HSCT) significantly improved the outcome compared to non-transplanted patients. In conclusion, our results emphasize that KMT2A-PTD should be considered as a potential adverse prognostic factor. Recently, a new subset of acute myeloid leukemia (AML) presenting a direct partial tandem duplication (PTD) of the KMT2A gene was described. The consequences of this alteration in terms of outcome and response to treatment remain unclear. We analyzed retrospectively a cohort of KMT2A-PTD-mutated patients with newly diagnosed AML. With a median follow-up of 3.6 years, the median overall survival was 12.1 months. KMT2A-PTD-mutated patients were highly enriched in mutations affecting epigenetic actors and the RTK/RAS signaling pathway. Integrating KMT2A-PTD in ELN classification abrogates its predictive value on survival suggesting that this mutation may overcome other genomic marker effects. In patients receiving intensive chemotherapy, hematopoietic stem cell transplantation (HSCT) significantly improved the outcome compared to non-transplanted patients. In the multivariate analysis, only HSCT at any time in complete remission (HR = 2.35; p = 0.034) and FLT3-ITD status (HR = 0.29; p = 0.014) were independent variables associated with overall survival, whereas age was not. In conclusion, our results emphasize that KMT2A-PTD should be considered as a potential adverse prognostic factor. However, as KMT2A-PTD-mutated patients are usually considered an intermediate risk group, upfront HSCT should be considered in first CR due to the high relapse rate observed in this subset of patients. [ABSTRACT FROM AUTHOR]

Simple Summary: DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of acute myeloid leukemia (AML) patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. A 4log reduction of NPM1 MRD was associated with a better outcome. DNMT3A negative patients who achieved a 4log reduction had a superior outcome to those who did not. However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identify a subgroup of patients at high risk of relapse. Minimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached ≥ 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse. [ABSTRACT FROM AUTHOR]

For the last 15years, imatinib mesylate (IM) has represented the gold standard treatment for chronic-phase chronic myelogenous leukemia (CP-CML); however, outcomes in the very long term remain unknown. We retrospectively analyzed the outcome of 418 IM first-line treated CP-CML patients followed in three reference centers over 15years in and outside of clinical trials, which is believed to represent the “real-life” care of such patients. Molecular analyses were standardized over the years. In case of intolerance or resistance or IM cessation and progression, all clinical data were collected and analyzed. After a median follow-up of 83 months (range 1–194), the overall survival (OS) rates were 91% and 82%, the progression-free survival (PFS) rates were 88.5% and 81%, and the event-free survival rates, including switching to another tyrosine kinase inhibitor, were 65% and 51%, respectively, at 5 and 10years. Thirteen patients (3%) entered blast crisis (BC) with a median survival of 2.2years after BC onset. Forty-nine percent of patients were in major molecular response at 1 year. Univariate analysis failed to detect any impact on survival of molecular response at 3 and 6 months. Sokal score had a significant impact on OS and PFS in a Cox model. Age had a significant impact on OS and PFS, mainly due to deaths in elderly patients unrelated to CML. Overall, 21% of patients reached a stable (≥1 year) molecular response 4 (MR4) and 6.5% reached MR4.5. At last follow-up, 63% of patients were still on IM and 19% were in treatment-free remission. We conclude that IM is an excellent therapeutic option providing impressive long-term OS rates. [ABSTRACT FROM AUTHOR]

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 × 10 9 /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.

