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Supplementary Figure 1 from Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Supplementary Table 1 from Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl–positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance. [Cancer Res 2008;68(23):9809–16]

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.

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.

As the impact of targeted next-generation sequencing (TNGS) on daily diagnosis has not been evaluated, we performed TNGS (46 genes) on lymphomas of unclear subtype following expert haematopathological review. The potential impact on patient care and modifications of final diagnosis were divided into major and minor changes according to the European Society of Medical Oncology (ESMO) guidelines. Among 229 patients [19 primary central nervous system lymphomas (PCNSL), 48 large B-cell lymphomas (LBCLs), 89 small BCLs (SBCLs), seven Hodgkin lymphomas (HL), 66 T-cell lymphomas], the overall concordance rate of histological and TNGS diagnosis was 89·5%. TNGS confirmed the histological diagnosis in 144 cases (62·9%), changed the diagnosis in 24 cases (10·5%) and did not help to clarify diagnosis in 61 cases (26·7%). Modifications to the final diagnosis had a clinical impact on patient care in 8·3% of cases. Diagnostic modifications occurred in all types of lymphoma except in PCNSL and HL; the modification rate was 14·6% in SBCL and 12·5% in LBCL. While comparing informative and uninformative cases, no differences were found in terms of DNA amplification, quality or depth of sequencing and biopsy type. The present study highlights that TNGS may directly contribute to a more accurate diagnosis in difficult-to-diagnose lymphomas, thus improving the clinical management in routine practice.

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.

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.

International audience; PurposeTo assess the incidence of BCR‐ABL kinase domain (KD) mutation detection and its prognostic significance in chronic phase chronic myeloid leukemia (CP‐CML) patients treated with tyrosine kinase inhibitors (TKIs).Patients and MethodsWe analyzed characteristics and outcome of 253 CP‐CML patients who had at least one mutation analysis performed using direct sequencing. Of them, 187 patients were early CP (ECP) and 66 were late CP late chronic phase (LCP) and 88% were treated with Imatinib as first‐line TKI.ResultsOverall, 80 (32%) patients harbored BCR‐ABL KD mutations. A BCR‐ABL KD mutation was identified in 57% of patients, who progressed to accelerated or blastic phases (AP‐BP), and 47%, 29%, 35%, 16% and 26% in patients in CP‐CML at the time of mutation analysis who lost a complete hematologic response, failed to achieve or loss of a prior complete cytogenetic and major molecular response, respectively.Overall survival and cumulative incidence of CML‐related death were significantly correlated with the disease phase whatever the absence or presence of a mutation was and for the latter the mutation subgroup (T315I vs P‐loop vs non‐T315I non‐P‐loop) (P

Early molecular response has been associated with clinical outcome in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The BCR-ABL1 transcript rate decline from baseline to 3 months has been demonstrated to be more predictive than a single BCR-ABL1 level at 3 months (M3). However, it cannot be used routinely because ABL1, as an internal gene control, is not reliable for BCR-ABL1 quantification above 10%. This study aimed to compare clinical outcome and molecular response of chronic phase CML patients, depending on the percentage of BCR-ABL1 transcript decrease from month 3 to month 6 using ABL1 as an internal control gene. Two hundred sixteen chronic phase CML patients treated with imatinib 400 mg for whom M3 and month 6 molecular data were available were included in the study. Associations with event-free (EFS), failure-free (FFS), progression-free (PFS), and overall survivals (OS) molecular response 4 log and 4.5 log were assessed. The percentage of BCR-ABL1 decline from month 3 to month 6 was significantly linked to the EFS and the FFS (p

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 &lt, 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.

International audience; 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.

RNA sequencing holds great promise to improve the diagnostic of haematological malignancies, because this technique enables to detect fusion transcripts, to look for somatic mutations in oncogenes, and to capture transcriptomic signatures 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 mRNA level, except for nonsense mutations which are subjected to mRNA decay. We conclude that targeted RNA sequencing might improve the diagnosis of haematological 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.

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 MR4.5 (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.

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 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.

