NPM1-mutated (NPM1-mut) acute myeloid leukemia (AML) is generally associated with a more favorable outcome, although the presence of additional gene mutations can influence patient prognosis. We analyzed intensively-treated adult NPM1-mut AML patients included in the HARMONY Alliance database. A newly developed risk classification, which included combinations of co-mutations in FLT3-ITD, DNMT3A, IDH1/IDH2, and TET2 genes, was applied to a training cohort of NPM1-mut AML patients included in clinical trials (n = 1001), an internal validation cohort more representative of real-world settings (n = 762), and an external validation cohort enrolled in UK-NCRI trials (n = 585). The HARMONY classification considered 51.8% of the NPM1-mut AML training cohort patients as favorable, 24.8% as intermediate, and 23.4% as adverse risk, with median overall survival (OS) of 14.4, 2.2, and 0.9 years, respectively; p < 0.001), thereby reclassifying 42.7% of NPM1-mut patients into a different European LeukemiaNet (ELN) 2022 risk category. These results were confirmed both in an internal and external validation cohort. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) showed the highest benefit in the NPM1-mut adverse-risk subgroup. The HARMONY classification provides the basis for a refined genetic risk stratification for adult NPM1-mut AML with potential clinical impact on allo-HSCT decision-making.

Despite advances for patients with acute leukemia health disparities limit access to diagnosis and treatment. Artificial Intelligence (AI) approaches may address some disparities. We retrospectively assemble a diverse, international cohort of 6206 leukemia patients from 20 centers to test an AI tool designed to support leukemia diagnosis using standard laboratory results. Executing the pretrained algorithm results in varying accuracy metrics. With confidence cutoff predictions, 2000-fold bootstrapped area under the curve (AUROC) metrics are 0.94 for acute myeloid leukemia (AML), 0.98 for the promyelocytic subtype and 0.84 for acute lymphoblastic leukemia. However, this cutoff excludes 70.8-92.5% of patients from predictions. We improve accuracy and robustness, while maintaining generalizability via an ensemble of Isolation Forest and Local Outlier Factor increasing AUROC for AML from 0.72 to 0.84 (hold-out test set, patients below confidence threshold), while excluding only 12.1% of patients. Furthermore, we retrain the algorithm for pediatric patients.

Background: Secondary acute promyelocytic leukemia (sAPL) is a very rare subtype of acute myeloid leukemia that develops following exposure to chemotherapy, radiotherapy, or immunosuppressive agents. The treatment results and survival outcomes of sAPL patients are still not precisely defined. Methods: A search of the Polish Adult Leukemia Group (PALG) database identified 29 cases of sAPL (median age 57 years; 62.1% female) among 437 APL patients (6.7%) diagnosed between 2006 and 2024. Each sAPL case was matched to a de novo APL patient by sex, age, year of diagnosis, and treatment protocol (LPA (Leucemia Promielocítica Aguda) 2005, LPA 2012, or LPA 2017). Results: All sAPL cases occurred following chemo- and/or radiotherapy, most commonly for breast cancer. The sAPL cases demonstrated higher CD15 expression than the de novo APL cases (median 27.6% vs. 7%, p = 0.04). The two groups exhibited comparable complete remission rates (82.8% sAPL vs. 75.9% de novo; p = 0.75) and early mortality rates (17.2% vs. 20.7%, p = 1.0). However, relapse-free survival was significantly shorter in sAPL (median 94.7 months vs. not reached; HR 7.23, 95% CI 1.63–32.02, p = 0.030), whereas overall survival did not differ significantly between groups. Multivariate analysis identified Eastern Cooperative Oncology Group performance status ≥3 and CD15 expression > 20% as independent predictors of inferior survival. Conclusions: These findings suggest that sAPL shares many clinical features with de novo APL but carries a higher risk of relapse, highlighting the need for further prospective studies and the potential implementation of tailored therapeutic strategies.

Background and Objectives: Primary myelofibrosis (PMF) is a rare hematologic malignancy with non-specific symptoms, causing diagnostic delays and missed diagnoses. Automated screening in heterogeneous electronic health records (EHRs) is challenging due to class imbalance, data sparsity, and incomplete labeling. We investigated two complementary objectives: (1) developing a screening algorithm using routine EHR data to identify PMF-risk patients for hematology consultation, and (2) assessing the applicability of positive-unlabeled (PU) learning. Methods: Using EHR data from 10 Polish hospitals, we evaluated several ensemble models and found that LightGBM achieved the best performance. Results: LightGBM achieved AP 20.83% (95% CI: 19.18-24.35%), sensitivity 45.52% (95% CI: 39.48-52.72%), and precision 14.72% (95% CI: 13.80-17.01%)-substantially exceeding expected prevalence (over 340-fold enrichment). The model captured interactions among RDW and PLT parameters revealing high-risk unlabeled patients with confirmed diagnoses (n = 5) or clinical profiles resembling PMF cases (n = 31), confirming PU problem. Conclusions: Although PU methods enhanced sensitivity, they reduced precision to levels exceeding real-world healthcare capacity. Ensemble learning enables disease identification in clinical settings.

BACKGROUND:Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by an increased risk of thrombotic complications due to a persistent pro-thrombotic and inflammatory state. Atrial fibrillation (AF) is a common cardiac arrhythmia and an established risk factor for thromboembolism; however, its impact on thrombotic complications in patients with MPN remains incompletely characterized. MATERIAL AND METHODS:This multicenter, real-world retrospective study analyzed 2,683 MPN patients diagnosed between 1976 and 2024 to investigate the impact of AF on thrombotic events, bleeding risk, and survival outcomes. RESULTS:Patients with MPN and AF (MPN-AF) had a higher rate of thrombotic events, with arterial thrombosis predominating (67% vs. 43%, p < 0.0001). Despite this arterial phenotype, 36.7% of MPN-AF patients did not receive antiplatelet therapy. Thrombosis rates remained high in AF patients despite cytoreductive therapy. Kaplan-Meier analysis revealed that AF was linked to significantly shorter overall survival (181 months vs. 265 months, HR: 1.86, 95% CI: 1.53-2.26, p < 0.0001), particularly in essential thrombocythemia and MF subtypes. CONCLUSION:Our findings highlight that the thrombotic profile of MPN patients with AF differs from that observed in the general AF population, with a clear predominance of arterial events. Conventional AF management focused on anticoagulation may incompletely address the predominantly arterial thrombotic phenotype observed in MPN. The treatment patterns observed in our cohort-with frequent reliance on anticoagulation alone and limited use of antiplatelet therapy-suggest a need for more disease-adapted antithrombotic considerations in this subgroup. Prospective studies are needed to clarify the contribution of inflammatory and vascular mechanisms to thrombotic risk in MPN-AF.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, life-threatening hematologic disease with diagnostic delays exceeding 5 years in 24% of cases. We developed and deployed an artificial intelligence algorithm analyzing structured and unstructured electronic health record data across 14 healthcare organizations in Poland. Screening of 1,307,140 patients identified 356 high-risk individuals; of 119 referred for flow cytometry, 13 were diagnosed (positive predictive value: 10.92%; 95% CI, 9.68%-12.30%), comparing favourably to 6.9% conventional screening hit rate. High-risk patients were significantly older (median 69.5 years) with elevated rates of fatigue (76.4% vs 29.19%), anaemia (72.2% vs 7.61%), and myelodysplastic syndrome (49.2% vs 0.24%; all p < 0.001). Only 2.25% presented with haemoglobinuria versus 45-62% in registry cohorts. Retrospective analysis revealed potentially preventable diagnostic delays of 74-1337 days. Monte Carlo feature selection identified Coombs-negative haemolysis and visit frequency as strongest predictors, supporting the potential utility of AI-assisted screening for identifying atypical PNH presentations.

The inclusion of nine myelodysplasia-related gene (MRG) mutations (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2) as adverse risk factors in the ELN risk classification has reshaped classification in acute myeloid leukemia (AML). AML with FLT3-ITD mutations and co-occurring MRG alterations is now classified to the ELN adverse risk group although supporting evidence remains limited. Among 4,078 patients with AML with available molecular information included in the HARMONY platform, 862 harbored FLT3-ITD mutations and underwent intensive chemotherapy. Of these, 171 (20%) exhibited co-occurring MRG mutations at diagnosis. In this cohort, MRGs were not independently associated with relapse-free survival (RFS) or overall survival (OS). In the FLT3-ITD/NPM1 co-mutated subgroup, MRG mutations were rare (9%) and showed no prognostic impact. Conversely, in FLT3-ITD/NPM1 wildtype AML, MRG mutations were predictive of shorter RFS (HR 1.37, 95%CI 1.01 – 1.88, p = 0.046) and OS (HR 1.34, 95%CI 1.02–1.74, p = 0.032) in multivariable analysis with survival times comparable to the ELN adverse risk category. The allelic ratio of FLT3-ITD did not further stratify OS and RFS in this subgroup. These findings suggest that the prognostic relevance of MRG mutations in FLT3-ITD AML is modulated by NPM1 co-mutational status and mirror findings in AML lacking FLT3-ITD.

Aim:The purpose of this study is to investigate whether IVIM-derived spleen parameters including the true diffusion coefficient (D) and perfusion fraction (f) can serve as non-invasive biomarkers to predict fibrotic transformation of the bone marrow. Methods:Eighteen patients with MPN and 18 healthy controls (HC) underwent abdominal IVIM diffusion-weighted imaging using 1.5 T MR unit. The values of spleen parameters of D and f were compared between patients with various forms of MPN, including 8 patients with myelofibrosis (MF), 10 patients without myelofibrosis (non-MF) and (HC). A Bayesian approach was adopted in the analysis, to assess whether the D and f coefficients, along with the spleen volume, can predict disease occurrence. Results:Comparing MF patients to HC, Bayesian analysis provided strong evidence for a difference in the f parameter (BF = 18.38), while the difference between MF and non-MF patients was weaker (BF = 1.42). There was no statistically significant difference between non-MF and HC (BF = 0. 64). There was no correlation between f parameter and spleen volume (CI= -0,43; 0,38). Logistic regression analysis with logkit function confirmed that f parameter was significant predictor of MF. Conclusions:The study demonstrates that IVIM measurements of spleen perfusion in both healthy individuals and MPN patients offer valuable predictive information regarding disease development and progression. The f parameter shows promise for predicting fibrotic transformation of the bone marrow in MPN patients regardless of the size of the spleen.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment option for a significant proportion of patients with acute myeloid leukemia (AML), and it is generally recommended when the relapse risk without allo-HSCT outweighs the estimated non-relapse mortality significantly. While current recommendations for allo-HSCT are based on risk groups, there is considerable heterogeneity within these individual categories. We analyzed 2550 intensively treated AML patients aged 18-70 with cytogenetic and next-generation sequencing data from the HARMONY Alliance database, who did not receive allo-HSCT in first complete remission (CR1). A non-parametric machine learning (ML) model based on Bayesian Additive Regression Trees (BART) integrated clinical variables and genomic aberrations to provide individualized outcome estimations. External validation was performed in a cohort of 714 patients enrolled in UK-NCRI trials. The predictive performance of the HARMONY ML model, measured by the area under the time-dependent receiver operating curve (AUC(t)), was superior to European LeukemiaNet (ELN)2022 risk classification in estimating 5-year overall survival (0.741 vs. 0.700), 5-year relapse-free survival (0.752 vs. 0.705), and 5-year cumulative incidence of relapse (0.742 vs. 0.708), which was confirmed in the external validation cohort. Notably, the model revealed substantial heterogeneity within ELN2022 risk groups, identifying a significant proportion of favorable-risk patients with a predicted 5-year CIR > 40%, who could potentially benefit from allo-HSCT in CR1. The HARMONY ML model provides individualized risk prediction in intensively treated adult AML patients and supports more tailored therapeutic decisions regarding allo-HSCT in CR1, which should be further advanced by integrating measurable residual disease in the future.

BACKGROUND:Acute myeloid leukemia (AML) harboring BCR::ABL1 is considered a separate diagnostic entity and is classified as adverse in the risk score of the European LeukemiaNet. However, its prognosis could change with the addition of tyrosine kinase inhibitors (TKI) to chemotherapy. METHODS:In this study, the authors interrogated Programa Español de Tratamientos en Hematología (PETHEMA) AML epidemiologic registry to shed light on the outcome of patients with AML harboring BCR::ABL1. RESULTS:Fifty-seven patients with newly diagnosed AML harboring BCR::ABL1 were included, 40 of them treated intensively. Fifteen patients received TKI and 19 did not receive TKI, median overall survival (mOS) was 15.7 months (95% CI, 3.9-27.6 months) and 12.3 months (95% CI, 0.4-24.1 months), respectively (p = .28). Median relapse-free survival was not reached versus 7.6 months (95% CI, 3-12.2 months) in patients who did or did not receive TKI (p = .029). Age, TKI treatment, allogeneic bone marrow transplantation and date of diagnosis were included in the Cox regression analysis, and no independent prognostic factors for OS were found. Comparison of BCR::ABL1 AML patients with the intensively treated global PETHEMA AML cohort (N = 2107) showed that patients who received a TKI had a median overall survival similar to that of the intermediate‑risk group. CONCLUSIONS:This study shows improved outcomes with the addition of TKI to chemotherapy and supports re-classification of AML patients harboring BCR::ABL1 in the intermediate-risk group. Nonetheless, given the small sample size, larger studies are needed to confirm these findings.

Chronic myeloid leukemia (CML) and Philadelphia (Ph)-negative myeloproliferative neoplasms (MPN) are generally distinct clonal disorders, with the co-occurrence of BCR::ABL1 rearrangement with concomitant Ph-negative MPN rarely reported. Here we describe the largest known international cohort of Ph-negative MPN and coexisting CML providing important insights into this rare clinical scenario. We performed an international, multicenter, retrospective analysis of patients with concomitant BCR::ABL1 rearrangement and Ph-negative MPN, identifying 61 cases from 30 centers in 7 countries, over a 29-year period (1996-2025). Thirty-one patients (50.8%) had Ph-negative MPN preceding CML, 18 patients (29.5%) had CML preceding Ph-negative MPN, and 12 patients (19.7%) had Ph-negative MPN and CML diagnosed simultaneously. We observed increased TKI resistance and myelofibrotic transformation, especially in patients initially diagnosed with Ph-negative MPN. In this group, 35.4% (n = 11) progressed to MF, 2 patients to blast-phase MPN, and 69.2% (n = 18) failed to achieve a complete cytogenetic response. The rare e1a2 BCR::ABL1 transcript was notably prevalent which is associated with TKI resistance and a more aggressive disease course in CML. We described superior survival in those with Ph-negative MPN preceding CML, with median OS not reached, compared with 277 months for CML preceding Ph-negative MPN and 100 months for those diagnosed simultaneously (p = 0.05).

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy where chemoresistance, particularly to daunorubicin (DNR), frequently leads to treatment failure and relapse. Current resistance prediction methods are often slow and inaccessible in clinical settings, delaying crucial treatment adjustments. There is an urgent need for rapid, reliable, and clinically accessible diagnostic tools to identify DNR resistance early, enabling personalized therapeutic strategies and improving patient outcomes. To address this problem, we developed a rapid flow cytometric assay to detect reduced intracellular DNR accumulation, a marker of drug resistance. This was complemented by FTIR spectroscopy, confocal microscopy, and proteomic profiling to validate findings and explore underlying mechanisms in drug-resistant and sensitive cells. The flow cytometry method demonstrated that the novel Sensitivity Index (S-index) accurately predicts DNR resistance after short drug exposure and correlates strongly with IC50 values. Complementary FTIR and confocal microscopy revealed distinct biochemical and structural differences between resistant and sensitive cells. Proteomic analysis suggests that increased drug metabolism contributes to DNR resistance, supporting the flow cytometry data. In summary, our findings demonstrate that flow cytometry provides a reliable and clinically accessible tool for targeted diagnostics, crucial for optimizing patient-specific treatment strategies based on rapid assessment of daunorubicin resistance in AML.

Acute promyelocytic leukemia (APL) is a highly curable leukemia characterized by life-threatening coagulopathy leading to hemorrhagic and thrombo-ischemic events. We analyzed the incidence, outcomes and risk-factors of thromboischemic events in a large series of 1210 patients with newly diagnosed APL reported to the PETHEMA registry. Therapy consisted on ATRA and chemotherapy (AIDA-based). Median age of patients was 46 years (range 2-90 years). Fifty-eight patients (5%) did not start AIDA regimen as they were unfit for chemotherapy, or they died early before initiating ATRA. A total of 195 (16%) patients developed thrombo-ischemic events, the most frequent being superficial-vein and/or central catheter-related (6.9%) followed by central nervous system (2.2%), deep-vein thrombosis (2.1%), pulmonary embolism (2.1%), acute myocardial infarction (1.6%), or other locations (1.2%). Thromboischemic events mostly occurred at diagnosis and during induction (4.0%, and 9.3%, respectively). Patients developing life-threatening thrombo-ischemic events (i.e, excluding superficial and/or catheter-related) at diagnosis/induction had 31% early death rate. Prolonged aPTT, age >40 years, ECOG more than 1, platelets > 25 x 109/L, and absence of bleeding at presentation were independent risk factors for life-threatening thrombo-ischemic events. Using these variables (1 point each) we developed and validated the Thromb-On risk score, identifying a high-risk group (3 to 5 points). The Thromb-On risk score was validated in a cohort of 585 patients treated since 2017 with arsenic trioxide plus ATRA (

BACKGROUND: Measurable residual disease (MRD) is increasingly recognized as a critical prognostic factor in AML for guiding therapeutic decisions and predicting patient outcomes. Multiparametric flow cytometry (MFC) is the most widely used MRD assessment method, with applicability in about 90% of AML patients (pts). However, obtaining accurate and reproducible MFC-MRD results requires substantial experience and expertise. Local MRD testing is commonly used in clinical practice and investigational trials but the correlation between MRD and outcomes may be confounded by interlaboratory variability. AIM: In this analysis, we evaluated the concordance between local and central MFC-MRD assessments and its impact on relapse-free survival (RFS) and overall survival (OS) in AML patients in first complete remission (CR1) using data from a prospective, multicenter, randomized phase 3 trial comparing two intensive chemotherapy induction regimens in AML (NCT03257241). PATIENTS AND METHODS: Pts with newly-diagnosed, untreated AML, ECOG performance status 0–2 and HCT-CI≤ 3 were randomized to Daunorubicin+Ara-C (DA-90) (n=220) or Daunorubicin+Ara-C+Cladribine (DAC) (n=219) induction chemotherapy (IC). Pts with >10% blasts in non-aplastic bone marrow at day 14 received early second IC with D-45 and DAC, respectively. Pts who achieved a CR/CRi/CRp (cCR) were offered IDAC consolidation with or without alloSCT according to predefined risk groups. Serial samples for multi-modality MRD assessment were collected at cCR after 1 or 2 inductions (MRD1), and after each consolidation cycle (MRD2-4). MRD evaluation using 6-8-colour MFC with LAIP-based analysis was performed at 16 local MFC labs and MRD-1 and MRD-2 FCS files were subsequently independently evaluated by a central reviewer. The ELN threshold of <0.1% for MRD negativity (MRD-) was used by the local laboratories and central reviewer. RESULTS: MRD results were available from local laboratories for 279 (87.7%) pts in cCR at MRD1 and for 267 (84%) pts at MRD2). Centrally reviewed MRD1 data were available for 173 (54.9%) pts in cCR and for 139 (43.7%) pts in MRD2. Reasons for missing MRD results from central review were: missing or inaccessible FCS files (n=69); insufficient cell acquisition or inadequate antibody panel selection identified by central assessment (n=54); lack of LAIP target by central review (n=22). Of the MRD1 results classified locally as MRD negative (MRD1-), 97.6% (81/83 pts) were concordant with central assessment. However, 74/90 pts (82.2%) considered MRD1+ by local evaluation were reclassified as MRD1- after central review, leading to an overall MRD1 concordance rate of 56.1%. The overall concordance between local and central evaluation of MRD2 was 70.7%, (96.7% and 22.4% concordance with central assessment for MRD2- and MRD2+ results, respectively). Cohen's kappa coefficient was 0.15 for MRD1 and 0.23 for MRD2, indicating poor agreement. Centrally reviewed MRD2 demonstrated strong prognostic value for overall survival, with a hazard ratio of 2.32 (95% CI: 1.1–5.1; p=0.034) in multivariable analysis adjusted for clinical factors. In contrast, MRD1 did not reach statistical significance (p=0.250). Locally assessed MRD1 and MRD2 results were not predictive of overall survival (p=0.829 and p=0.523, respectively) in the cohort with centrally reviewed data. This was also the case when multivariable analysis included all MRD results submitted by local laboratories. CONCLUSIONS: These findings underscore the critical need for expert central verification of MFC-MRD results in multicenter AML trials, as local MRD analysis showed limited prognostic value and substantial discordance with centralized evaluation.

Background:Mixed-phenotype acute leukemia (MPAL) is a rare and heterogeneous subtype of acute leukemia, associated with unfavorable outcomes. MPAL is defined by the presence of more than 20% blasts and bi- or trilineage assignment based on strong immunophenotypic markers, with specific subcategories characterized by BCR::ABL1, KMT2A, ZNF384, or BCL11B rearrangements. This review aims to summarize current knowledge and challenges in the diagnosis and management of MPAL. Methods:Data were synthesized primarily from meta-analyses and original studies, with a particular emphasis on the roles of immunophenotyping, cytogenetics, and novel targeted therapies from 1985 to the present. Results:MPAL accounts for 1%-5% of acute leukemias, with B/myeloid (59%) and T/myeloid (35%) subtypes being the most prevalent. Cytogenetic abnormalities are identified in up to 90% of cases, predominantly complex karyotypes. Molecular investigations have identified frequent mutations in genes such as RUNX1, DNMT3A, IDH1/2, NOTCH1, and FLT3, particularly enriched in T/myeloid MPAL. Adverse prognostic factors include KMT2Ar, elevated leukocyte counts, extramedullary disease, and bilineage disease biology. Generally, the prognosis for adults is poorer than for the pediatric population. No standardized treatment strategy has been established. Retrospective analyses indicate superior complete remission rates and overall survival with ALL-based regimens, and allogeneic hematopoietic stem cell transplantation remains crucial for improving survival. Recently, hybrid regimens such as FLAG-IDA and CLAG-M have demonstrated promising efficacy with acceptable toxicity. Targeted therapies are emerging options, although lineage switch under selective therapeutic pressure remains a concern. Conclusions:MPAL remains a significant challenge in diagnosis and treatment. Advances in molecular characterization have enhanced classification techniques and have the potential to inform personalized treatment strategies. Considering the rarity and heterogeneity of MPAL, extensive prospective multicenter trials are imperative to develop evidence-based therapeutic protocols.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy characterized by a complex clinical presentation. Comprehensive characterization at diagnosis is essential for proper classification and therapeutic decisions. Immunophenotype and/or immunohistochemistry are currently mandatory diagnostic criteria for confirming this neoplasm. However, large series describing marker features at diagnosis are scarce. Material This multinational retrospective study performed by PALG and PETHEMA groups registered BPDCN patients from July 1999 to 2025 (EPI-BLAS project). Local data for clinical presentation, morphology, immunophenotype, and immunohistochemistry was collected from participating centers and reviewed centrally. BPDCN classification was made locally based on WHO criteria applicable at the time of diagnosis. Results A total of 257 patients diagnosed with BPDCN were included in the multinational PETHEMA and PALG registry. Most were male (78%), median age was 66 years old (range, 15-92), bone marrow involvement (>5% blasts) was documented in 75% (177/237, 20 data not available [NA]), and peripheral blood in 62% (103/165, 92 NA). Regarding extramedullary involvement, 197 (81%) out of 242 with available data had skin involvement, and 49% (131/237, 20 NA) had lymphadenopathy. A lumbar puncture was performed at diagnosis in 77% (168/217, 40 NA) patients and CNS infiltration was documented in 48 (29%) of them. Disease was restricted to the skin in (10%) 25 patients. Immunophenotypic and/or immunohistochemical information was available in 236 patients. Immunohistochemistry (IHQ) studies were conducted on 178 patients (115 skin biopsies, 47 bone marrow biopsies, 13 lymph nodes, 1 salivary gland, and 1 spleen specimen). Flow cytometry (FC) analyses were performed in 244 patients (179 bone marrow aspirates, 26 peripheral blood samples, 16 skin biopsies, 21 cerebrospinal fluid (CSF) samples, and 2 lymph nodes). In 6 additional patients, the specific technique used could not be determined due to insufficient documentation. Skin biopsies from IHQ showed in most cases positive expression of CD4 (94/96, 98%, 19 NA), CD56 (92/99, 93%, 16 NA), CD123 (66/66, 100%, 49 NA), CD304 (1/1, 100%, 114 NA), and TCL1 (6/7, 86%, 108 NA). CD303 and TCF4 expression were never assessed. Negative expression for CD3, CD14, CD19, CD34, lysozyme and myeloperoxidase (MPO) were found in 94% (79/84, 31 NA), 100% (6/6, 109 NA), 100% (2/2, 113 NA), 98% (55/56, 46 NA), 92% (11/12, 77 NA) and 98% (55/56, 45 NA), respectively. When performed, FC analysis of bone marrow detected BPDCN cells in 95% of cases (170/179), and the vast majority showed positivity for CD4 (132/141, 94%, 29 NA), CD56 (117/147, 80%, 23 NA), CD123 (143/147, 97%, 23 NA), CD304 (12/14, 86%, 156 NA), and TCL1 (22/22, 100%, 148 NA). CD303 was positive in 29% (2/7, 163 NA). TCF4 expression was never assessed. CD3, CD14, CD19, CD34, lysozyme, and MPO were consistently negative in 98% (89/91, 79 NA), 98% (96/98, 52 NA), 95% (112/118, 52 NA), 74% (100/135, 35 NA), 75% (3/4, 166 NA), and 93% (102/110, 60 NA), respectively. In 20% (30/149, 21 NA) of patients, more than one leukemic population was identified by FC in bone marrow, potentially representing different stages of plasmacytoid dendritic cells (pDC) differentiation. Among 23 BPDCN patients with ≤5% morphological blast cells in bone marrow and with available FC analyses, 18 patients (78%) showed BPDCN cells (median 1.95% blasts, range 0.01% to 20%), 2 (10%) showed pDC with normal immunophenotypic profile; and 5 (22%) showed no evidence of pDCs or BPDCN. Conclusion Our large registry study in this rare disease showed skin and bone marrow involvement in the vast majority of patients. Half of BPCN patients had lymph node involvement, and 29% CNS disease. IHQ and FC analyses showed positivity for CD123, CD4 and CD56 in almost all samples. CD304 and TCL1 were also positive in almost all cases, but these surface antigen markers were only tested in a minority of samples. FC analyses could detect BPDCN cells in most cases without bone marrow morphological infiltration, highlighting the role of FC assessment in this tissue. The consistent implementation of well-defined markers, particularly those outlined in the WHO 2022 classification, could further strengthen diagnostic precision of BPDCN. Disclosures The EPI-BLAS registry was partially funded by Menarini.

ELN recommendations for AML diagnosis, genetic risk stratification and treatment have been updated in 2022. Accordingly, FLT3-ITD+ AML is now included in the intermediate-risk category, regardless of the allelic ratio (AR), and capillary electrophoresis (CE) is the recommended detection method. In two large trials combining intensive chemotherapy with the FLT3 inhibitors midostaurin (RATIFY) and quizartinib (QuANTUM-First), the AR threshold to define FLT3-ITD+ was conventionally set at 0.05 and 0.03, respectively. CE analysis requires high level of expertise specifically to detect FLT3-ITD microclones (FLT3-ITDm, AR: >0.01,<0.05), whose clinical role is not yet well established. Here, we leveraged the EHA AML-SWG framework to explore diagnostic evaluation methods and prognostic impact of FLT3-ITDmin AML. In a first methodological part, results of CE from 63 FLT3-ITD+patient samples were blindly analyzed by 7 European labs, and reproducibility of results was assessed. The intraclass correlation coefficient (ICC) was computed as an index of interrater reliability of AR data. Overall, we observed a good ICC for ITD length determination (95%CI=0.52–0.92), but a less satisfactory ICC for AR estimation (95%CI=0.06–0.63). Next, we compared NGS and CE for FLT3-ITD status determination, by running in parallel an additional set of 48 FLT3-ITD+ AML cases. Results showed a significant correlation of AR/VAF (r=0.923,p<0.001), and a 94% concordance overlap, with only 3/48 cases with AR<0.02 detected by CE, but not by NGS. Next, we enrolled AML patients diagnosed between 2017 and 2022 with FLT3-ITD AR<0.05 by CE, and a comparator cohort of cases with AR>0.05 treated with standard chemotherapy without FLT3-inhibitors, or with less-intensive strategies. A total of658 patients with FLT3-ITD+ AML were included in our study. Of these, 212 (32%) had FLT3-ITDm (AR range:0.01-0.04), whereas 446 (68%) had an AR>0.05 (0.05-15.26). Median age at AML onset was 59 years (range 18-94). Patients with microclones were older (62 vs 58 years, p<0.001), and had a less proliferative phenotype. AML with FLT3-ITDm were also more frequently classified as “secondary” with regards to disease ontogeny (14% vs 8%, p=0.012), and had a higher frequency of myelodysplasia-related (MR) genes mutations (60% vs 30%, p<0.001), whereas no difference was noticed for NPM1. A total of 161 patients (24%, median age 76 years) were deemed unfit and were managed with less-intensive approaches, while 497 patients (76%) received intensive chemotherapy. Among the latter, complete response (CR) was achieved in 88% of cases with AR>0.05 vs. 74% of cases with FLT3-ITDm (p<0.001). CR achievement was associated with younger age (p<0.001), presence of NPM1 (p=0.018), absence of DNMT3A (p=0.035) and MR-genes (p=0.011) mutations, FLT3-ITD AR >0.05 (p<0.001), and ELN 2017/22 favorable/intermediate vs adverse categories (p=0.015/0.045). With a median follow-up of 30 months (18-41), 3-year overall survival (OS) was 58% in intensively treated patients, censoring for transplant. The multivariable analysis (MVA) for OS, including clinical and genetic variables, and ELN 2022 categories, showed that increasing age and WBC, together with NPM1 mutation had an independent prognostic role (HR=1.03, 1.03 and 0.63 respectively, p<0.001, 0.021 and 0.012, respectively). Overall, 136 intensively treated patients (27%) relapsed at a median of 8 months (range 1-56) from AML diagnosis, leading to a 3-year disease-free survival (DFS) of 47%, when censoring for transplant, with no impact of FLT3-ITD AR (HR=0.9, p=0.6). The MVA identified increased WBC and presence of NPM1mutation as independent predictors of DFS (HR=1 and 0.61, p=0.007 and 0.017, respectively). Of cases with available FLT3-ITD status at relapse (n=33), 45% remained positive, of which 64% were originally FLT3-ITDm. Dissecting their FLT3-ITD clonal make-up, the majority (91%) recapitulated the same clone harbored at onset (18% also acquiring additional clones, and 27% losing prior clones), whereas a new clone was detected in 9% of cases. Finally, the clonal burden expanded in all cases (median 0.027 vs 0.686, p<0.001), with 89% of FLT3-ITDm patients relapsing with AR >0.05. Our data show that FLT3-ITDmare associated with older age, secondary-type AML and higher frequency of MR-gene mutations vs AML with FLT3-ITD >0.05. The potential benefit of FLT3 inhibitors in patients with FLT3-ITDm needs to be evaluated in randomized clinical trials.