High-throughput sequencing plays a pivotal role in hematological malignancy diagnostics, but interpreting missense mutations remains challenging. In this study, we used the newly available AlphaMissense database to assess the efficacy of machine learning to predict missense mutation effects and its impact to improve our ability to interpret them. Based on the analysis of 2073 variants from 686 patients analyzed for clinical purpose, we confirmed the very high accuracy of AlphaMissense predictions in a large real-life data set of missense mutations (AUC of ROC curve 0.95), and provided a comprehensive analysis of the discrepancies between AlphaMissense predictions and state of the art clinical interpretation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The objective of this study was to assess the clinical impact and financial costs of next-generation sequencing (NGS) in 5 categories of pediatric and adult hematological cancers. NGS prescriptions were prospectively collected from 26 laboratories, with varied technical and reporting practice (all or only significant targets). Impact was defined by the identification of (1) an actionable mutation, (2) a mutation with prognostic and/or theranostic value, and/or (3) a mutation allowing nosological refinement, reported by local investigators. A microcosting study was undertaken in 4 laboratories, identifying the types and volumes of resources required for each procedural step. Individual index prescriptions for 3961 patients were available for impact analysis on the management of myeloid disorders (two thirds) and, mainly mature B, lymphoid disorders (one third). NGS results were considered to impact the management for 73.4% of prescriptions: useful for evaluation of prognostic risk in 34.9% and necessary for treatment adaptation (actionable) in 19.6%, but having no immediate individual therapeutic impact in 18.9%. The average overall cost per sample was 191 € for the restricted mature lymphoid amplicon panel. Capture panel costs varied from 369 € to 513 €. Unit costs varied from 0.5 € to 5.7 € per kb sequenced, from 3.6 € to 11.3 € per target gene/hot-spot sequenced and from 4.3 € to 73.8 € per target gene/hot-spot reported. Comparable costs for the Amplicon panels were 5-8 € per kb and 10.5-14.7 € per target gene/hot-spot sequenced and reported, demonstrating comparable costs with greater informativity/flexibility for capture strategies. Sustainable funding of precision medicine requires a transparent discussion of its impact on care pathways and its financial aspects., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Current guidelines for patients with chronic lymphocytic leukemia (CLL) recommend mutation status determination of the clonotypic IGHV gene before treatment initiation to guide the choice of first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2 × 300 bp sequencing, which restricts their use for routine practice. The cost of the commercial kits also represents an important drawback. We present a new method called Next-CLL, a ready-to-use strategy to evaluate IGHV gene mutation status using any NGS device (including 2 × 150 bp sequencers) in routine diagnostic laboratories. The performance of Next-CLL was validated on genomic DNA and cDNA obtained from 80 patients with CLL at diagnosis. Next-CLL identified a productive clone in 100% of cases, whereas PCR with Sanger sequencing led to a 12.5% failure rate. Next-CLL had 100% concordance with the reference technique for IGHV gene identification and allowed assessment of the IGHV mutation status from the leader sequence, following international guidelines. Comparing a large retrospective series of samples, analyzed by using Sanger sequencing (n = 773) or Next-CLL (n = 352), showed no bias in IGHV usage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straightforward workflow for IGHV analysis in routine practice to assess clonal architecture and prognosis of patients with CLL., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) after conditioning with a sequential association of fludarabine, amsacrine, and cytosine arabinoside (FLAMSA) followed by a reduced-intensity conditioning regimen has emerged for patients with high-risk acute myeloid leukemia (AML), especially in refractory or relapsing patients. Here we aimed to address retrospectively the impact of pretransplantation minimal residual disease (MRD) by flow cytometry on the outcomes of high-risk AML patients who underwent allo-HSCT after sequential FLAMSA-busulfan (FLAMSA-Bu)-based conditioning regimens. We included 165 high-risk AML patients who underwent transplantation after FLAMSA-BU in this retrospective single-center "real life" study. All patients received in vivo T cell depletion with antithymocyte globulin (5 mg/kg). MRD detection was based on a leukemia-associated immunophenotype using the European LeukemiaNet recommendations, with a threshold of .1%. Univariate and multivariate analyses were performed using R version 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria). With a median follow-up of 4.0 years post-transplantation, the median overall survival (OS) was 54.9 months. Overall, 41 patients (24.8%) relapsed post-transplantation, with a resulting cumulative incidence of relapse (CIR) of 26.7% at 2 years and 34.0% at 5 years. Detectable MRD preceding allo-HSCT and refractory status were associated with worse median OS and CIR rates compared with patients without detectable MRD; however, OS was not significantly different between pre-HSCT MRD-positive and refractory patients (median, .7 year versus 2.0 years; P = .3). Conversely, pre-HSCT MRD negativity was associated with a reduced 2-year CIR. Neither European LeukemiaNet risk stratification nor age had a significant influence on OS. In the multivariate analysis, only pre-HSCT MRD positivity and lower conditioning regimen intensity were significantly associated with a poorer OS. The cumulative incidence of extensive chronic graft-versus-host disease at 2 years was 26.15%. The estimated nonrelapse mortality (NRM) of the entire cohort at 2 years was 23.1%, with age and unrelated donor identified as risk factors for higher NRM. Our data ahow that FLAMSA-Bu conditioning did not reverse the pejorative effect of detectable pre-HSCT MRD, suggesting that such patients should be offered alternative strategies before HSCT to reach deeper remission., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR 4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1 IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance., (© 2022. The Author(s).)