Aims: The absolute number of chronic phase CML patients (pts) reaching the treatment-free remission (TFR) criteria has been substantially increased by the use of second-generation TKI (TKI2), initiated since diagnosis, comparing to Imatinib first-line. However, the relative rate of unsuccessful TFR (i. e. pts loosing their MMR after TKI2 cessation) still remains around 50% at 2 years and beyond, whatever the TKI2 was. The aim of this study is to analyse the rate of successful TFR in pts receiving Nilotinib (Nilo) or Dasatinib (Dasa) first-line obtaining the appropriate criteria. Methods: Observational retrospective study in 3 reference centers of the French group of CML lead between 2010 and 2021. Eligible pts were CP CML pts initiating either Nilo 300 mg BID or Dasa 100 mg daily since diagnosis, until cessation for sustained MR4.5 (i.e. ≥2 years on ≥4 datapoints). Data were retrospectively collected according to the national regulations with pts' information. All pts were assessed and followed according to ELN recommendations 2009, 2013 and 2020 along treatment and to the recommendations from the French group of CML (D. Rea et al., Cancer 2018) for TFR. In this regard, the TKI2 was resumed in case of loss of MMR. All BCR-ABL1 assessments were performed in the 3 reference laboratories, standardised and expressed in % (IS) with ≥32,000 copies of ABL1 as control. All patients were harbouring major BCR-ABL1 transcripts. The primary endpoint was the survival without loss of MMR after TKI2 cessation. The secondary endpoints were the kinetics of MMR loss, and the identification of factors influencing MMR loss. Results: Seventy-two pts were reported (47 Nilo, 25 Dasa) with 57% females with a median age at diagnosis of 48 (36.75-61.25) years. The median follow-up since diagnosis was 9.26 (3.75-13.75) years (8.8 for Nilo and 9.47 for Dasa p=ns) and after TKI2 cessation 3.94 (0.7-8.8) years (3.92 for Nilo and 3.90 for Dasa p=ns). Sokal scores were 42% Low, 41% Intermediate, 17% High in Nilo and 39% L, 25% I and 35% H in Dasa pts (p=ns). ELTS scores were 50% L, 22% I, 9.5% H (18.5% Uk) in Nilo and 46.5% L, 28.5% I and 3.5% H (21.5% Uk) in Dasa pts (p=0.95). Five (9%) pts harboured ACA at diagnosis in the Nilo group and 2 (7%) in the Dasa group (p=1.00). The median time from TKI2 initiation to sustained MR4.5 was 19 (3.12-36) months in the Nilo group and 16 (6.3-39) months in the Dasa group (p=0.644). The duration of sustained MR4.5 until cessation was 3.04 (1.5-9.3) years for Nilo and 2.65 (1.11-7.95) for Dasa (p=0.96). The median dosing of Nilo was 600 (300-800) mg daily and 80 (20-100) mg at TKI2 cessation. None of these patients switched to another TKI during the follow-up. TKI2 cessation occurred after 60.5 (43-74.5) months in the Nilo group and 68 (39-90) months in the Dasa group (p=0.581). Thirty-seven pts out of 47 (79%) were BCR-ABL1 undetectable at Nilo cessation 18/25 (72%) at Dasa cessation (p=0.60). At M3 after discontinuation, 58% of pts remained undetectable after Nilo cessation and 30.4% after Dasa cessation (p=0.05).The median survival of pts without loss of MMR was not reached in the Nilo group, and was 14 (4.73-NR) months in the Dasa group, (p=0.042) as analysed by the KM method (Figure 1.). Two patients died (1 Nilo, 1 Dasa) from competing events (solid tumours) after unsuccessful TFR. Twenty-eight pts (14 Dasa, 14 Nilo) restarted their TKI2 after MMR loss and all regained ≥ MMR after 3 months of Dasa at a median dose of 75 (40-100) mg daily and all except one (who regained MMR at M12) after resumption of Nilo at a median dose of 350 (300-600) mg daily. Univariate analysis identified pts with H+I Sokal (as compared to low) as an unfavourable factor for successful TKI2 cessation [HR=0.35 (0.15-0.83), p=0.017] and type of TKI2 (Nilo as reference vs Dasa) was discriminant [HR=2.1 (1.01-4.35), p=0.047]. Multivariate analysis identified the type of TKI2 as a significant factor impacting on TFR outcome [HR 2.11 (0.97-4.55], p=0.05]. Conclusions: As it is likely that no prospective head-to-head comparison will be performed in this setting, on this limited series of pts, we conclude that the outcome of TFR seems to be different according to the TKI2 used since diagnosis, suggesting the impact of distinct biological variables modified by the type of TKI2 on the long run (such as immunological system, BM micro-environment, others) on TFR outcome. Figure 1 Figure 1. Disclosures Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Etienne: Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Aims Life expectancy of CML pts optimally responding to tyrosine kinase inhibitors (TKI) is close to that of the general population and recently, TFR has been acknowledged as a new goal of CML management. TKI discontinuation in the view of TFR requires the achievement of deep and long-lasting molecular responses (MR). The gold standard BCR-ABL mRNA quantification technology and MR definitions rely on internationally standardized (IS) RT-qPCR but atypical transcripts located outside the Major-BCR region, harbored by 1-2% of pts, cannot be expressed on the IS scale. Thus, most trials and clinical practice recommendations prevent such pts from attempting TFR. The Fi-LMC group retrospectively collected real-life observations to assess TFR likelihood in this rare population. Methods Data from CML pts with precise characterization of atypical transcripts in whom any line TKI was stopped for any reason but after at least 2 years of undetectable molecular residual disease (UMRD) by individualized non-standardized RT-qPCR were collected. RT-qPCR sensitivity varied depending on transcript type and local molecular biology laboratory. TFS was estimated by the Kaplan-Meier method. Relapse was analyzed using the cumulative incidence function, relapse being as UMRD loss at any time and any level during follow-up (FU). Results Our series comprised 16 adult CP CML pts with atypical BCR-ABL fusions including 12 males (75%). Median age at CML diagnosis was 56 years (range: 21-75) and that at TKI discontinuation was 67 years [range: 29-82]. Sokal score was low, intermediate and high in 7, 8 and 1 pts, respectively. ELTS score was low and intermediate in 10 and 4 pts, respectively and unknown in 2. Most pts expressed e19a2 (n=6) followed by e6a2 (n=4), b3a3 (n=3), b2a3 (n=2) and e8a2 (n=1). Seven pts discontinued imatinib, 4 stopped dasatinib, 4 nilotinib and 1 bosutinib. Number of lines of therapy was 2 in 8 pts, 1 in 5 pts and 3 in 3 pts. Median TKI treatment duration before discontinuation was 64 months (range: 31-218) and median duration of UMRD was 41 months (range: 21-168). The median FU after TKI discontinuation was 68 months (range: 3-149). Five pts experienced relapse leading to TKI resumption. Four relapses occurred within 3-6 months and included 2 loss of hematologic response in CP, 1 loss of hematologic response in accelerated phase CML and 1 molecular recurrence with BCR-ABL transcripts up to 1.5%. One relapse occurred at 49 months and consisted in loss of a complete cytogenetic response. These 5 pts resumed TKI and regained UMRD within 6 months, including 1 pt who died in UMRD from non-CML-related cause at the age of 82 years and 1 pt who rapidly failed a 2 nd TKI discontinuation attempt. In 1 additional pt, BCR-ABL transcripts became detectable intermittently with maximum transcript level of 0.15% and TKI was not resumed. The median FU of pts who remained treatment-free was 68 months (range: 8-149). Overall, the 5-year cumulative incidence of relapse regardless of whether TKI was resumed was 41.6% (95% confidence interval: 21.9%-78.7%) (Figure 1). The 5-year TFS rate was 65.2% (95% confidence interval: 40.3%-90.2%) (Figure 2). Conclusions Our observational study of TKI discontinuation in CML pts with atypical BCR-ABL transcripts is the largest reported so far. While effort must be made for proper assessment of deep MR, preliminary results suggest that TFS pattern might favorably compare with that obtained in pts with Major-type BCR-ABL transcripts. However, relapses may be more aggressive and caution is required in order to avoid loss of hematologic responses and progression. Whether the type of atypical fusion gene influences TKI discontinuation outcome, as well as other potential prognostic factors, need to be determined in a larger series. Figure 1 Figure 1. Disclosures Charbonnier: Novartis: Speakers Bureau; Incyte: Speakers Bureau. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Etienne: Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

