Francesco Buccisano

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disorder caused by a defect of glycosylphosphatidyl‐anchored proteins, leading to an uncontrolled complement‐mediated hemolysis. The advent of complement inhibitors in clinical practice radically changed patients' outcomes and survival. Despite this significant progress, numerous unmet needs persist in clinical practice. A group of Italian experts explored the main challenges currently associated with PNH diagnosis and monitoring and reached a consensus on screening, diagnosis, initial work‐up, and monitoring. The following core questions were examined: (i) Which patients need to be tested? (ii) Which test to use, and how do we define positivity? (iii) Which workup is requested for patients with a newly detected PNH clone? (iv) Which monitoring in untreated patients? (v) Which monitoring in C‐terminal‐inh and C‐proximal inh? Each specific clinical question was framed according to the GRADE format. Overall, flow‐cytometry for PNH detection was recommended in patients presenting with two or more suggestive signs or symptoms and/or with hematologic conditions known to be associated with PNH clones, including DAT‐negative hemolysis, idiopathic venous thrombosis (particularly at atypical sites), and primary bone marrow dysfunction (such as aplastic anemia or myelodysplastic syndrome). A high‐sensitivity flow cytometry test in neutrophils and erythrocytes should be preferred (lower limit of quantification 0.01%). Baseline assessment of disease activity (i.e., LDH > 1.5×ULN and PNH signs/symptoms), thrombotic risk, bone marrow (BM) features and imaging for organ dysfunction are suggested. Monitoring comprises blood counts and hemolytic markers with frequency depending on anemia severity and symptoms, as well as annual PNH clone retesting, BM reevaluation in case of cytopenias, and constant education about infectious risk and breakthrough hemolysis in patients on treatment.

Summary: Venetoclax (VEN) plus hypomethylating agents (HMAs) represents the standard of care for newly diagnosed acute myeloid leukaemia (AML) patients unfit for intensive chemotherapy, but prospective real‐world observational data outside randomized clinical trials remain limited. The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) AML2320 is a prospective, multicentre, observational study (NCT04589728) including 193 newly diagnosed unfit AML patients receiving VEN plus azacitidine or decitabine between November 2020 and December 2021. Primary end‐point was overall survival (OS); secondary end‐points included composite complete remission (cCR), disease‐free survival and safety. Median age was 74 years with 42% of patients being ≥75 years. After completing cycle 4, cCR was achieved in 73% of the patients, with 54% responding before cycle 2. After a median follow‐up of 23 months, median OS was 13.0 months. cCR achievement within cycle 4 correlated with longer OS (19.1 vs. 9.1 months, p = 0.001). Patients receiving 400 mg VEN without azoles had improved OS compared with those on reduced doses with azoles (18.2 vs. 11.4 months, p = 0.015). An anchored matching‐adjusted indirect comparison with the phase III VIALE‐A trial (NCT02993523) showed comparable median OS (14.8 months vs. 14.9 months; p = 0.6). The GIMEMA AML2320 trial confirmed the efficacy of VEN/HMAs in a prospective, real‐world unfit population. Early remission was associated with improved outcomes.

Purpose: The aim of this review is to summarize transcriptomic studies using single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq to investigate leukemic stem cells (LSCs) population in acute myeloid leukemia (AML). Methods: Literature for inclusion was identified through a PubMed search. Twenty-seven studies were analyzed, covering various aspects of LSCs biology, including epigenetic, transcriptional, and translational gene regulation, metabolic reprogramming or cellular differentiation. Results: The reviewed studies highlight the complexity of LSCs, including their quiescent state, state-to-state cellular plasticity, and metabolic reprogramming from glycolysis to lipogenesis. Comparative analyses of matched diagnostic and post-treatment samples provide insights into the dynamic behavior of LSCs during therapy. The findings also identify potential therapeutic targets and strategies to selectively eradicate LSCs. Conclusion: Single-cell RNA sequencing technologies offer unprecedented resolution for studying LSCs in AML, revealing cellular heterogeneity and dynamic evolution within these critical populations. Despite these advances, standardized criteria for LSCs identification are still needed, and longitudinal studies at clinically relevant time points are essential to fully understand their role in disease progression and therapy resistance. Such approaches may ultimately uncover novel therapeutic vulnerabilities and improve clinical management of AML.

Background: The level of measurable residual disease (MRD) is one of the most important features correlating depths of response and long‐term outcomes in multiple myeloma (MM) and MRD evaluation is currently the gold standard tool for assessing treatment response. Nevertheless, reproducibility across laboratories is a major concern, as discrepancies among results make comparability impractical. Aims: herein, we report preliminary results from the “Italian MM‐MRD network” project. Patients & Methods: MRD in bone marrow (BM) samples have been measured from newly diagnosed MM patients using next‐generation flow‐cytometry (NGF) or next‐generation sequencing (NGS) approaches in different laboratories. Results: The NGF workgroup (7 laboratories) implemented the Euro‐Flow Standard‐Operating‐Protocol to reach minimum 1 × 10 ⁻⁵ sensitivity. The inter‐operator retrospective study (Stage 1) showed high inter‐center concordance in monoclonal plasma cells detection (ICC = 0.90, p < 0.001), whereas moderate concordance was observed in the inter‐laboratory correlation (Stage 2) in in‐vivo samples (ICC = 0.63, p < 0.001), reaching a median limit‐of‐detection (LOD) and limit‐of‐quantification (LOQ) of 8 × 10−6 and 2 × 10 ⁻⁵ , respectively. Greater variability was also observed in the analysis of other BM cell populations. The NGS workgroup (4 laboratories) employed a targeted amplicon‐based approach to detect clonotypic IGH/IGK gene rearrangements in diagnostic samples, subsequently used to track MRD in mock samples. The experimental design was divided into three quality‐control (QC) rounds, focused on finding a shared strategy for clonotype identification (QC1: 100% concordance among centers), or quantifying MRD in mock samples (concordance: 81% [QC2]; 91% [QC3]). The 10 ⁻⁵ ‐sensitivity level was successfully reached in most of tested dilutions (QC2: 19/20 = 95%; QC3: 19/23 = 83%). Conclusion: Overall, this pilot study provided preliminary data for MRD harmonization across Italian centers, paving the way for an expanded network, aiming at reducing variability, improving comparability, and enabling broader use of MRD‐monitoring in clinical practice.

Measurable residual disease (MRD) monitoring has become a critical component in the management of acute myeloid leukemia (AML), to inform prognosis, guide therapy, and serve as a key end point in clinical trials. The 2025 update of the MRD guideline provides a comprehensive and refined framework for MRD assessment, aligned with the European LeukemiaNet (ELN) 2022 genetic risk classification. Developed by members of the ELN AML MRD Working Party, the guidelines incorporate expert consensus determined through a 2-stage Delphi round. They address the clinical implementation of MRD methodologies, technical considerations, integration into clinical trials, and future directions. Importantly, MRD recommendations are tailored to individual prognostic and genetic subgroups. A new qualitative MRD response category, designated as optimal, warning, or high risk of treatment failure, has been introduced to facilitate contextual interpretation of the MRD burden and its clinical relevance. Notably, ultrahigh-sensitivity next-generation sequencing–based MRD assessment is now recommended for FLT3 internal tandem duplication–mutated AML after intensive chemotherapy and before allogeneic hematopoietic cell transplantation. A total of 56 recommendations were formulated, with 53 achieving a high level of consensus (≥90%). These updated guidelines represent a major step forward toward harmonizing MRD assessments in AML and enhancing its clinical utility across diverse treatment settings.

CPX-351, a novel liposomal formulation of cytarabine and daunorubicin, represents the standard of care in fit patients with acute myeloid leukemia with myelodysplasia–related changes (AML-MRC) and therapy-related AML (t-AML). Considering its better safety profile than conventional intensive chemotherapy, we investigated its cost-to-benefit ratio, in terms of overall survival and of mortality, in a large multicentric series of AML-MRC and t-AML receiving CPX-351 outside clinical trials between 2019 and 2022. Patients were classified as fit or unfit for intensive chemotherapy through a comprehensive evaluation of age, comorbidities, and performance status by adopting Italian Society of Hematology/Italian Society of Experimental Hematology/Gruppo Italiano per il Trapianto di Midollo Osseo (SIE/SIES/GITMO) criteria. Disease risk was defined according to the European LeukemiaNet 2017 classification. Before treatment start, 328 of 403 (81.4%) patients were classified as fit and 75 of 403 (18.6%) as unfit. Three hundred and ninety-six had a full genetic/cytogenetic profile, with 17 (4%) being categorized as favorable risk, 162 (41%) intermediate risk, and 217 (55%) adverse risk according to European LeukemiaNet 2017. After induction, 230 of 403 (57.1%) patients achieved complete remission, with no differences between fit (57.3%) and unfit (56%) patients. However, the 2 groups significantly differed in terms of survival (median overall survival, 18 months vs 8 months for fit and unfit patients, respectively) and of 28- and 100-day mortality (4.6% vs 10.7% at 28 days and 14.3% vs 32% at 100 days for fit and unfit patients, respectively). In conclusion, the SIE/SIES/GITMO criteria distinguished patient subgroups with different short- and long-term outcomes after treatment with CPX-351. The update or design of dedicated fitness criteria could represent a future and valid strategy to optimize the use of this specific treatment.

Background: Perianal infections (PIs) are a serious threat in patients with acute myeloid leukemia (AML). While CPX-351 is designed to reduce gastrointestinal toxicity, its impact on the incidence of PIs is unknown. This study aims to evaluate the incidence and characteristics of PIs in a cohort of CPX-351-treated AML patients. Methods: We enrolled 22 adult patients diagnosed with secondary AML receiving CPX-351 between May 2020 and July 2025 at Policlinico Tor Vergata Hospital. Statistical analysis used descriptive statistics and multivariate analysis. Results: The incidence of PIs in the cohort was 31.8%. Microbiological cultures from the lesions commonly yielded Klebsiella pneumoniae and Enterococcus species. The development of a PI was associated with a significantly longer hospital stay (mean, 49.6 vs. 37.7 days; p = 0.034). An increased odds ratio of having PIs was noted for mucositis and positive rectal swabs (17.961, p = 0.062; 5.554, p = 0.391, respectively), with two patients (28.5%) having a positive pre-infection swab for Klebsiella pneumoniae. Surgical intervention was guided by patient pain levels and hematological criteria. Surgical patients had significantly higher pain levels (p = 0.001) and a platelet count greater than 20 × 10⁹/L (p = 0.028). All patients were alive at 30 days, with low rates of septic shock (14.2%, n = 1) and no infection-related mortality or recurrence. Conclusions: Despite CPX-351’s known reduced gastrointestinal toxicity, our study showed a significantly higher incidence of PIs compared to literature data. While the outcomes were favorable, PIs led to prolonged hospitalization. Routine rectal swab surveillance could be a valuable tool for risk stratification and preemptive strategies.

Background: Perianal infections (PIs) are a serious threat in patients with acute myeloid leukemia (AML). While CPX-351 is designed to reduce gastrointestinal toxicity, its impact on the incidence of PIs is unknown. This study aims to evaluate the incidence and characteristics of PIs in a cohort of CPX-351-treated AML patients. Methods: We enrolled 22 adult patients diagnosed with secondary AML receiving CPX-351 between May 2020 and July 2025 at Policlinico Tor Vergata Hospital. Statistical analysis used descriptive statistics and univariate analysis. Results: The incidence of PIs in the cohort was 31.8%. Microbiological cultures from the lesions commonly yielded Klebsiella pneumoniae and Enterococcus species. The development of a PI was associated with a significantly longer hospital stay (mean, 49.6 vs. 37.7 days; p = 0.034). A trend was noted between the presence of PIs and mucositis (p = 0.05) and positive rectal swabs (p = 0.09), with two patients (28.5%) having a positive pre-infection swab for Klebsiella pneumoniae. Surgical intervention was guided by patient pain levels and hematological criteria. Surgical patients had significantly higher pain levels and a platelet count greater than 20 x 10^9/L. All patients were alive at 30 days, with low rates of septic shock (14.2%, n=1) and no infection-related mortality or recurrence. Conclusions: Despite CPX-351′s known reduced gastrointestinal toxicity, our study showed a significantly higher incidence of PIs compared to literature data. While the outcomes were favorable, PIs led to prolonged hospitalization. Routine rectal swab surveillance could be a valuable tool for risk stratification and preemptive strategies.