We report a case of donor-derived leukemia (DDL) occurring 34 months after double-unit cord blood transplantation (CBT). Molecular analysis using short tandem repeat (STR) sequences proved the acute myeloid leukemia (AML) to be of dominant cord blood origin. Karyotype was normal and molecular analysis showed WT1 and EVI1 overexpression. Cytological and molecular remission were achieved with only induction and consolidation chemotherapy. Relapse occurred after 6 years of remission from one clone with only WT1 overexpression. Potential etiologies for donor cell leukemogenesis in the recipient are discussed, including occult leukemia in the donor or genetic predisposition to hematologic malignancies, impaired immune surveillance, induced or inherited stromal abnormalities, transformation of donor cells during engraftment via altered signals of the host tissues, and fusion of donor cells with residual leukemic cells leading to acquisition of oncogenes. Although cases of DDL occurring after umbilical CBT have already been reported, very few cases have been described arising after double-unit CBT. DDL cases following CBT previously described in the literature have been reviewed., (© 2022. The Author(s).)

WT1 overexpression is frequently identified in acute myeloid leukemia (AML) and has been reported to be a potential marker for monitoring measurable residual disease (MRD). We evaluated the use of postinduction WT1 MRD level as a prognostic factor, as well as the interaction between postinduction WT1 MRD response and the effect of allogeneic stem cell transplantation (allo-SCT) in the first complete remission (CR). In the ALFA-0702 trial, patients with AML, aged 18 to 59, had a prospective quantification of WT1 MRD. The occurrence of a WT1 MRD ratio >2.5% in bone marrow or >0.5% in peripheral blood was defined as MRDhigh, and ratios below these thresholds were defined as MRDlow. The prognostic value of MRD after induction chemotherapy was assessed in 314 patients in first CR by comparing the risk of relapse, the relapse-free survival (RFS), and the overall survival (OS). Interaction between MRD response and the allo-SCT effect was evaluated in patients by comparing the influence of allo-SCT on the outcomes of patients with MRDhigh with those with MRDlow. The results showed that patients with MRDhigh after induction had a higher risk of relapse and a shorter RFS and OS. The MRD response remained of strong prognostic value in the subset of 225 patients with intermediate-/unfavorable-risk AML who were eligible for allo-SCT, because patients with MRDhigh had a significantly higher risk of relapse resulting in worse RFS and OS. The effect of allo-SCT was higher in patients with MRDlow than in those with MRDhigh, but not significantly different. The early WT1 MRD response highlights a population of high-risk patients in need of additional therapy., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Molecular tests have become an indispensable tool for the diagnosis and prognosis of hematological malignancies and are subject to accreditation according to the International Standard ISO 15189. National standardization of these techniques is essential to ensure that patients throughout France benefit from the same care. We report here on the experience of the GBMHM ( Groupe des Biologistes Moléculaires des Hémopathies Malignes ). By organizing External Evaluation of Quality (EEQ) programs and training meetings, the GBMHM has contributed to improvement and standardization of molecular tests in 64 French laboratories. A retrospective analysis of the quality-control results of 11 national campaigns spanning 10 years was performed for the 3 most frequently prescribed tests: BCR-ABL1 , JAK2 V617F, and lymphoid clonality. For each test, particular attention was placed on comparing methodologies and their evolution throughout the period. The establishment of the BCR-ABL1 , JAK2 V617F, and lymphoid clonality EEQ programs and the associated training meetings have initiated a process of collective standardization concerning the methods of implementation ( JAK2 V617F) and the interpretation and formulation of results (lymphoid clonality). In addition, it resulted in objective improvement in technical performance ( BCR-ABL1 ). Our evaluation of the impact of these EEQ programs demonstrates that it is possible to obtain reproducible values across different laboratories in France by applying national recommendations. To our knowledge, this is the first publication that evaluates the impact of a national quality assurance program on improving molecular results in hematology., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

RNA sequencing holds great promise to improve the diagnostic of hematological malignancies, because this technique enables to detect fusion transcripts, to look for somatic mutations in oncogenes, and to capture transcriptomic signatures of nosological entities. However, the analytical performances of targeted RNA sequencing have not been extensively described in diagnostic samples. Using a targeted panel of 1385 cancer-related genes in a series of 100 diagnosis samples and 8 controls, we detected all the already known fusion transcripts and also discovered unknown and/or unsuspected fusion transcripts in 12 samples. Regarding the analysis of transcriptomic profiles, we show that targeted RNA sequencing is performant to discriminate acute lymphoblastic leukemia entities driven by different oncogenic translocations. Additionally, we show that 86% of the mutations identified at the DNA level are also detectable at the messenger RNA (mRNA) level, except for nonsense mutations that are subjected to mRNA decay. We conclude that targeted RNA sequencing might improve the diagnosis of hematological malignancies. Standardization of the preanalytical steps and further refinements of the panel design and of the bioinformatical pipelines will be an important step towards its use in standard diagnostic procedures., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 transcript as a result of reciprocal translocation between chromosome 9 and 22. The most common transcripts subtypes are e13a2 (b2a2) and e14a2 (b3a2). The prognostic impact of the type of BCR-ABL1 transcript has been the subject of controversies over time. In the imatinib era, several studies have suggested a deeper and faster response in patients expressing e14a2. However, the impact on response after first line therapy with a second-generation tyrosine kinase inhibitor, nilotinib, is unknown. We retrospectively evaluated 118 patients newly diagnosed with chronic phase CML and treated frontline with nilotinib inside or outside clinical trial in five French centers. Only patients expressing e14a2 or e13a2 transcripts alone were analyzed. At baseline, 55.3% expressed e14a2, 44.7% expressed e13a2. The median age was 51 years and median follow-up was 49 months. Relative risks of CML at diagnosis were similar according to the ELTS score ( p = .87). Complete hematological response and complete cytogenetic response rates were similar among groups. Patients expressing e14a2 transcripts compared to e13a2 transcripts had deeper and faster molecular responses, when considering MMR (100% vs 84.1%, p = .007) with a median time of 6.7 and 17.1 months or MR 4.5 (100% vs 59.9%, p = .005) with a median time of 39.7 and 70.9 months, respectively. A sustained treatment free remission was observed in 10/10 patients with e14a2 versus 1/3 with e13a2 transcript ( p = .04). In conclusion, even treated with nilotinib first line, patients with chronic phase CML expressing BCR-ABL1 e13a2 transcript have a lower rate of deep molecular responses., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare.

In the context of chronic myeloid leukemia (CML) resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL1 tyrosine kinase domain (TKD) mutations still remain the sole biological marker that directly condition therapeutic decision. These recommendations aim at updating the use of BCR-ABL1 mutation testing with respect to new available therapeutic options and at repositioning different testing methods at the era of next generation sequencing (NGS). They have been written by a panel of experts from the French Study Group on CML (Fi-LMC), after a critical review of relevant publications. TKD mutation testing is recommended in case of treatment failure but not in case of optimal response. For patients in warning situation, mutation testing must be discussed depending on the type of TKI used, lasting of the treatment, kinetic evolution of BCR-ABL1 transcripts along time and necessity for switching treatment. The kind and the frequency of TKD mutations occasioning resistance mainly depend on the TKI in use and disease phase. Because of its better sensitivity, NGS methods are recommended for mutation testing rather than Sanger's. Facing a given TKD mutation, therapeutic decision should be taken based on in vitro sensitivity and clinical efficacy data. Identification by sequencing of a TKD mutation known to induce resistance must lead to a therapeutic change. The clinical value of testing methods more sensitive than NGS remains to be assessed., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Introduction: EVI1 (MECOM)-positive acute myeloid leukemia (AML) cells have shown in vitro sensitivity to all-trans-retinoic acid (ATRA) by inducing differentiation, cell death, and decreased leukemic engraftment., Methods: In this pilot study, we investigated the response to ATRA in 13 high-risk AML patients with overexpression of EVI1., Results: Seven of the 13 patients (53.8%) achieved complete remission (CR), and response can be combined with a decreased of the leukemia stem cell pool., Conclusion: These primary results tend to confirm in vitro results and suggest that addition of ATRA might be of benefit in the treatment of patients with EVI1-positive AML.

Background: Outcome of patients with mutation of the FLT3 tyrosine kinase domain (FLT3-TKD) in acute myeloid leukemia (AML) remains controversial., Patients and Methods: Herein, we present a retrospective study of 126 newly diagnosed patients with AML performed in 2 French centers., Results: FLT3-TKD mutations represented 12.7% of patients, whereas FLT3-internal tandem duplication (ITD) mutation was observed in 20.6% of AML cases and 1.6% of patients harbored both anomalies. At diagnosis, FLT3-TKD and FLT3-ITD were associated with higher peripheral leukocytes count and a higher blast count in bone marrow (P < 10 -4 ). Mutations of the NPM1 gene were frequently associated to FLT3-TKD (68.7%) and FLT3-ITD (61.5%) mutations compared with FLT3 wild type (WT) patients (P < 10 -4 ). Patients with both FLT3-TKD and NPM1 mutations (n = 12; 9.5%) showed a favorable outcome. Interestingly, mutations in NPM1 gene lost their favorable prognostic when not associated with FLT3-TKD both in univariate and multivariate analyses., Conclusion: Our data suggest that FLT3-TKD mutations should be routinely determined at the time of diagnosis. In association with NPM1 mutations, patients should follow the therapeutic schedule of favorable-risk patients with AML., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2 , TNFAIP3 and MYD88 , common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent ( TNFAIP3 and MYD88 ) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia., (Copyright© 2017 Ferrata Storti Foundation.)

Bone marrow necrosis (BMN) in acute leukemia is a rare histopathological entity at the time of initial diagnosis. However, it represents an important diagnostic and prognostic challenge. Two cases of BMN are reported: a 44-year-old patient with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) and a 27-year-old man with FAB-M5 acute myeloid leukemia (AML) who both presented with bone marrow failure and extensive necrosis. From these clinical cases, we conducted a brief review of the literature.

Background: Nilotinib is now recommended for patients with newly diagnosed chronic myeloid leukaemia in chronic phase and leads to important rates of molecular response 4·5 log (MR(4·5)), allowing the prospect of therapy cessation. However, most patients do not reach this criterion and nilotinib is taken for lengthy periods, resulting in chronic or late-onset adverse events. Nilotinib combined with interferon might further increase rates of MR(4·5), avoid late side-effects, and allow therapy cessation. In a phase 2 trial we aimed to assess the feasibility, safety, and deep molecular response of the combination of nilotinib (600 mg daily) and peginterferon alfa-2a in newly diagnosed patients with chronic-phase chronic myeloid leukaemia (CML)., Methods: In a non-randomised, open-label, phase 2 trial, we enrolled adult patients (age ≥18 years) without any organ failure who had BCR-ABL-positive, chronic-phase CML, at diagnosis. After a priming procedure with 90 μg per week of peginterferon alfa-2a alone for a month, we gave patients peginterferon alfa-2a 45 μg per week combined with nilotinib 600 mg daily until 24 months after interferon initiation. The primary endpoint was the cumulative incidence of MR(4·5) at 12 months after initiation of peginterferon alfa-2a. Data were analysed by a modified intention-to-treat principle. This trial is registered at the European Clinical Trials Database (EudraCT), number 2010-019786-28., Findings: Between March 24, 2011, and Sept 27, 2011, we enrolled 42 patients. One patient withdrew consent before receiving any study treatment so was excluded from analysis; 41 patients received treatment with peginterferon alfa-2a and nilotinib. At 12 months, seven (17%) patients had achieved MR(4·5). Haematological and hepatic adverse events were frequent-with grade 3-4 neutropenias occurring in ten (24%) patients, grade 3-4 thrombocytopenias occurring in ten (24%) patients, grade 3-4 cholestatic events occurring in seven (17%) patients, and grade 3-4 elevations in aspartate aminotransferase or alanine aminotransferase occurring in three (7% patients-particularly during the first 3 months. However, 30 (73%) patients remained on interferon therapy at 1 year. Three grade 3-4 cardiac events (7% of patients, all coronary stenoses) occurred at later timepoints., Interpretation: The combination of peginterferon alfa-2a resulted in good molecular responses in patients. Despite substantial toxic effects, most patients remained on the study drugs for more than a year. This combination should now be tested in a randomised controlled trial., Funding: Novartis Pharma., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The response definitions proposed by the European Leukemia Net (ELN) have been recently modified. We evaluated the new criteria for de novo imatinib (400 mg/d) chronic phase chronic myeloid leukemia (CP-CML) patients. Response status according to the 2009 and 2013 criteria were determined in 180 unselected patients. Outcome of the subgroups of patients were then compared. The 180 patients were classified as optimal responders (OR2009; n = 113, 62.7%), suboptimal responders (SOR2009; n = 47, 26.1%) and failures (FAIL2009; n = 20, 11.1%) according to the 2009 ELN criteria and optimal responders (OR2013; n = 77, 42.7%), warnings (WAR2013; n = 59, 32.7%), and failures (FAIL2013; n = 44, 24.4%) according to the 2013 ELN criteria. No difference in terms of outcome was observed between OR2009 patients who became WAR2013 when compared with OR2013 patients. When compared with FAIL2009 patients, SOR2009 patients who became WAR2013 had better EFS, FFS, PFS, and OS. No difference was observed in PFS or OS in SOR2009 patients who became FAIL2013. The 2013 ELN response status criteria have improved patients classification in terms of response status. However, in our patient population this improvement is related to a better definition of failure rather than that of optimal response for CP-CML patients treated with IM frontline therapy., (© 2014 Wiley Periodicals, Inc.)