Rational: Patients with t-AML are those previously treated for primary malignancy and are categorized with poor prognosis. t-AML treatment options include intensive chemotherapy (IC), hypomethylating agents (HMA) or low dose cytarabine (LDC). However, survival remains poor and allogeneic hematopoietic cell transplantation (Allo-HCT) could lead to better outcomes. The aim of this analysis is to assess the outcome of all consecutive t-AML pts from two regional centers in France, based on treatment intensity, and to identify prognostic factors. Patients and Methods: between January 2006 and December 2019, 112 adult pts diagnosed with t-AML occurring after treatment of solid tumors (ST; n=72), hematologic malignancies (HM; n=34) or autoimmune diseases (AID; n=6). Pts received chemotherapy and/or radiotherapy or high dose chemotherapy followed by autologous hematopoietic cell transplantation (Auto-HCT) in some. All eligible t-AML pts received intensive chemotherapy (IC, n=55) with the combination of anthracycline and cytarabine in 48 pts and CPX-351 in 7 pts. Unfit pts received non-IC (9 LDC, 24 HMA). The third study group (24 pts) received best supportive care (BSC). Fifteen pts underwent HSCT in first CR. Statistical analysis: We performed a descriptive analysis of the baseline characteristics and each treatment group was compared with a Kruskal-Wallis test and a Pearson's Chi-square test. Survival curves (Kaplan-Meier method) were compared with a log-rank test. Univariate and multivariate analyses on survival, relapse and NRM were done using Cox regression, Gray test, Fine & Gray regression. The bilateral level of significance was set at 5%. All analyses and graphics were made under the R program (v3.5.1). A pair-matched control analysis with de novo AML was performed only on pts who received IC matched for age, cytogenetics and ELN 2010 classification. Results: A total of 112 t-AML pts were identified, 56 were males (50%) with a median age of 68 yrs (19-87) at t-AML diagnosis. Before t-AML, 72 pts had ST (64.5%), 34 HM (30.5%) and 6 AID (5%). Forty-six pts (41%) received chemotherapy alone for their primary cancer, 17 (15%) radiotherapy alone, 49 (44%) radio chemotherapy, 17 (15%) auto-HCT and 6 (5%) long-term methotrexate for AID. Median interval from treatment of previous ST or HM to t-AML diagnosis was 4.7 years in ST, 6.6 years in HM and 21 years in AID (p=0.03). At t-AML diagnosis, 42% of pts presented an unfavorable karyotype and 44% an unfavorable ELN 2010 classification. Regarding molecular alterations, FLT3-ITD, FLT3-TKD, NPM1 and IDH1/2 mutations were observed in 6.9, 2.3, 11.5 and 2.3% of pts respectively. MECOM1 was overexpressed in 25.5% of cases. Among treated pts (n=88), 43 pts (49%) achieved CR: 4 from 33 (12%) in non-IC pts (3 in HMA and 1 in LDC) and 39 from 55 (71%) in IC pts. Fifteen pts (17%) underwent Allo-HCT with a median interval between AML diagnosis and Allo-HCT of 4.7 mo (2.6-17.5). The best response after transplantation was CR in 10 pts; at the last follow up, 4 pts are alive (3 in CR and 1 in relapse) and 11 died (5 from relapse, 5 from TRM causes and 1 from another subsequent malignancy). Overall, with a median follow up of 5.5 mo (0-144), the median OS and DFS were 9 mo (5.9-13.5) and 6.3 mo (5.3-10.3) respectively (Figure). There was a significant difference between the 3 treatment groups concerning OS (p Conclusion: The prognosis of t-AML remains poor and our study showed the advantage of using intensive chemotherapy whenever possible and, in comparison with de-novo AML, a higher NRM and a lower incidence relapse. Figure 1 Figure 1. Disclosures Nicolini: Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

