Interleukin-33 (IL-33) is a central regulator of immune responses and inflammation, and genetic variation in IL33 and its receptor IL1RL1 (ST2) is strongly linked to disease susceptibility, notably asthma. Emerging evidence suggests that IL-33 also influences hematopoietic processes and platelet biology, indicating functions beyond canonical immunity. We investigated two missense variants identified in a patient with unexplained thrombocytopenia: a rare novel IL33 variant, c.385T>C (p.Tyr129His) and the common IL1RL1 variant c.1501_1502CA>AG (p.Gln501Arg), previously associated with reduced IL-33 signaling. Structural modeling revealed that IL-33 Y129H disrupts a conserved hydrogen bond within the IL-33/ST2/IL-1RAcP ternary complex, destabilizing receptor engagement. Functional assays confirmed markedly reduced binding affinity and biological activity, establishing Y129H as a loss-of-function variant. The IL1RL1 Q501R variant affects the Toll/IL-1 receptor (TIR) domain, critical for recruiting adapter proteins such as MyD88. Modeling revealed pronounced perturbation in a peripheral helix of the TIR domain, potentially impacting adapter recruitment and downstream signaling, providing a mechanistic basis for its protective association with asthma. Together, these findings provide structural and functional insights into clinically relevant IL33 and IL1RL1 variants. Their simultaneous occurrence in a patient with thrombocytopenia further supports a potential role for this pathway in platelet homeostasis and stress-responsive hematopoiesis.

Introduction: Acute myeloid leukemia (AML) post myeloproliferative neoplasm (MPN) have very poor prognosis and are often excluded from most clinical trials. Methods: We retrospectively collected data from 166 patients, including 156 with available treatment data and 83 with NGS data treated in France and USA. Results: 2022 ELN risk categories were favorable, intermediate and adverse in 3 (2%), 17 (13%), and 110 (85%), respectively. Overall response rate was 57%, 20%, and 25% in patients treated by intensive chemotherapy (IC), hypomethylating agents (HMAs) and BSC (including low-intensity treatments as hydroxyurea and low-dose cytarabine), respectively. A total of 31 (22%) patients underwent allogeneic stem cell transplantation (ASCT). Median overall survival (OS) was 7.2 months without significant difference between IC and HMA (9.5 and 8.6 months, respectively). OS was significantly improved in patients allografted (6.7 vs. 1.3 months, respectively, p < 0.001). Even though 2017 and 2022 ELN risk categories were not prognostic for OS, we observed a prognostic impact on OS of Lindsley’s classifier (p = 0.014). In multivariate analysis, JAK2 mutation was associated with worse OS (p = 0.037), whereas SRSF2 showed a trend toward adverse prognosis (p = 0.057). Among functional groups, only spliceosome mutations predicted poor prognosis (p = 0.015). Conclusion: We confirmed poor prognosis of AML post-MPN. Classical prognostic classification was not validated in our cohort. We observed poor outcome using IC or HMA encouraging us to propose new clinical trials in this specific subgroup. Only ASCT was able to improve prognosis.

Introduction:Histiocytic sarcoma (HS) is a rare neoplasm derived from non-Langerhans histiocytic cells, exceptionally arising from B-ALL. Methods:We present the case of a child with high-risk B-ALL with PAX5 P80R mutation. Results:Despite initial remission, a chemoresistant paravertebral mass was identified as HS. A shared IGK/TCRB rearrangements and PAX5 alterations between the leukaemic and histiocytic clones suggested transdifferentiation driven by PAX5. A somatic MAP2K1 mutation in the HS component prompted selumetinib treatment, leading to a rapid response. Conclusion:This case underscores the role of PAX5 in lineage plasticity and highlights the potential of targeted MEK inhibition in MAPK-driven HS arising from B-ALL. Trial Registration:The authors have confirmed clinical trial registration is not needed for this submission.

ABSTRACT:Intensified chemotherapy regimens have improved outcomes in adults with Philadelphia chromosome-negative (Ph-) B-cell acute lymphoblastic leukemia (B-ALL), yet relapse remains a major cause of treatment failure and death. Blinatumomab is a T-cell engager that demonstrated marked activity in relapsed and measurable residual disease-positive (MRD+) B-ALL, as well as in frontline consolidation for MRD- patients. The phase 2 GRAALL-2014/B-QUEST substudy evaluated the integration of blinatumomab into consolidation and maintenance therapy for adults with high-risk (HR) B-ALL. Between 2015 and 2020, 489 adults aged 18 to 59 years with newly diagnosed Ph- B-ALL were enrolled, and among these 259 were classified as HR (presence of KMT2A-r, IKZF1 deletion, or end-of-induction MRD ≥ 10-4). A total of 94 patients with HR were enrolled in the QUEST study (2018-2020) and received up to 5, 28-day cycles of blinatumomab during consolidation and maintenance. In addition, 90 patients with HR who were treated before QUEST activation without blinatumomab served as controls in a post hoc analysis. Baseline characteristics were comparable between groups. Blinatumomab consolidation significantly improved MRD clearance, reduced relapse, and prolonged survival. At 5 years, cumulative incidence of relapse, disease-free survival (DFS), and overall survival were 23%, 68%, and 79% in the blinatumomab group compared with 49% (P = .001), 42% (P = .001), and 60% (P = .03) in controls. Patients eligible for allogeneic hematopoietic stem cell transplantation (alloHSCT) derived overall benefit. However, no clear additional DFS advantage was observed among those who ultimately underwent transplantation. These findings support the frontline integration of blinatumomab and warrant prospective evaluation of transplantation strategies in this setting. This trial was registered at www.clinicaltrials.gov as #NCT03709719.

