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Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Background The early diagnosis of Sezary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. Objectives Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. Methods This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. Results The presence of KIR3DL2+ Sezary cells (SCs) ≥ 200 μL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells Conclusions KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sezary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sezary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 μL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

Le diagnostic de la neoplasie myeloproliferative que represente la LMC, ainsi que la determination de sa phase evolutive et de son score pronostique necessitent: 1) un examen clinique mesurant l’eventuelle splenomegalie; 2) un hemogramme retrouvant une hyperleucocytose avec basophilie et myelemie harmonieuse; 3) unmyelogramme montrant une hyperplasie granulocytaire; 4) un caryotype medullaire et/ou FISH identifiant le chromosome de Philadelphie par translocation (9;22) et/ou le gene de fusion BCR-ABL1 codant pour la proteine tyrosine-kinase a l’origine de la maladie; 5) un polymerase chain reaction (PCR) caracterisant qualitativement et quantitativement le transcrit de fusion BCR-ABL1.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10 6, 1.08±0.11 × 10 5, 1.03±0.10 × 10 4, 1.02±0.09 × 10 3, 1.04±0.10 × 10 2 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP (P=0.003). With a median follow-up of 39.2 months (6.3-67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations (P=0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations (P=0.014), and a worse progression-free survival (PFS) for T315I mutations (P=0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

Philadelphia chromosome (Ph) acute lymphoblastic leukemia-positive (ALL) is a subgroup of ALL with a poor prognosis. We sought to evaluate the results of allogeneic hematopoietic stem cell transplantation (HSCT) in this situation. From 1992 to 2000, 121 patients with Ph- positive ALL enrolled in one of the three main French prospective ALL chemotherapy trials and receiving allogeneic HSCT were reported to the registry of the French Society of Bone Marrow Transplantation (SFGM-TC). The median age was 35 years (range, 1-53). In all, 76 patients received HSCT in first complete remission and 45 in a more advanced disease stage. Minimal residual disease was evaluated just before the graft: 35 patients of the 52 patients in first remission had persistent BCR-ABL transcript detectable while 17 had no detectable minimal residual disease. Bone marrow and/or peripheral blood samples from 94 patients were submitted for reverse transcriptase polymerase chain reaction analysis at variable points after transplantation. Estimated 2-year survival and relapse rate for patients in CR1 were 50 and 37%, respectively, significantly better than for patients with more advanced disease (P=0.0001 and 0.01, respectively). There was no difference in survival or in relapse rates in terms of the donor used. Relapse was the most common cause of treatment failure. Hematological status at the time of transplant and the occurrence of acute graft-versus-host disease (GvHD) were the only two prognostic factors identified for relapse (P=0.02 and 0.02, respectively). Detection of BCR-ABL transcripts after transplantation had a predictive value on relapse occurrence. Finally, donor lymphocyte infusions were successfully used to treat some relapses. The graft-versus-leukemia effect of HSCT in Ph-positive ALL appears to be supported by (1) the lack of prognostic significance of pretransplant BCR-ABL transcript detection, (2) the efficacy of donor lymphocyte infusions in cases of relapse, and (3) the role of GvHD as protecting against relapse.

Identification of a rare e8a2 BCR-ABL fusion gene in three novel chronic myeloid leukemia patients treated with imatinib

Imatinib mesylate is the sole BCR-ABL tyrosine kinase inhibitor approved as first-line treatment of accelerated-phase (AP) chronic myeloid leukemia (CML). Indication was based on the STI571 0109 study, in which imatinib favorably compared to historical treatments in patients failing prior therapies. The relevance of these results to currently newly diagnosed AP-CML patients remains unknown. We evaluated the benefit of imatinib in 42 newly diagnosed AP-CML patients. In all, 16 patients had hematological acceleration without chromosomal abnormalities in addition to the Philadelphia chromosome (ACAs; HEM-AP), 16 solely had ACAs (ACA-AP) and 10 had hematological acceleration plus ACAs (HEM-AP + ACA). Major cytogenetic responses were achieved in 93.7% of HEM-AP patients, 75% of patients with ACA-AP (P=NS) and 40% of patients with HEM-AP + ACA (P=0.0053). The 24-month failure-free survival rate was 87.5% in HEM-AP patients, 43.8% in ACA-AP patients and 15% in HEM-AP + ACA patients (P=0.022). The 24-month estimate of progression-free survival was 100% in HEM-AP patients, 92.8% in ACA-AP patients and 58.3% in HEM-AP + ACA patients (P=0.0052). In conclusion, frontline imatinib allows favorable outcomes in HEM-AP and ACA-AP patients but appears insufficient for patients with HEM-AP + ACA. Broader-target and/or more potent BCR-ABL tyrosine kinase inhibitors alone or in combination may be considered in this setting.

Imatinib dose escalation for chronic phase–chronic myelogenous leukaemia patients in primary suboptimal response to imatinib 400 mg daily standard therapy

Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Prospective multicentric molecular study for poor prognosis fusion transcripts at diagnosis in adult B-lineage ALL patients: the LALA 94 experience

Internal tandem duplications (ITDs) of the FLT3 gene have been observed in about 35% of APL cases. If FLT3-ITD is associated with a worse outcome in patients with acute myeloid leukemia (AML) in general, its prognostic value in acute promyelocytic leukemia (APL) is still a matter of debate. We investigated incidence, associated clinical features, and prognostic implication of FLT3-ITD, but also FLT3-D835 point mutation and N-Ras or K-Ras mutations in 119 APL patients, all prospectively enrolled in the two consecutive APL-93 and APL-2000 trials. Mutation incidences were 38, 20, and 4%, for FLT3-ITD, FLT3-D835, and Ras, respectively. The presence of FLT3-ITD was associated with high white blood cell count, high Sanz index, M3-variant subtype, and V/ S PML- RAR alpha isoforms. Complete remission (CR), induction death, and death in CR rates were not affected by FLT3 or Ras mutations, as well as cumulative incidence of relapse. However, a trend for a shorter overall survival (P = 0.09) was observed in FLT3-ITD patients, because of a very poor postrelapse survival (P = 0.02). This feature, which has been also reported in patients with AML in general, is suggestive of an underlying genetic instability in FLT3-ITD patients, leading to the acquisition of additional unknown bad-prognosis gene mutations at relapse.

Despite modern regimen of chemotherapy, one-third of children with acute lymphoblastic leukaemia relapse. Recent studies have shown that a high level of minimal residual disease (MRD) at the end of induction is associated with an increased risk of relapse. We have developed and validated a real-time PCR method (RQ-PCR) to quantify MRD. We monitored 57 patients using IgH and TCR (Vdelta2Ddelta3) genes rearrangements as PCR targets. RQ-PCR was performed with a primer and a TaqMan probe designed to consensus sequences in VH segments in combination with one allele specific oligonucleotide primer complementary to the junctionnal region. A sensitivity of 10(-4) was reached for 72% of the IgH alleles (n = 50) and for 54,5% of the Vdelta2Ddelta3 alleles (n = 22). We compared the results with those obtained by competitive PCR in 53 patients: no discordance between the two methods was observed. Seventeen patients were found positive (32%) and 27 negative (51%) with both techniques and 9 children (17%) were positive only with TaqMan technology. RQ-PCR is more sensitive and more specific than competitive PCR. We thus propose that RQ-PCR might be used in first intention for MRD analysis.

Sustained major molecular response in the absence of any antileukaemic therapy after dasatinib treatment and autologous peripheral blood stem cell transplantation in a patient with imatinib-resistant myeloblastic-phase chronic myeloid leukaemia

Hypercoagulable states have been reported to be associated with venous leg ulcers. In an attempt to investigate the prevalence of hypercoagulable states in patients with venous leg ulcers, we performed a prospective case-control study for the presence of coagulation defects in such patients, including resistance to activated protein C (APC), factor V Leiden mutation and a newly described mutation in factor II. Results were compared with those obtained in a control group. APC resistance was found in four of 33 patients tested, but only one was found to be heterozygous for the factor V Leiden mutation. Factor II mutation was found in two of 30 patients tested. Our findings show that the prevalence of coagulation abnormalities is not different in patients with venous leg ulcers from controls in our study, suggesting that only selected patients with venous ulcers and a history of recurrent deep venous thrombosis should be investigated for the presence of coagulation defects.

The TAL1 gene is disrupted by translocation or deletion (tal(d)) in up to 30% of T-cell acute lymphoblastic leukaemia (T-ALL), leading to aberrant transcriptional activation, as a SIL-TAL1 fused transcript in tal(d). It has been suggested that TAL1 transcription occurs in approximately 50% of a T-ALLs without apparent rearrangement. SIL-TAL1 was positive in 15/60 (25%) of T-ALL, whereas wild-type TAL1 transcripts were detected in all 13 SIL-TAL1 and in 19/43 (44%) T-ALL without SIL-TAL1. To investigate the cellular origin of TAL1 we exploited the fact that GATA1 and TAL1 are co-ordinately expressed in non-lymphoid haemopoietic cells, whereas only the latter is found in T-ALL. GATA1 was detected in 10/23 (43%) TAL1-negative T-ALLs but in 17/19 (89%) 'unexplained' TAL1-positive cases, suggesting a common non-lymphoid cellular origin. Immunocytochemical analysis with a TAL1-specific monoclonal antibody showed nuclear expression in the blasts of 10/34 (29%) cases, including 8/10 SIL-TAL1+ and two RT-PCR TAL1+, SIL-TAL1- cases. In the remaining cases TAL1 expression was restricted to a minor population (5%) of larger, strongly TAL1-positive cells which comprised erythroid cells, CD34+ CD3- precursors and an unidentified TAL1+ CD45- population which morphologically resembled monocytes/macrophages. We therefore suggest that appropriate diagnostic evaluation of T-ALL should include molecular detection of SIL-TAL1 transcripts and in situ immunocytochemical detection of TAL1 protein expression by leukaemic blasts. This approach will enable accurate analysis of the prognostic significance of TAL1 deregulation in T-ALL.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorders have recently been observed during treatment of rheumatoid arthritis and dermatomyositis with low-dose methotrexate.A patient with psoriasis developed a B-cell lymphoproliferative disorder during long-term treatment with low-dose methotrexate. The lymphoid cells expressed EBV latent membrane protein 1, and the EBV viral genome was present as demonstrated by in situ hybridization. Evaluation for EBV clonality showed that the lymph node contained clonal EBV DNA. Polymerase chain reaction studies confirmed that the B-cell lymphoproliferative disorder was mainly monoclonal, suggesting that the disorder arose from a single EBV-infected B-cell clone.Lymphoproliferative disorders associated with Epstein-Barr virus in which the clinicopathological presentation is similar to those occurring in patients after transplantation may be observed in patients with psoriasis treated with methotrexate. While it is impossible to rule out a fortuitous occurrence of an EBV-associated lymphoproliferative disorder and psoriasis treated with methotrexate in the same patient, EBV appears to be critical in the pathogenesis of the lymphoproliferative disorder in this patient.

Granulomatous slack skin disease (GSS) is a rare disorder characterized by bulky cutaneous lesions and epithelioid and giant cell granulomas with destruction of the dermal elastic tissue. We detail the observation of a 29-year-old man with clinical and histological features of GSS. Pendulous skin tumors were associated with typical clinical and immunohistochemical aspects of mycosis fungoides and with clonal rearrangement of the V gamma T-cell receptor gene in lesional skin. This case report supports cutaneous T-cell lymphoma as a cause of GSS.

Acute myelomonocytic leukemia with bone marrow eosinophilia (AML-M4Eo in the French-American-British FAB] classification) is frequently associated with pericentric inversion of chromosome 16, inv(16)(p13q22). Recently, the molecular cloning of teh breakpoints has led to the identification of the two fused genes, CBFB on 16q and MYH11 on 16p. We have analyzed 24 patients with AML-M4Eo at diagnosis and 47 patients with AML of other FAB subtypes, by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the CBFB/MYH11 fusion mRNAs. Three types of fusion mRNAs were detected in 22 samples of AML-M4Eo (type A, n = 20; type C, n = 1; and type D, n = 1). Among these 22 positive samples, inv(16) was found in the 20 cytogenetically studied cases. No fusion transcript was detected in two patients with AML-M4Eo and in patients with other types of AML. These results confirm that CBFB/MYH11 transcripts (with a predominant type A form) are present in most cases of inv(16) AML. Moreover, detection of the hybrid transcript is closely associated with the finding of abnormal bone marrow (BM) eosinophils in AML-M4Eo as it is not found in other, FAB subtypes of AML, including AML-M4. To assess the presence of type A CBFB/MYH11 fusion transcripts in five AML-M4Eo patients in remission, we designed a sensitive assay combining nested PCR and allele-specific amplification (NPASA). Residual leukemia cells were detected in four patients who were in remission from 4 to 22 months, but not in one patient in long-term remission (5 years). The clinical relevance of persistent CBFB/MYH11 fusion transcripts in remission remains to be established by studying a large prospective series of patients. NPASA provides a useful and sensitive tool for the detection of minimal residual disease in inv(16) AML and, potentially, in other leukemias associated with translocations that result in a predominant fusion transcript.

Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V-(D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.

PURPOSE: Most studies using various reverse-transcription polymerase chain reaction (RT-PCR) techniques reported that the detection of the AML1-ETO fusion transcript was a common finding in long-term complete remission (CR) in acute myeloid leukemia (AML) with t(8;21) translocation. However, larger prospective studies with interlaboratory quality control may be important to investigate more precisely the clinical usefulness of studying minimal residual disease with RT-PCR in t(8;21) AML. PATIENTS AND METHODS: We collected 223 marrow samples from 51 patients with t(8;21) AML diagnosed in five centers and tested all samples by two different RT-PCR techniques (a nested technique and a one-step technique, with a sensitivity of 10−6 and 10−5, respectively) in two different laboratories. RESULTS: Samples from 14 patients in long persistent CR (median follow-up duration, 112 months) were taken at least twice, and all were PCR-negative by both techniques. Samples were prospectively taken from 37 patients after achievement of first CR and/or second CR, before intensive consolidation treatment, and every 3 to 6 months after completion of therapy. Patients who converted to PCR negativity with the one-step technique (60%) or both techniques (48%) after CR achievement had a longer CR duration than those with persistently positive PCR results (two-sided log-rank test, P = .0001). Patients who became PCR-negative with the one-step technique before intensive consolidation (23%) had a lower relapse rate (11% v 72%) and a longer CR duration than those who remained persistently PCR-positive at that point (two-sided log-rank test, P = .0015). CONCLUSION: Patients with AML with t(8;21) in long-term remission were all PCR-negative. In prospectively studied patients, a good correlation was found between negative PCR results and absence of relapse. Early negative results with the one-step RT-PCR technique, before consolidation treatment, seemed to carry an especially good prognosis, suggesting that RT-PCR analysis could help in choosing the type of consolidation therapy in patients with t(8;21) AML.

Background: Epstein-Barr virus (EBV)-associated lymphoproliferative disorders have recently been observed during treatment of rheumatoid arthritis and dermatomyositis with low-dose methotrexate. Observation: A patient with psoriasis developed a B-cell lymphoproliferative disorder during long-term treatment with low-dose methotrexate. The lymphoid cells expressed EBV latent membrane protein 1, and the EBV viral genome was present as demonstrated by in situ hybridization. Evaluation for EBV clonality showed that the lymph node contained clonal EBV DNA. Polymerase chain reaction studies confirmed that the B-cell lymphoproliferative disorder was mainly monoclonal, suggesting that the disorder arose from a single EBV-infected B-cell clone. Conclusions: Lymphoproliferative disorders associated with Epstein-Barr virus in which the clinicopathological presentation is similar to those occurring in patients after transplantation may be observed in patients with psoriasis treated with methotrexate. While it is impossible to rule out a fortuitous occurrence of an EBV-associated lymphoproliferative disorder and psoriasis treated with methotrexate in the same patient, EBV appears to be critical in the pathogenesis of the lymphoproliferative disorder in this patient. Arch Dermatol. 1997;133:867-871

Abstract 2506 Introduction: The development of massively parallel sequencing technologies has led to the identification of somatic DNA methyltransferase 3A (DNMT3A) gene mutations in acute myeloid leukemia (AML), with the highest frequency being found in cytogenetically normal (CN) AML. DNMT3A mutations have been suggested to predict poor clinical outcome in AML, but only few data are available on their prognostic significance within CN-AML. The aim of this study was to determine the frequency, the main associated features, and the prognostic significance of DNMT3A mutations in CN-AML. Patients and methods: This retrospective study was performed in 123 young adult patients (16–60 years) with previously untreated primary CN-AML and enrolled on two concomitant protocols of the Acute French Leukemia Association (ALFA), the ALFA-9801 and ALFA-9802 trials. DNMT3A mutations were screened on genomic DNA by PCR and direct Sanger sequencing. We focused our screening on the 3 conserved domains of DNMT3A (the proline-tryptophane-tryptophane-proline (PWWP) domain, the ADD-type zinc finger domain, and the C5-methyltransferase domain), corresponding to exons 8–9 and 11–23. The patients were also assessed for the presence of FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), NPM1, CEBPA, WT1, IDH1, and IDH2 mutations. Results: Thirty-eight DNMT3A mutations were identified in 36 of the 123 (29%) patients. These alterations consisted of 36 nucleotide substitutions and 2 frameshift deletions. Thirty out of 36 (83%) nucleotide substitutions affected the amino acid residue R882 (R882H, n = 21; R882C, n = 7; R882P, n = 2), 5 represented other missense alterations, and 1 was a nonsense mutation. Two patients exhibited 2 heterozygous missense mutations in different exons, and one patient had a homozygous missense mutation. DNMT3A mutated and wild-type cases did not differ in terms of age, gender, and white blood cell (WBC) count at presentation. DNMT3A mutations were strongly associated with the French-American-British (FAB) subtypes M4/M5 (P =.0002) and the presence of NPM1 mutations (P =.0006), and tended to often co-occur with IDH1R132 mutations (P = .09). In the entire cohort, complete remission rate was found lower in DNMT3A mutated patients than in DNMT3A wild-type patients, but without reaching statistical significance (80% vs 90%, P =.24). DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P =.02) and overall survival (5-year OS: 23% vs 45%, P =.02) compared to DNMT3A wild-type patients. We next performed subgroup analysis according to the NPM1/FLT3-ITD genotypes. In patients with the non-favorable genotypes, that is NPM1 mutated/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD negative (n = 86), 18 (21%) had a concomitant DNMT3A mutation. In this high-risk subgroup of CN-AML, DNMT3A mutations conferred a worse clinical outcome (5-year EFS: 0% vs 23%, P =.02; 5-year OS: 14% vs 37%, P =.06). In patients with the favorable genotype NPM1 mutated/FLT3-ITD negative (n = 37), 18 (49%) were found to display a concomitant DNMT3A mutation. Within this favorable subgroup, patients carrying a DNMT3A mutation had a significantly inferior EFS and OS compared to DNMT3A wild-type patients (5-year EFS: 25% vs 65%, P =.01; 5-year OS: 29% vs 76%, P =.02). Furthermore, in multivariate analysis including age, WBC count, NPM1/FLT3-ITD genotypes, and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS (hazard ratio = 2.29; 95% CI, 1.42 to 3.70; P =.0007) and OS (hazard ratio = 2.34; 95% CI, 1.37 to 4.00; P =.002). Conclusion: DNMT3A mutations are one of the most common gene mutations in CN-AML and independently predict poor clinical outcome. Testing for DNMT3A mutations could help further improve risk stratification in CN-AML. Disclosures: No relevant conflicts of interest to declare.