We analysed the prognostic significance of minimal residual disease (MRD) level in adult patients with acute myeloid leukemia (AML) treated in the randomized gemtuzumab ozogamicin (GO) ALFA-0701 trial. Levels of WT1 and NPM1 gene transcripts were assessed using cDNA-based real-time quantitative PCR in 183 patients with WT1 overexpression and in 77 patients with NMP1 mutation (NPM1mut) at diagnosis. Positive WT1 MRD (defined as > 0.5% in the peripheral blood) after induction and at the end of treatment were both significantly associated with a higher risk of relapse and a shorter overall survival (OS). Positive NPM1mut MRD (defined as > 0.1% in the bone marrow) after induction and at the end of treatment also predicted a higher risk of relapse, but did not influence OS. Interestingly, the achievement of a negative NPM1mut MRD was significantly more frequent in patients treated in the GO arm compared to those treated in control arm (39 % versus 7% (p=0.006) after induction and 91% versus 61% (p=0.028) at the end of treatment). However, GO did not influence WT1 MRD levels. Our study supports the prognostic significance of MRD assessed by WT1 and NPM1mut transcript levels and show that NPM1 MRD is decreased by GO treatment.

Sustained imatinib treatment in chronic myeloid leukemia patients can result in complete molecular response allowing discontinuation without relapse. We set out to evaluate the frequency of complete molecular response in imatinib de novo chronic phase chronic myeloid leukemia patients, to identify base-line and under-treatment predictive factors of complete molecular response in patients achieving complete cytogenetic response, and to assess if complete molecular response is associated with a better outcome. A random selection of patients on front-line imatinib therapy (n=266) were considered for inclusion. Complete molecular response was confirmed and defined as MR 4.5 with undetectable BCR-ABL transcript levels. Median follow up was 4.43 years (range 0.79-10.8 years). Sixty-five patients (24%) achieved complete molecular response within a median time of 32.7 months. Absence of spleen enlargement at diagnosis, achieving complete cytogenetic response before 12 months of therapy, and major molecular response during the year following complete cytogenetic response was predictive of achieving further complete molecular response. Patients who achieved complete molecular response had better event-free and failure-free survivals than those with complete cytogenetic response irrespective of major molecular response status (95.2% vs. 64.7% vs. 27.7%, P=0.00124; 98.4% vs. 82.3% vs. 56%, P=0.0335), respectively. Overall survival was identical in the 3 groups. In addition to complete cytogenetic response and major molecular response, further deeper molecular response is associated with better event-free and failure-free survivals, and complete molecular response confers the best outcome.

The BCR-ABL T315I mutation confers resistance to currently licensed tyrosine kinase inhibitors in chronic myelogenous leukemia. However, the impact of this mutation on survival in early stages of disease, in chronic phase, has never been detailed. Using matched pair analysis, a cohort of 64 patients with chronic phase chronic myelogenous leukemia harboring a T315I mutation and resistant to imatinib mesylate was compared to a similar cohort of 53 chronic phase patients resistant to imatinib, but with no detectable T315I mutation, in the pre-ponatinib era. These patients were matched according to age at diagnosis, interval between disease diagnosis and start of imatinib treatment, and duration of imatinib therapy. Kaplan-Meier survival analyses demonstrated the significant negative impact of the presence of the T315I mutation on overall survival (since imatinib-resistance: 48.4 months for T315I(+) patients versus not reached for T315I(-) ones; P=0.006) and failure-free survival (since imatinib-resistance: 34.7 months for T315I(+) patients versus not reached for T315I(-) patients; P=0.003). In addition, Cox proportional hazard models adjusted on overall survival demonstrated the negative influence of the T315I mutation (P=0.02, HR=2.54). These results confirm early assumptions concerning the poor prognosis of chronic phase chronic myelogenous leukemia patients with the T315I mutation who are not eligible for allogeneic transplantation, and demonstrate the need for more therapeutic options.

This report aims to more accurately define the frequency of the involvement of SRC Family Kinases (SFKs) in imatinib- and dasatinib-resistant CML patients. Clinical samples were analysed during in vivo treatment. We confirmed the high frequency of SFKs involvement in Tyrosine kinase inhibitor-resistant CML (52% of the cases) and even further in progressive disease and blast crises (60% of the cases). The SFKs deregulation is also observed in patients harboring BCR-ABL mutations. In T315I and F317L mutated patients, CML-resistance appears to be promoted by SFKs kinase protein reactivation once the BCR-ABL mutated clone has decreased on Omacetaxine., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: The onset of a BCR-ABLT315I mutation during the course of chronic myelogenous leukemia (CML) on tyrosine kinase inhibitors (TKIs) usually results in poor survival, and therapeutic options remain few in the absence of any allogeneic donor., Patients and Methods: We have investigated the affect of subcutaneous omacetaxine (OMA, or homo-harringtonine) cycles on unmutated and T315I-mutated BCR-ABL transcripts in a series of 8 TKI-resistant chronic-phase CML patients and we have addressed the question of whether the administration of OMA could resensitize patients to TKIs. Patients were regularly monitored for total disease burden and for BCR-ABLT315I transcripts using a new quantitative sensitive technique (sensitivity threshold, 0.05%), for up to 27 cycles of OMA., Results: Overall, patients demonstrated hematologic, cytogenetic, or molecular improvement. An initial rapid decline and a sustained disappearance of T315I-mutated transcripts were observed in 50% of patients, after a median of 10.5 cycles (range, 3-27 cycles) of OMA. As the unmutated leukemic burden reduction was modest, 2 patients were submitted to nilotinib after 9 months of sustained BCR-ABLT315I transcripts negativity on OMA and mutated transcripts remained undetectable after a median follow-up of 12 months on nilotinib challenge., Conclusion: We suggest that OMA (ie, a non-targeted therapy) might provide a better disease control allowing the disappearance of the mutated clone probably elicited by the clone deselection after TKI release, and/or a preferential activity of OMA on the T315I-mutated cells through unknown mechanisms. These observations suggest that OMA could allow a safe TKI rechallenge in patients with resistant chronic-phase CML.

The treatment of chronic myeloid leukemia (CML) has considerably evolved since imatinib mesylate has been introduced as a new therapeutic weapon for this disease. The 5-year updated results of the IRIS study confirmed that imatinib mesylate is the best first line therapy for chronic phase CML with an overall survival of 90%. Responses improve with time and complete cytogenetic and major molecular levels reach 87 and 70% respectively at 5 years. However, despite these remarkable improvements, new problems arise as sub-optimal responses, imatinib-resistances with recently identified BCR-ABL protein point mutations, responsible for a variety of therapeutic consequences : imatinib dose increase, alternative treatments with second generation tyrosine kinase inhibitors (TKIs : dastinib, nilotinib) or allogeneic stem cell transplantation. The treatment of accelerated and blastic phases relies on imatinib +/- conventional chemotherapy, ideally followed by allogeneic stem cell transplantation, as newly developed TKIs are currently evaluated within this frame. Finally, BCR-ABL(T315I) mutations remain a new therapeutic critical challenge as none of the three TKIs (imatinib, nilotinib, dasatinib) can efficiently control such diseases.

We analyzed 27 CML patients treated with imatinib (IM) who developed a BCR-ABLT315I mutation. These patients had poor prognostic features: High or intermediate Sokal index (82%), and lack of CCyR under IM (59%). At T315I discovery, patients were in advanced phase (59%), with clonal evolution (84%). Median time since diagnosis was 39 months, and progression occurred 13 months after IM initiation, regardless of disease phase. Overall survival since IM initiation was 42.5 months for chronic, and 17.5 months for advanced phases, and all patients progressed. This mutation seems related to or (partially?) responsible for progression and poor survival.

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNF alpha interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with beta-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation.