Abstract Among NPM1‐mutated acute myeloid leukemia (AML) (NPM1mut), a distinct subtype has been described with an immunophenotypic profile resembling acute promyelocytic leukemia (APL‐like). In this retrospective multicenter study including 384 NPM1mut AML patients, we identified 95 (24.7%) cases exhibiting an APL‐like immunophenotype. This subset was characterized by significant abnormalities in coagulopathy markers (D‐dimer, D‐dimer/fibrinogen ratio, and disseminated intravascular coagulation [DIC] score). The cumulative incidence of vascular events at 30 days was significantly higher in the APL‐like group compared to the non‐APL‐like group (30.5% vs. 10.1%, P < 0.001). Notably, a higher cumulative incidence of early death due to vascular complications (within 30 days) was observed in the APL‐like group (6.3% vs. 0.35% in controls; P = 0.00015). In multivariate analysis, the APL‐like immunophenotype was the only significant factor associated with vascular‐related early death (hazard ratio [HR] = 19, P = 0.0063). There was a significantly higher rate of IDH1/2 mutations in APL‐like (68.3%) compared to non‐APL‐like (18.3%, P < 0.001) cases. We validated these clinical and molecular findings in an independent validation cohort of 302 NPM1mut patients enrolled in the acute myeloid leukemia study group (AMLSG) 09‐09 clinical trial, which included the administration of all‐trans retinoic acid (ATRA) to all patients and a randomization for gemtuzumab ozogamicin. In this cohort, the APL‐like immunophenotype was associated with events occurring within the first 15 days but did not influence mortality, likely due to protocol‐driven patient selection. Our findings have important clinical implications that warrant the development of studies exploring disease‐tailored clinical measures to mitigate the risk of early vascular events, as in current APL management.

We report the case of a stage 4 mantle cell lymphoma patient, resistant to conventional first- and second-line therapies, treated with CAR-T cells. The therapy was initially efficacious, with an appreciable CAR-T cells expansion in vivo. However, downregulation of CD19 expression on malignant B cells led to sudden therapy failure.

Tissue Factor (TF)pos-platelets represent a subset of the platelet population. Recently, this subset has been shown to predict cardiovascular mortality in patients with coronary artery disease, establishing it as a biomarker useful for thrombotic risk stratification. Accurate quantification of TFpos-platelets by flow cytometry requires strict standardization of pre-analytical handling, staining procedures, and instrument settings, particularly in multicenter studies where technical variability may affect their measurement. This protocol describes a harmonized workflow for whole-blood flow cytometry assessment of circulating TFpos-platelets, designed to ensure reproducibility across laboratories with different technical infrastructures. The procedure includes standardized blood collection and whole-blood fixation to preserve the in vivo platelet phenotype. Two different methods for sample preparation are provided according to local laboratory capabilities: shipment of fixed samples to the Core Laboratory for centralized staining, acquisition, and analysis for centers without flow cytometry facilities, or local staining and acquisition for centers equipped with flow cytometry instrumentation and trained personnel. The assessment of the percentage of TFpos-platelets is achieved by direct labeling with anti-TF Star Fluor 488 and anti-CD41 PerCP-Cy5.5 antibodies to identify the target protein and the platelet population marker, respectively. Flow cytometer harmonization is achieved either through a shared acquisition template for identical cytometer models or through bead-based alignment for different platforms. The workflow further incorporates a standardized gating approach and centralized data analysis to enable reliable comparison of TFpos-platelet measurements across sites. Representative results demonstrated that this workflow supports reproducible quantification of TFpos-platelets across the validated multicenter acquisition settings. This protocol may facilitate broader application of TFpos-platelet assessment in thrombotic risk stratification and support wider standardization of platelet flow cytometry in translational and clinical research.

Background. The aim of intensive induction treatment in acute myeloid leukemia (AML) is the achievement of complete remission (CR). After the establishment of “3+7” regimen as the standard induction, several efforts were pursued for improvement through different modalities, among which the increase of anthracycline and cytarabine doses. All these approaches provided a survival advantage in specific subgroups, generally offset by increased toxicity. Since 2017, novel agents have received approval for frontline treatment of selected patient categories, changing the uniform management to a targeted diversification of therapeutic approaches: the incorporation of the FLT3 inhibitor Midostaurin for FLT3-mutated patients, the use of CPX-351 for therapy-related AML and AML with myelodysplasia-related changes, the implementation of Gemtuzumab ozogamicin prominently in patients with favorable-risk cytogenetics. Nonetheless, induction treatment is still conceived as a single treatment block, the response to which is appreciated only 3-4 weeks after its completion. We previously provided evidence that the kinetics of blast reduction in the peripheral blood (PB) during the first days of “3+7” course might reflect AML chemosensitivity. The analytical approach was based on the quantification of leukemia-associated aberrant immuno-phenotype(s) (LAIP) by multi-parameter flow cytometry (MFC) in PB before starting the induction on day 1 (baseline), and daily thereafter up to day 8. We defined the “clearance of peripheral blast cells” (PBC) as the logarithmic ratio between the absolute blast count on the day of chemo start and the values measured daily. In an exploratory cohort of 61 patients, followed by prospective analysis in the of NILG 02-06 randomized clinical trial, we showed the potential of PBC to predict the achievement of CR and measurable residual disease (MRD) negative status. Rationale. We reasoned that an early appraisal of chemosensitivity based on PBC might support the modulation of induction regimen. Accordingly, we envisioned to continue standard induction in patients where PBC data predict a good response, while in patients with a low clearance of PB blasts, immediate switching to an intensified induction could be offered. Such a PBC-driven approach is meant to increase the rate of CR, but with lower overall toxicity, by early identification of patients with lower probability to achieve CR. The second component of this personalized approach regards the early assessment of disease-associated risk: based on the correlation of PBC with survival and MRD, PBC-low patients will be considered as high-risk and early allocated to allogeneic HSCT. The rationale is to avoid delaying HSCT and prevent delivering of additional chemotherapy courses that are predicted to have very low chance to improve the response depth. Study design. AMELIORATE is a phase 3, randomized, trial aiming to personalization of treatment in FLT3-mutated AML (EudraCT number 2019-003936-21). The study is sponsored by GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) and co-funded by MYNERVA (MYeloid NEoplasms Research Venture AIRC). Key eligibility criteria are a diagnosis of untreated AML according to WHO 2016 and the presence of FLT3 mutation, either ITD and/or TKD. PBC is calculated on day 4 of induction as a logarithmic ratio between the absolute PB LAIP+ cell count on day 1 (baseline) and on day 4. A threshold of 2.0 log is decisional to assign patients to different arms. Patients with PBC >2 (PBC-high) complete the standard induction course and are managed according to standard practice. Patients with PBC≤2 (PBC-low) are randomized at day 4 between a standard (analogue to PBC-high) and an experimental approach providing two main modifications: the immediate switch to intensified induction with high-doses cytarabine (1500 mg/m2 bid on days 5, 6 and 7); and the early allocation to high-risk category, to be refined later, based on ELN stratification and post-induction MRD status. All patients receive Midostaurin as per standard practice. Event free survival (EFS) is selected as the primary endpoint. Recruitment. Between April 2020 and June 2025, 28 study sites adhered to the trial and 147 FLT3-mut pts were enrolled so far. Based on the expected distribution of patients into PBC-high and PBC-low category, a total of 172 subject is to be recruited to include 86 PBC-low patients suitable for randomization to the conventional and experimental arm

Introduction: Despite the approval of novel agents that have significantly improved long-term survival rates for multiple myeloma (MM) patients undergoing autologous stem cell transplant (ASCT), most patients eventually relapse. The failure to achieve or maintain bone marrow (BM) minimal residual disease (MRD) negativity is a recognised adverse prognostic factor for progression-free survival (PFS) and overall survival (OS). Contamination of stem cell apheresis by clonal plasma cells may also affect prognosis, though data remain limited. Methods: We conducted a prospective, single-centre observational study including 100 newly diagnosed MM patients eligible for ASCT and treated with bortezomib-based triplet induction. MRD was assessed both on BM and apheresis samples using multiparameter flow cytometry (MFC-MRD) with a sensitivity of 10−5. Results: Clonal plasma cells were detected in 22 apheresis samples (aMRD+), all of which were associated with BM MRD positivity. Patients with aMRD+ had inferior pre-ASCT responses (≥VGPR: 10% vs. 63%, p = 0.005) and worse post-ASCT BM MRD negativity rates (4% vs. 49%, p = 0.048). After a median follow-up of 52.4 months, aMRD+ was associated with shorter progression-free survival (median 38.5 vs. not reached, p = 0.007) and overall survival (median 60 months vs. not reached, p = 0.003). Conclusions: Contamination of the apheresis product is associated with persistent BM disease and poorer outcomes. Combined MRD assessment in both bone marrow and apheresis may improve risk stratification in MM patients undergoing ASCT.

IntroductionRecently, a flow cytometric (FC) based test has been developed for detection of circulating fetal cells to replace the less accurate and reproducible Kleihauer-Betke test.FC test is easier to perform, it can distinguish the origin of fetal cells, but it is expensive and available in highly specialized laboratories. We evaluated the introduction of high-performance liquid chromatography (HPLC) approach as initial screening to identify patients who need an additional FC test to better discriminate the nature of haemoglobin-F (HbF) positive cells.MethodsBlood samples from 130 pregnant women suspected to have fetomaternal haemorrhage were analysed with HPLC and FC methods. The cut-off for HbF HPLC concentration was calculated. Statistical analyses for the evaluation of HPLC as a screening method were performed. The positivity cut-off of HbF to be used as decision-making value to continue the investigation was calculated.ResultsAn excellent agreement (R2>0.90) was observed between the percentage of HbF obtained by HPLC and the percentage of fetal cells detected by FC. Results obtained from each assay were compared to define the HPLC threshold below which it is not necessary to continue the investigations, confirming the maternal nature of the HbF positive cells detected. Our study demonstrated that a cut-off of 1.0% HbF obtained by HPLC was associated with the lowest rate of false negative results in our patient cohort.ConclusionsThis study provides a new FMH investigation approach that possibly leads to a reduction in times and costs of the analysis.

Measurable residual disease (MRD) is a powerful predictor of outcome in acute myeloid leukemia. In the early phases of treatment, MRD refines initial disease risk stratification and is used for the allocation to allogeneic transplant. Despite its well-established role, a relatively high fraction of patients eventually relapses albeit achieving MRDneg status. The aim of this work was to assess specifically the influence of baseline features and treatment intensity on the predictive value of an MRDneg status, particularly focusing on MRD2, measured after two consecutive chemotherapy cycles. Among baseline features, younger MRD2neg patients (<55 years) had a significantly longer disease-free survival (median not reached) compared to their older counterparts (median 25.0 months, P=0.013, hazard ratio=2.08). Treatment intensity, specifically the delivery of a high dose of cytarabine in induction or first consolidation, apparently had a pejorative effect on the outcome of MRD2neg patients compared to standard dose (P=0.048, hazard ratio=1.80), a finding also confirmed by the analysis of data extracted from the literature. The combination of age and treatment intensity allowed us to identify categories of patients, among those who reached a MRD2neg status, characterized by significantly different disease-free survival rate. Our data showed that variables such as age and intensity of treatment administered can influence the predictive value of MRD in patients with acute myeloid leukemia. In addition to underscoring the need for further improvement of MRD analysis, these findings call for a reasoned application of MRD data, as currently available, to modulate consolidation therapy on adequately estimated relapse rates.

PDF file - 54K, MFI values indicating basal and EPO and G-CSF induced activation of ERK1/2, STAT5, p38 MAPK, and caspase-3 in MDS subpopulations. Correlation between EPO response in vitro and in the clinic

PDF file - 48K, Flow cytometric analysis of STAT5 activation after G-CSF and EPO stimulation

Background: Flow-cytometry is considered crucial for monitoring multiple myeloma (MM) patients, in particularly for evaluation of minimal residual disease (MRD) [1]. Routinely MRD on bone marrow aspirate is the optimal method for assessing depth of response and identifying higher risk of relapse [2]. There are also emerging data about the prognostic role of peripheral plasmacell count on flow-cytometry at diagnosis of MM and after treatment [3]. However, so far limited information is available on the use of flow cytometric MRD for the research of clonal plasma cells on stem cell apheresis as well as its clinical significance. Aim: Our purpose is to determine whether the contamination of apheresis product with clonal plasma cells predicts the risk of MM disease progression or impacts on patients’ overall survival (OS). Methods: We retrospectively analysed 75 newly MM patients diagnosed at our Center between July 2017 and November 2021. The multiparameter flow cytometry, with a sensitivity of 10(-5), was applied after induction therapy on both bone marrow samples and on apheresis product in order to evaluate the MRD status. Results: The median age was 60 years and all the patients were treated with triplet induction regimens (89% VTD, 5% VRD, 4% KRD, 2% PAD), followed by autologous stem cell transplantation (ASCT). Stem cell harvest was performed following cyclophosphamide and G-CSF mobilization. Sixty-two patients (91%) received maintenance therapy with lenalidomide and 6 (8%) with daratumumab. Among 75 patients included in the study, 64% and 36% of them achieved MRD positivity and negativity on bone marrow (mMRD), respectively. MRD status on apheresis (aMRD) was negative in 61 patients (81%) and positive in 14 (19%). All patients with positive aMRD had a concomitant mMRD positivity. The median follow-up was 28 months (range, 7 to 58) and progression free survival (PFS) at 24 months was 90% (95% confidence interval [CI], 86 to 94) versus 50% (95% CI, 35 to 65) for the patients aMRD-negative versus those aMRD-positive, respectively (p < 0.001). The OS at 24 months was 98% (95% CI, 96 to 100) in the aMRD negative cases and 74% (95% CI, 61 to 87) in the aMRD-positive (p=0.007). According to mMRD status, PFS at 24 months was 94% (95% CI, 88 to 100) and 75% (95% CI, 68 to 82) in the mMRD negative and positive, respectively (p=0.16). There was no statistically significant difference between the two groups according to other prognostic factors (ISS disease stage and cytogenetic profile) and maintenance therapy. Univariate analysis identified only the aMRD positivity as risk factor for reduced PFS. Conclusion: PFS data showed that the rate of progression was higher in the group of aMRD- positive patients, suggesting a potential deleterious role for graft contamination. Instead, mMRD status had not a significant impact on PFS. These results need to be confirmed in a larger prospective study with quadruplet induction regimen daratumumab-based, in order to provide a larger applicability of aMRD monitoring.[1] Moor I. et al. Peripheral flow-MRD status at the time of autologous stem cell collection predicts outcome in multiple myeloma. Bone Marrow Transplant. 2018 Dec;53(12):1599-1602. [2] Turner R. et al. The Utility of Euroflow MRD Assessment in Real-World Multiple Myeloma Practice. Front Oncol. 2022 May 18;12:820605. [3] Li J. et al. Prognostic value of circulating plasma cells in patients with multiple myeloma: a meta-analysis. PLoS ONE. 2017;12:e0181447.