Abstract FLT3 internal tandem duplications (FLT3-ITD) are major genetic events in acute myeloid leukemia (AML). Although the clinical impact of FLT3-ITD “macroclones” (allelic ratio [AR] ≥0.05) is well established, the significance of low-level FLT3-ITD subclones (“microclones”) remains uncertain. We conducted a post hoc analysis of 1733 patients with newly diagnosed AML enrolled in the Backbone Intergroup 1 trial (ClinicalTrials.gov identifier: NCT02416388). Using next-generation sequencing (NGS), we detected FLT3-ITD microclones (AR between 0.0004 and 0.05) in 17.4% of patients without FLT3-ITD macroclones. Microclones and macroclones (low and high AR) were independently associated with increased relapse risk (cause-specific hazard ratio, 1.50 [95% confidence interval (CI), 1.18-1.91]; 1.98 [1.50-2.62]; and 2.33 [1.69-3.22], respectively) after adjustment for age, white blood cell count, other gene mutations, midostaurin treatment, and allogeneic hematopoietic stem cell transplantation. At 2 years, the cumulative incidence of relapse reached 42.5% (95% CI, 37.0-47.9) in patients with macroclones, 45.1% (38.3-51.6) in patients with microclones, and 29.4% (26.6-32.3) in patients without FLT3-ITD. In NPM1-mutated AML, both microclones and macroclones were associated with higher levels of measurable residual disease (MRD) and increased relapse risk, without independent impact on overall survival after adjustment for MRD. An analysis of paired samples further revealed that 41.8% of relapses in patients with FLT3-ITD microclones at diagnosis were associated with a macroclone at relapse. These findings challenge current risk stratification models and support the integration of NGS-based FLT3-ITD detection into the diagnostic and prognostic workflow for AML. Prospective trials addressing the management of patients with FLT3-ITD microclones are warranted, as is their consideration in future European LeukemiaNet guidelines.

The addition of midostaurin (MIDO) to intensive chemotherapy (IC) improves survival in younger adults with FLT3-mutated acute myeloid leukemia (AML); however, real-world data in elderly patients (≥ 60 years) are limited. This large, retrospective, multicenter study from three European registries (PETHEMA, FILO, DATAML) evaluated MIDO+IC (n = 194) versus IC alone (n = 371) in 565 patients with FLT3-mutated AML aged ≥ 60 years (median age 67.5 years; 35.6% ≥ 70 years). MIDO+IC was associated with lower day-60 early death (8.2% vs. 21.4%, p < 0.0001) and higher composite complete remission (CRc) rates (78.9% vs. 63.1%, p < 0.0001). After a median follow-up of 46.0 months, median overall survival (OS) was 24.2 months for MIDO+IC versus 8.7 months for IC (p < 0.0001), with 5-year OS rates of 40.6% vs. 12.9%, respectively. Event-free survival (EFS; median 13.5 vs. 4.6 months; 5-year EFS: 36.0% vs. 10.1%) and relapse-free survival (RFS; median 20.2 vs. 8.0 months; 5-year RFS: 45.4% vs. 15.7%) were also significantly improved (both p < 0.0001). The 5-year cumulative incidence of relapse was lower with MIDO+IC (47.8% vs. 67.1%, p < 0.001). In multivariate analyses, midostaurin was an independent favorable prognostic factor for CRc (aOR 1.97 [95% CI: 1.29-2.98]), OS (aHR 0.46 [95% CI: 0.36-0.58]), EFS (aHR 0.49 [95% CI: 0.39-0.60]), and RFS (aHR 0.47 [CI: 0.36-0.62]) (all p ≤ 0.002). These benefits were confirmed by propensity score matching. This large real-world study demonstrates that combining midostaurin with IC significantly improves remission rates and survival outcomes in elderly patients with FLT3-mutated AML, supporting its consideration in this population.

ABSTRACT:The Group for Research in Adult Acute Lymphoblastic Leukemia (GRAALL)-2014 trial evaluated an intensive, age-adapted protocol for adults aged 18 to 59 years with Philadelphia chromosome negative acute lymphoblastic leukemia. The trial was motivated by findings from the previous GRAALL-2005 study, which reported excessive toxicity from pediatric-inspired therapy in older patients and no added benefit from allogeneic hematopoietic stem cell transplantation (allo-HSCT) among those with an early favorable response to treatment. Thus, the GRAALL-2014 protocol aimed to reduce treatment-related toxicity in patients aged ≥45 years and to limit allo-HSCT to patients with poor measurable residual disease (MRD) responses. A total of 743 patients were included, and outcomes were compared with those of GRAALL-2005 trial. The GRAALL-2014 study demonstrated reduced early mortality and higher complete remission rates in patients aged ≥45 years. MRD-guided transplantation decisions reduced allo-HSCT indications by ∼50%. Although older patients experienced a higher cumulative incidence of relapse, no significant difference in disease-free survival (DFS) was observed compared with historical cohorts across age subgroups. The overall 4-year DFS was 57.1% (95% confidence interval [CI], 53.4-61.1). Notably, 4-year overall survival improved significantly, from 65.5% (95% CI, 61.7-69.8) to 71.7% (95% CI, 67.7-76.0) in younger patients (P = .031) and from 49.6% (95% CI, 43.5-56.5) to 59.5% (95% CI, 53.5-66.3) in older patients (P = .011). These findings highlight the value of individualized treatment strategies that balance efficacy and safety. Future studies should investigate the integration of immunotherapy to further reduce treatment intensity and improve outcomes. This trial was registered at www.clinicaltrials.gov as #NCT02617004 and #NCT02619630.

We report a large cohort of 95 adult patients with acute myeloid leukemia (AML) harboring NUP98 rearrangements (NUP98r). Patient characteristics included a young age (median 50 years [IQR 38-64]), 20% of therapy-related AML, a high WBC count (median 52×109/L), normal karyotype in 32%, FLT3-ITD in 48% and WT1 mutations in 34%. NUP98::NSD1 fusion was the most common (54%), and these patients were significantly younger (41 y vs. 61 y), had more de novo AML (94% vs. 64%), higher rates of normal karyotypes (56% vs. 4.5%), FLT3-ITD (76% vs. 18%) and WT1 mutations (50% vs. 16%) than other NUP98r AML. The median overall survival (OS) for the entire cohort was 15.2 months (95% CI, 11.9-20.8) and event-free survival was 5.8 months (2-7.5). Among patients treated intensively (n = 73), age (HR = 2.7), FLT3 inhibitor therapy (HR = 0.45) and hematopoietic stem cell transplant (HR = 0.5) influenced OS in univariate analysis. Compared with NUP98 wild-type (WT) AML, NUP98r patients had a prognosis more similar to that of NUP98 WT ELN adverse patients whether initially classified as intermediate (20.3 months [11.7-30.2]) or adverse (15.7 months [13.5-42.9]). However, treatment with FLT3 inhibitors improved prognosis, with median OS not reached and 5-year OS of 53.3%, approaching that of intermediate-risk patients.

The E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL) promotes positive selection and antigen responses in mouse T lymphocytes by ubiquitinating ZAP70. Conversely, mouse CBL and CBL-B mutually redundantly regulate SYK ubiquitination and B cell receptor signaling. Here we studied individuals with somatically homozygous CBL loss-of-function variants in leukocytes. Human CBL is largely redundant for the development and function of human T cells. Conversely, B cell development is altered at the immature stage, with a tenfold increase in transitional cells, enhanced survival of autoreactive clones and impaired tolerance manifested by autoantibody production. B cell maturation is intrinsically impaired by reduced apoptosis and dysregulated B cell receptor signaling. CBL deficiency impairs humoral immunity by limiting memory B cell formation and reducing class switching and somatic hypermutation. Consequently, antigen-specific B cell generation and adaptive immune memory are disrupted, predisposing individuals to infection. Human CBL is critical for B cell development and function but redundant for T cell biology.

Abstract B‐cell acute lymphoblastic leukemia (B‐ALL) is a heterogeneous malignancy driven by diverse genetic alterations. Among these, CEBP family genes and ZEB2 are recurrently involved, yet the spectrum of genomic mechanisms and their clinical impact remain incompletely defined. Integrated genomic analyses of a cohort of 992 Philadelphia‐negative adult B‐ALL patients revealed multiple mechanisms of enhancer hijacking‐mediated deregulation of CEBPA, CEBPB, CEBPD, and CEBPE, including IGH and several non‐IGH fusions, as well as noncoding mutations in regulatory regions. Combined with gene expression analysis, we identified three distinct subtypes, defined by co‐occurring CEBP and ZEB2 p.H1038R alterations (CEBP/ZEB2, n = 18 cases); isolated CEBP alterations (CEBPalt, n = 43), associated with frequent IKZF1 deletions and FLT3 deregulation; and isolated ZEB2 p.H1038R mutation (ZEB2alt, n = 15), associated with various additional genomic hits targeting ZEB2 and enhancing mutant ZEB2 expression. The three subtypes exhibited distinct clinical features, including age distribution (patients with CEBP/ZEB2 and ZEB2alt B‐ALL were younger) and sex bias (female and male predominance in CEBPalt and ZEB2alt, respectively). Early treatment responses and outcomes also differed: patients with CEBP/ZEB2 B‐ALL had a favorable early response, in contrast to patients with ZEB2alt B‐ALL, who had high levels of minimal residual disease and a dismal prognosis. Collectively, our findings define CEBP and ZEB2 alterations as drivers of genetically and clinically distinct subtypes of adult B‐ALL and provide a rationale for subtype‐specific risk stratification. Preclinical experiments in CEBPalt B‐ALL patient‐derived xenografts demonstrated sensitivity to FLT3 inhibition, highlighting a potential therapeutic vulnerability.

IntroductionMRD assessment in AML provides a quantitative approach to define a deeper remission status and refine the risk assessment of postremission relapse. Currently, the two most extensively validated methodologies are multiparameter flow cytometry-based MRD (MFC-MRD) and molecular MRD (Mol-MRD) assessed by quantitative PCR (qPCR). The latest update of the ELN recommendations on MRD includes new technical guidance for standardized MFC-MRD and Mol-MRD analyses, MRD thresholds, definitions of MRD response, and recommendations for clinical application. In this context, we evaluate the clinical utility of MRD assessment using MFC and qPCR in patients with NPM1-mutated AML.MethodsBetween January 1, 2015, and September 30, 2024, 506 adult patients (≥18 years) with NPM1-mutated AML were included in the DATAML registry. Immunophenotyping at diagnosis was available for 383 patients from Toulouse University Hospital and 123 patients from Bordeaux University Hospital. Among them, 306 patients received a standard intensive chemotherapy regimen. 270 patients achieved complete remission (CR/CRi), and 194 of these were monitored for post-induction bone marrow minimal residual disease (MRD1) using both MFC-MRD and qPCR techniques.MFC analysis of leukemic cells was performed on fresh bone marrow samples. For leukemic bulk analysis, the antibody panel included CD34, CD13, CD38, CD7, CD33, CD56, CD117, HLA-DR, and CD45, as recommended by ELN. MFC-MRD data were analyzed using the FlowSOM algorithm (Vial et al., Cancers, 2021). The threshold for MRD1 positivity was defined as ≥0.1% for MFC-MRD and ≥2% for low-level (LL) Mol-MRD.Results194 NPM1-mutated AML patients in first CR/CRi after induction chemotherapy were included. Main co-mutations were: DNMT3A (38%), FLT3-ITD (34%) IDH1 (18%), IDH2 (14%) and FLT3-TKD (14%). 31 patiens (16%) had NPM1/DNMT3A/FLT3-ITD mutations.The median follow-up (FU) was 40 months. In patients with positive MFC-MRD, 1-y and 3-y OS was 83% and 67%, vs 93% and 82% in patients with negative MFC-MRD (Hazard Ratio [HR]: 2.39, p=0.0056). The 3-y cumulative incidence of relapse estimation (CIR) was 55% for MFC-MRD positive patients vs 30% for MFC-MRD negative patients (HR: 3.17, p<0.0001).In patients with LL Mol-MRD, 1-y and 3-y OS rates were 83% and 59% vs 93% and 85% in patients with negative Mol-MRD (HR: 3.52, p=0.0001). The 3-y CIR estimation was 58% in LL Mol-MRD positive patients vs 30% in Mol-MRD negative patients (HR: 3.01, p<0.0001).The combination of Mol-MRD and MFC-MRD identified four distinct patient groups: double negative (-/-; n=74, 38%), MFC-MRD positive only (-/+; n=49, 25%), Mol-MRD positive only (+/-; n=41, 21%), and double positive (+/+; n=30, 15%). At 3 years, OS estimates were 90%, 77%, 72% and 48% respectively in these 4 subgroups (p<0.0001). CIR estimates were 18%, 45%, 49% and 71% respectively (p<0.0001). In multivariate analysis, both LL Mol-MRD positivity and MFC-MRD positivity were independently and significantly associated with inferior OS (HR: 3.59, p<0.001; HR: 2.16, p=0.014, respectively) and increased CIR (HR: 2.82, p<0.001; HR: 2.30, p=0.001, respectively) whereas DNMT3Am, FLT3-ITD or allo-HSCT did not retain statistical significance.ConclusionIn NPM1 mutated AML, where transcript levels typically decline rapidly after induction, FlowSOM assisted MFC-MRD yields prognostic information that is complementary to qPCR. Combining both assays at post-induction (MRD1) delineates four clinically distinct groups, ranging from a double-negative cohort with excellent outcomes (3-year OS=90%, CIR 18%) to a double-positive cohort at very high risk (3-year OS=48%, CIR 71%). MFC-MRD positivity at MRD1 remained independently associated with inferior OS and higher relapse, even after adjusting for molecular MRD. Although larger prospective studies are needed, these findings strongly suggest that MFC-MRD may not be redundant in NPM1-mutated AML. Notably, when integrated with qPCR, it could improve the accuracy of predicting early relapse. These results support the incorporation of dual-modality MRD monitoring into prospective testing.

Supplemental figure 1. Flow chart for sample availability of patients included in our study and their outcome. Supplemental figure 2. Supplemental figure 3. Size of LSC nodes in CD34+CD38- subpopulation in AML-CD34+ and AMLCD34- populations. Mann-Whitney test. Supplemental figure 4. LSC phenotypic profiles according to MRD status. Normalized MFI of CD34, CD38, CD45RA, CD135 and CD133 in LSC nodes used to measure LSC MRD. Mann-Whitney tests. Supplemental figure 5. Landmark analysis at 6 months of the impact of HSCT according to MRD status. Supplemental figure 6. Evaluation of immunophenotypic markers in chemoresistant and chemosensitive AML patients at diagnosis. Supplemental figure 7. Evaluation of LSC phenotypic markers in clinically relevant AML patients in relation to Figure 4.

Introduction: FLT3 internal tandem duplications (ITD) are among the most frequent genetic alteration, found in approximately 30% AML, and are associated with a high risk of relapse and death. Measurable residual disease (MRD) NGS-based monitoring of FLT3-ITD mutations offers a new opportunity to detect leukemic cells during treatment. Here we aimed to retrospectively assess the prognostic value of FLT3-ITD MRD monitoring using a high sensitivity NGS-based approach in FLT3-ITD AML patients included in the AML-treated with ponatinib trial, a pan-BCR::ABL kinase inhibitor. Methods: The AML-ponatinib trial (Clinicaltrial ID: NCT02428543) included AML patients positive for FLT3-ITD by fragment analysis in complete remission. Patients then received consolidation courses (2 to 3) with the combination of ponatinib 30 mg per day and Cytarabine (HDAC for the 18-55y cohort A and IDAC for the 55-70y cohort B). Ponatinib was used as a weak FLT3 inhibitor (FLT3 IC50: 12.6 nM). In the first part of the work, FLT3-ITD MRD was monitored using 3 different techniques: 2 custom amplicon-based NGS, one according to Blätte et al (Leukemia, 2019) (AB technique), a second developed by the Toulouse team (Toulouse technique) and the last technique was a capture-based technique (capture technique) including the full sequence of FLT3 gene. For these 3 techniques, the detection and the quantification of the FLT3-ITD clones was performed using FiLT3R algorithm (4f569307). These 3 assays were compared to the reference FDA approved FLT3-ITD MRD NGS kit (IVS; Invivoscribe). Of note the 700 ng DNA input was similar for all the tests except for Toulouse (200ng). Results: Using 72 samples, the comparison between the 3 in-house techniques and the IVS kit showed the following results: 99%, 97% and 93% concordance for the AB technique, capture technique and Toulouse technique, respectively. Regarding the strict similarity between the AB technique with IVS, the same sensitivity (10-5), the good median average reads obtained on the NextSeq 1000 (Illumina) and the advantage of FiLT3r algorithm (identification of the sequence and breakpoint of the different clones), we decided to apply the AB technique to the samples of the PONATINIB-AML trial. Forty-seven patients were included, 27 in cohort A and 20 in cohort B. Median age was 57.8y (28-70), sex ratio was 0.4. NPM1 mutation was present in 36 patients (76.6%). FLT3 MRD was assessed in 40 out of 46 FLT3-ITD patients (87%). After induction (inclusion, timepoint 1 (TP1)), 25 (62.5%) and 20 (50%) of the patients achieved FLT3 MRD1 negativity at the threshold of 10-4 and 10-5 respectively. With a median follow-up of 4.1 years, FLT3 MRD negativity was associated with a strong benefit in overall survival (OS) (median survival not reached vs 1.2y in MRD1 positive patients, p=0.0003) with a 5y OS of 82.8% (95% CI:60.1-93.2). Sequential follow-up was performed in 31 patients with MRD post induction (TP1) and post consolidation 1 (TP2) with a threshold of 10-4. Median RFS was not reached, 0.8y and 0.35y for patients with neg/neg, pos/neg and pos/pos FLT3 MRD TP1/TP2 respectively (p=0.0001). NPM1 MRD was assessed at TP2 in 29 out of 36 NPM1 mutated patients (80%). Median RFS was not reached and 1y in NPM1 MRD negative and positive patients respectively (p=0.013). At TP2, MRD was assessed using NPM1 and FLT3 in 34 evaluable patients. No patients had NPM1 neg and FLT3 pos MRD. Median RFS was unreached, 1.2y and 0.2y for NPM1/FLT3 neg/neg, pos/neg and pos/pos MRD respectively (p=0.0001). Twenty-two patients relapses and 14 were evaluated for FLT3 MRD. Only 2 of them were negative, 9 had the same clone and 3 had a new FLT3-ITD clone emphasizing the major role of FLT3-ITD clones to trigger the relapse. Conclusion: In conclusion, using an external quality control based on 72 samples, we are able to identify a very good FLT3-ITD MRD technique and to validate the methodology to assess FLT3-ITD MRD with a level of sensitivity of 10-5/10-4. In this post hoc study, FLT3-ITD was a major predictor of OS and RFS, particularly if negative at TP1. Patients with delayed later negativity had lower OS and RFS. The negativity of NPM1 and FLT3-ITD MRD at TP2 is predictive of a better RFS. Our data strongly support the use of FLT3-ITD MRD to monitor FLT3-ITD AML patients.

The France Genomic Medicine Plan (PFMG) 2025 has established very high throughput genome sequencing (HTS; including paired germline/tumor WGS 60x, WES 150x, and RNAseq) in clinical care for patients with cancer within the framework of 12 indications since March 2020. Two of the predefined indications apply to pediatric malignancies and led to 1475 prescriptions until October 2024, i.e. 48% of those in all cancer patients. Prescription in the context of “treatment failure” (all tumor types) represents 64% of prescriptions. Initially established within the MOSCATO-01 and MAPPYACTS studies, a multidisciplinary national clinical molecular tumor board (CMTB) evaluates the conclusions of all HTS reports to propose personalized therapeutic interventions to clinical prescribers including information on available clinical trials. HTS identified at least one somatic molecular alteration considered potentially relevant for 90% of prescriptions at “treatment failure” (487/546). At least one adapted targeted therapy was recommended for 77% of the discussed cases (366/471). Recently, the multicenter study MAPPYACTS 2 (NCT05691608) accompanies PFMG to analyze the survival benefit of novel therapies identified by the CMTB. This study also allows the implementation of ancillary projects such as circulating biomarkers. For 5 pediatric cancers types for which HTS has been validated at diagnosis (leukemia, neuroblastoma, brain tumor, rare tumors, osteosarcomas), the contribution of HTS compared to tumor molecular analyses carried out within routine care is currently being evaluated via the PFMG vs Standard of Care CARE-Seq study. In the field of oncogenetics, analyses of “incidental” constitutional data from the HTS provides important insights allowing the discovery of a predisposition gene for cancers. Other more fundamental studies will include an analysis of VUS-type variations with integration of AI (same as the PCM4U adult project with classifier) ​​in pediatric tumors or exploration of molecular mechanisms of oncogenesis, or treatment resistance including the study of non-coding RNAs, the study of the structure around telomeres, centromeres, or tumor type-specific alterations. The clinical ‘pre-indications’ are subjected to a medico-economic analysis to determine which will eventually be covered by the French Health Insurance System and become ‘clinical indications’. The integrative molecular approach including HTS in standard of care constitutes a crucial tool for developing national or international collaborative research studies aimed at improving the care of children and adolescents with cancer. Ludovic Lacroix, Gaelle Pierron, Julien Masliah Planchon, Marc Barritault, Adrien Buisson, Alain Viari, Tiphaine Adam-De-Beaumais, Gaelle Tachon, Valery Attignon, Helene Cavé, Helene Lapillonne, Pascale Flandrin-Gresta, Eric Delabesse, Sylvie Tondeur, Mathilde Laubert, Isabelle Rochet, Imene Hezam, Silvana Beatriz Dangiolo, Samuel Abbou, Pablo Berlanga, Jacques Grill, Franck Bourdeaut, Natacha Entz-Werle, Pierre Leblond, Nicolas André, Gabriel Revon-Riviere, Antony Ceraulo, Marion Strullu, Pierre Rohrlich, Arnaud Petit, Stephane Ducassou, Lea Guerrini-Rousseau, Claudia Pasqualini, Gudrun Schleiermacher, Nadege Corradini, Birgit Geoerger. Very high throughput genome sequencing for pediatric cancers in France [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1204.

Introduction: Prognosis in relapsed acute myeloid leukemia (AML) patients (pts) is dismal, with no standard chemotherapy. Intermediate (I) or high (H)-dose cytarabine (DAC)-based regimens are common salvage treatments (Döhner, Blood 2022) leading to around 50% CR/CRi (Megías-Vericat, AJH 2018). Venetoclax (VEN)-azacitidine (AZA) is a front-line standard of care for unfit pts but is also widely used in pts with molecular relapse, primary induction failure (PIF) or morphologic relapse (Rel). In this study, we report the largest cohort of pts with AML treated with VEN-AZA compared to salvage I/HDAC-based intensive chemotherapy (IC) in first Rel. Methods: Inclusion criteria were 18-75y old AML pts, first Rel after front line IC, treated by I/HDAC-based IC or VEN-AZA between January 2015 and September 2023. Exclusion criteria were 3rd drug added to VEN-AZA, molecular Rel, CR1/CRi1 after 2 induction courses. Results: 347 pts from 9 centers treated with VEN/AZA (n=125, 36%) or I/HDAC (n=222, 64%) were included. 191 (55%) were male, median age was 63y (IQR 54.4-70; range 20-75) and 57y (IQR 45-63; range 19-73) in VEN-AZA and I/HDAC groups (p<0.0001), respectively; 29 (23.4%) and 15 (6.8%) pts had secondary AML, cytogenetic risk was intermediate in 83 (68%) and 172 (77.5%) (p=0.03); 17 (14.3%) and 82 (41%) pts had a NPM1 mutation (p<0.0001), in VEN-AZA and I/HDAC groups, respectively. Induction chemotherapy was 3+7 including 15 (4.3%) CPX-351. All pts were in CR1/CRi1 and post remission strategy was I/HDAC-based for 245 (74.7%) pts whereas other pts received non-IC. Finally, 49 (39.2%) and 39 (17.5%) pts underwent an allogeneic stem cell transplantation (HSCT) in CR1/CRi1 (p<0.0001), in VEN-AZA and I/HDAC groups, respectively. All pts relapsed after a median delay of 12 months (IQR 7-22; range 1-226), 73 (21%) pts ≤6 months and 274 (79.0%) pts >6 months. Performans status (PS) at relapse was 0-1 in 247 (86.7%) pts and ≥2 in 38 (13.3%) pts. Median WBC at relapse were 3x109/l (IQR 1.9-7.1; range 0.3-265.7). There was no significant difference between the 2 groups for these characteristics. Finally, at relapse, 76 (60.8%) and 147 (66.2%) pts obtained a CR2/CRi2 in VEN-AZA and I/HDAC-based salvage groups, respectively (p=0.35). Pts received a median of 2 cycles of VEN-AZA (IQR 2-5; range 1-21), best response has been considered whatever the time. Early death during first month occurred in 18 (14.4%) and 20 (9.0%) pts in VEN-AZA and I/HDAC groups, respectively (p=0.15). Bridge to transplant has been obtain for 31 (24.8%) and 120 (54.1%) pts in VEN-AZA and I/HDAC group, respectively (p<0.0001). Multivariate logistic regression for factors associated with CR2/CRi2 included gender, sAML, cytogenetic, FLT3-ITD and NPM1, consolidation in CR1/CRi1, previous HSCT, age at Rel, VEN-AZA or I/HDAC, PS at relapse. Independent factors associated with CR2/CRi2 were female (OR 0.55, CI95% 0.32-0.94, p=0.03) and PS 0-1 at Rel (OR 0.35, CI95% 0.17-0.76, p=0.008). After a median FU for alive pts from Rel of 21 months, median OS was 15 and 16 months; 1y-OS was at 51.7% and 55.3%, in VEN-AZA and I/HDAC groups (p=0.19), respectively. Cox model for factors associated with OS included also time to relapse and HSCT in CR2/CRi2 and found secondary AML (aHR 1.73, CI95% 1.18-2.56, p=0.006), adverse cytogenetic risk (aHR 1.52, CI95% 1.07-2.16, p=0.02), time to relapse >6 months (aHR 0.38, CI95% 0.28-0.52, p<0.0001) and HSCT in CR2/CRi2 (aHR 0.28, CI95% 0.21-0.39, p<0.0001). Median LFS was 14 months in both groups; 1y-LFS was at 55.5% and 52.9%, in VEN-AZA and I/HDAC groups (p=0.95), respectively. Cox model for factors associated with LFS were time to relapse >6 months (aHR 0.37, CI95% 0.23-0.59, p<0.0001) and HSCT in CR2/CRi2 (aHR 0.39, CI95% 0.27-0.57, p<0.0001). Conclusion: This cohort is characterized by first relapse including three quarters of relapse > 6 months, no PIF, no previous exposure to HMA for AML and mostly intermediate risk cytogenetics. Salvage treatment allowed high CR2/CRi2 rates, close to those observed in front-line for VEN-AZA and at the upper limit for salvage treatment based on I/HDAC. These high response led to interesting bridge to transplant rates in both groups and long LFS and OS. These results raise the question of prospective evaluation of non-intensive chemotherapy in first relapse for AML patients.

Supplementary Table 1. Karyotypes of LH-ALL patients at diagnosis. Supplementary Table 2. Somatic variants detected in LH-ALL patients at diagnosis. Supplementary Table 3. ARCH related variants detected in LH-ALL patients at remission. Supplementary Table 4. Minimal residual disease values of remission samples used for mutation analysis. Supplementary Table 5. Somatic alterations in cell populations from diagnostic samples (BMMC) based on single-cell analyses. Supplementary Table 6. Somatic alterations in FACS-sorted cell populations from diagnostic samples. Supplementary Table 7. Panel of genes for targeted sequencing. Supplementary Table 8. Single cell DNA amplicons for genotyping and LOH analyses. Supplementary Table 9. Single cell DNA amplicons for B-ALL clono-specific IG/TR detection. Supplementary Table 10. ADT-seq panel and spike-in antibodies. Supplementary Table 11. Single cell sequencing metrics.

IntroductionResults from pivotal clinical trials evaluating the combination of intensive chemotherapy (IC) with inhibitors of IDH1, IDH2, menin, FLT3 and BCL2 are expected in the coming years. These results could transform the therapeutic landscape for AML patients (pts) fit for IC. However, although some of these studies are placebo-controlled, interpreting phase 3 results in the field of AML is often tricky since consolidation treatments may include allo-HSCT, which has a very different mechanism of action, highly effective in preventing relapse, but also potentially toxic. Furthermore, salvage treatments including targeted agents and/or allo-HSCT for induction failure or relapse may also be curative. Traditional evaluation criteria such as OS, EFS, and RFS, are therefore potentially influenced by both therapeutic interventions. We postulate that any new molecules added to IC must be effective enough to significantly increase the proportion of patients who are cured after first-line treatment without the need for allo-HSCT in first CR or additional salvage therapy. Therefore, we propose to define a composite endpoint to analyze the specific impact of new drugs in the frontline treatment of AML: the salvage-free, transplantation-free survival (STFS). In this study, we evaluated this endpoint in a cohort of pts treated with IC with a special focus on subgroups for which significant progress is expected soon.MethodsInclusion criteria for the AML cohort: ND AML between 01/2010 and 12/2022, ≥18y, treated with IC. Exclusion criteria: pts treated in clinical trial assessing novel drugs. Inclusion criteria for the APL cohort (used as control): ≥18y, between 01/2010 and 12/2022, first-line treatment including arsenic trioxide (ATO) and ATRA. EFS, RFS and OS were calculated according to ELN 2017 definition. STFS was measured from day 1 of induction chemotherapy to the date of induction failure after 1 or 2 cycles, salvage therapy, morphologic or molecular relapse, allo-HSCT, or death from any cause, whichever occurs first (or the date of last contact if no event).Results1966 AML pts were included: median age, 62y; female sex (n=897, 46%), secondary AML (n=428, 22%), PS 0-1 (n=1549, 81%), median WBC (8.6 G/L), cytogenetic risk: favorable (n=174, 9%), intermediate (n=1322, 67%), adverse (n=470, 24%), ELN 2017: favorable (n=195, 20%), intermediate (n=163, 16%), adverse (n=629, 64%); mutations: NPM1 (n=530, 31%), FLT3-ITD (n=338, 20%), FLT3-TKD (n=94, 7%), IDH1 (n=116, 9%), IDH2 (n=126, 10%), KMT2Ar (n=57, 3%). CR/CRi rate was 83% after 1 or 2 IC cycles. Day-30 and -60 death rates were 6 and 8%. Allo-HSCT was performed in 561 pts (35%) during CR1 and in 244 pts (27%) after relapse. The APL cohort included 100 pts: age 53y, WBC ≥ 10 G/L: 16%, CR rate 93% after ATO/ATRA based-therapy, d30 and d60 death: 6% and 8%, 3 relapses, 0 HSCT.The median FU was 91 months. For the whole AML cohort, median OS, EFS, RFS and STFS were 31, 15, 22 and 6 months. In CBF-AML, median OS, EFS, RFS and STFS were not reached (NR, 74% at 5y), NR (57% at 5y), NR (58% at 5y) and 12 months (35% at 5y). In the NPM1mut without FLT3-ITD subgroup, OS, EFS, RFS and STFS were 109 (60% at 5y), 49 (46% at 5y), 69 (52% at 5y) and 22 months (37% at 5y). In pts with FLT3-ITD, median OS, EFS, RFS and STFS were 25, 12, 16 and 5 months. In pts with IDH1 mutation and wild type FLT3, median OS, EFS, RFS and STFS were 54, 16, 20 and 7 months. In pts with IDH2 mutation and wild type FLT3, median OS, EFS, RFS and STFS were 48, 17, 17 and 8 months. In pts with KMT2Ar, median OS, EFS, RFS and STFS were 23, 13, 14 and 4 months.In the APL cohort, 5y-OS, EFS, RFS and STFS were 86%, 85%, 91% and 84%.ConclusionThis study shows that there is considerable room for improvement in the outcome of AML patients, even in the favorable subgroups, who often require additional therapy to achieve a cure. The impact of truly effective first-line treatment is illustrated in APL patients, who previously had a similar prognosis to other AML patients before the era of PML-RARA directed therapies. Improving STFS should become a key objective in clinical trials assessing new targeted agents in combination with IC in AML.

Rationale. Young adults with Philadelphia chromosome–negative (Ph-negative) B-cell acute lymphoblastic leukemia (B-ALL) have benefited from intensified frontline strategies, but still face a 30–40% relapse risk and increased toxicity. Blinatumomab, a bispecific T-cell engager, was recently approved for frontline consolidation based on two key studies: the phase 3 ECOG-E1910 trial, showing benefit in patients with favorable measurable residual disease (MRD) response to chemotherapy, and the phase 2 BLAST study, supporting use in poor MRD responders. The phase 2 GRAALL-2014/B-QUEST study, nested within the GRAALL-2014/B trial, evaluated blinatumomab consolidation in high-risk patients defined by poor end-of-induction (EOI) MRD1 response (≥10-4), or presence of KMT2A rearrangement (KMT2A-r) or IKZF1 intragenic deletion (IKZF1del). This final report also includes a non-randomized comparison with high-risk patients from GRAALL-2014/B not enrolled in QUEST. Patients and methods. Between December 2015 and December 2020, the GRAALL-2014/B trial enrolled 489 patients aged 18–59 with Ph-negative B-ALL. Of these, 455 (93.1%) achieved complete remission (CR), including 259 classified as high-risk (HR). The QUEST substudy began in October 2018, enrolling HR patients without central nervous system involvement at diagnosis and in sustained CR at the start of consolidation 2 (week 12). Blinatumomab was given as a bridge to allogeneic hematopoietic stem cell transplant (allo-HSCT) in very high-risk (VHR) patients (MRD1 ≥10-3 and/or MRD2 ≥10-4 before consolidation 2), or as up to five cycles during consolidation and maintenance in those not eligible for transplant. The primary objective was to improve 3-year disease-free survival (DFS) from 50% to 65% (alpha 5%, power 90%). Ninety-five patients were enrolled in QUEST between October 2018 and December 2020, with 94 evaluable. An internal control cohort included 90 similar patients treated before QUEST activation (December 2015–October 2018) without blinatumomab. Results. Baseline characteristics were comparable between the QUEST and control cohorts, including the frequency of KMT2A-r (17% vs 23%, p=0.36), IKZF1del (40% vs 39%, p=0.99), and MRD1 ≥10-4 (73% vs 70%, p=0.29). By design, all patients were in continuous CR at the start of consolidation 2, with no difference between groups in the proportion requiring a second induction to achieve CR. The rate of MRD2 ≥10-4 prior to consolidation 2 was also similar (45% vs 39%, p=0.54). However, the rate of complete MRD response at the end of consolidation 2 was significantly higher in the blinatumomab-treated group (72% vs 55%, p=0.041). The primary endpoint was met, with a 3-year disease-free survival (3y-DFS) of 70% (95%CI[60–78], lower limit above 50%). In contrast, the control cohort demonstrated a 3y-DFS of 48% (95% CI[38–58]), consistent with the historical benchmark used for comparison. Following blinatumomab, the 5-year cumulative incidence of relapse (CIR), DFS, and overall survival (OS) were 23% (95% CI: 16–33), 68% (95% CI: 58–76), and 79% (95% CI: 69–86), respectively, significantly better than outcomes in the control cohort (CIR: SHR 0.41, 95% CI: 0.25–0.68; DFS: HR 0.48, 95% CI: 0.31–0.75; OS: HR 0.54, 95% CI: 0.32–0.93). DFS benefit was consistent across subgroups defined by age (<45 vs ≥45 years), gender, and white blood cell count (<30 G/L, ≥30 G/L). Among HR features, a benefit in DFS was observed in patients with MRD1 ≥10-4 and IKZF1del, while no significant benefit was seen in those with KMT2A-r. Although the proportion of allo-HSCT–eligible patients was similar between groups (50/94 [53%] vs 51/90 [57%], p=0.66), significantly more patients received a transplant after blinatumomab (88% [44/50] vs 65% [33/51], p=0.009). Overall, blinatumomab consolidation improved outcomes in VHR patients (DFS: HR 0.54, 95% CI: 0.31–0.95). Whether this benefit stems from blinatumomab itself, increased transplant rates, or a combination of both remains to be clarified. Conclusion. In conclusion, this study supports the use of blinatumomab consolidation in high-risk Ph-negative B-ALL patients. Building on these findings and to further refine risk-adapted treatment strategies, the ongoing GRAALL-2024 trial prospectively randomizes allo-HSCT in patients with poor oncogenic characteristics or EOI MRD response, but who achieve undetectable MRD after blinatumomab.