Aims: The onset of blast crisis (BC) in initially chronic phase (CP) CML patients that entered treatment-free remission (TFR) after TKI is an exceptional event, however, there is emerging evidence that this may occur in such patients (pts), although the pathogenesis remains unclear to date. Methods: Anonymous clinical case retrospective data collection from patients' datafiles, after written agreement of living patients, centralisation of available frozen nucleic acid collection from diagnosis and from blast crisis and reanalysis by Next-Generation Sequencing of samples (ASXL1, ASXL2, BCOR, CALR, CBL, CEBP alpha , CSF3R, DNMT3A, EP300, ETNK1, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2 JAK2, KIT, , KRAS,MPL, NPM1, NRAS, PHF6, PTPN11 , RAD2, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2 genes analysed) on Illumina platform. CNV analysis were performed using VisCAp or in-house pipelines and/or by Multiplex Ligation Dependant Probe Amplification (MLPA). ABL1 tyrosine kinase domain mutations were screened by NGS on cDNA or directly on DNA. BCR-ABL1 transcripts are expressed in % (IS) with at least 32,000 copies of ABL1 as control. All patients discontinued their TKI after 2 years of MR4.5 and a TKI was resumed in case of MMR loss. Results: Along 15-year experience of TFR in our country and ≥ ~800 patients experiencing a TKI cessation attempt, informations from 4 (~0.5%) TFR CML patients entering BC have been collected. All patients harboured Major BCR transcripts. The chronic phase characteristics are mentioned in Table 1. All these long-lasting CP CML pts were ELTS risk score low, and 1 was harbouring ACA at diagnosis. One pt was mutated for ASXL2 and 2 mutations for EP300, found again at BC. Three pts had IFN-a prior to imatinib for all. Three out of 4 lost their MMR after a first cessation attempt at 12, 10 and 3 months after cessation. Pt #1 experienced a 2 nd cessation attempt 52 months after re-initiation of TKIs and entered lymphoid BC 6 months after a second resumption of TKI for a 2 nd MMR loss. The BC characteristics are mentioned in Table 2. Three out of 4 BC were lymphoid, one had ACA different from those at CP diagnosis. Two pts explored had multiple mutations in Runx1, U2AF1, EP300 and ASXL2 genes, not present at CP diagnosis, in addition to multiple ABL1 mutations in 2 out of 4 pts (2 T315I, 3 P-Loop mutations). All pts underwent chemotherapy + various TKI leading to complete remission (CR) in all and 3 out of 4 pts could be allotransplanted in CR, one relapsed shortly after transplant, and a second one 34 months after transplant. Overall 3 pts are alive with 1 in controlled relapse. Conclusions: The onset of BC after TFR for sustained deep molecular response remains an exceptional event and is probably not induced by this therapeutic procedure. These 4 cases underline the need for a sustained long-lasting molecular follow-up of pts in TFR, although the majority of these BC seem sudden. Figure 1 Figure 1. Disclosures Bauduer: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

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.

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

Purpose This study assessed the prognostic impact of postinduction NPM1-mutated ( NPM1m) minimal residual disease (MRD) in young adult patients (age, 18 to 60 years) with acute myeloid leukemia, and addressed the question of whether NPM1m MRD may be used as a predictive factor of allogeneic stem cell transplantation (ASCT) benefit. Patients and Methods Among 229 patients with NPM1m who were treated in the Acute Leukemia French Association 0702 (ALFA-0702) trial, MRD evaluation was available in 152 patients in first remission. Patients with nonfavorable AML according to the European LeukemiaNet (ELN) classification were eligible for ASCT in first remission. Results After induction therapy, patients who did not achieve a 4-log reduction in NPM1m peripheral blood-MRD (PB-MRD) had a higher cumulative incidence of relapse (subhazard ratio [SHR], 5.83; P < .001) and a shorter overall survival (OS; hazard ratio [HR], 10.99; P < .001). In multivariable analysis, an abnormal karyotype, the presence of FLT3-internal tandem duplication (ITD), and a < 4-log reduction in PB-MRD were significantly associated with a higher relapse incidence and shorter OS. In the subset of patients with FLT3-ITD, only age, white blood cell count, and < 4-log reduction in PB-MRD, but not FLT3-ITD allelic ratio, remained of significant prognostic value. In these patients with nonfavorable AML according to European LeukemiaNet, disease-free survival and OS were significantly improved by ASCT in those with a < 4-log reduction in PB-MRD. This benefit was not observed in those with a > 4-log reduction in PB-MRD, with a significant interaction between ASCT effect and PB-MRD response ( P = .024 and .027 for disease-free survival and OS, respectively). Conclusion Our study supports the strong prognostic significance of early NPM1m PB-MRD, independent of the cytogenetic and molecular context. Moreover, NPM1m PB-MRD may be used as a predictive factor for ASCT indication.

To the editor: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), characterized by the t(15;17) translocation resulting in the PML-RARA fusion transcript. The use of all-trans retinoic acid (ATRA) and/or arsenic has revolutionized the treatment of this disease, which

Minimal residual disease is now a powerfull surrogate marker to assess response to chemotherapy in acute myeloid leukemia (AML). In younger adults, NPM1 MRD has recently demonstrated to be a favorable predictive marker for EFS and OS independently of fms-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD) status. However, there is very few datas regarding predictive value of NPM1 MRD in elderly patients treated with intensive chemotherapy. Moreover, numerous studies have suggested the negative impact of DNMT3a mutation in NPM1 AML patients, especially in those with concurrent FLT3-ITD mutation. In this study, we aimed to investigate the impact of DNMT3a status on post induction NPM1 MRD1 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 (Lyon, Lille) were analyzed retrospectively. Median age of the entire cohort was 66.1 years old (range 60-78.2). An FLT3-ITD mutation was evidenced in 52 of 138 patients (37.6%) with a median FLT3-ITD AR of 0.53 (range, 0.05-3). With a median follow-up of 19.61 months (0.07-128.4), the overall CR rate was 89.9% with no influence of DNMT3a or FLT3 mutational status on the probability of CR. In this elderly cohort of NPM1mut patients, a 4log reduction of NPM1 bone marrow (BM) MRD1 was associated with better outcome (median OS: NR vs 13.4 months, HR=0.35, p 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 very high risk of relapase, despite good molecular responses. As hematopoietic stem cell transplantation (HSCT) might improve OS in elderly patients, DNMT3a positive AML elderly patients should be considered for HSCT or post induction maintenance strategies, even within the favorable ELN risk group. Figure Disclosures Sujobert: Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding.