An Automated Cartridge-Based Microfluidic System for Real-Time Quantification of BCR::ABL1 Transcripts in Chronic Myeloid Leukemia: An Italian Experience Alice Costanza Danzero, Enrico Marco Gottardi, Fabrizio Quarantelli, Ciro Del Prete, Alessandra Potenza, et al. International Journal of Molecular Sciences, 2025 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR::ABL1 fusion gene, resulting from a reciprocal translocation between chromosomes 22 and 9. Quantification of BCR::ABL1 transcript levels in peripheral blood by RT-qPCR represents the gold standard for molecular response (MR) monitoring, providing essential clinical information on treatment efficacy. Xpert® BCR-ABL Ultra is a fully automated in vitro diagnostic test that quantitatively detects e13a2 and e14a2 BCR::ABL1 transcripts using a single-use cartridge that integrates RNA extraction, cDNA synthesis, nested real-time PCR, and signal detection within a rapid, closed, and user-friendly system. In this study, we evaluated Xpert® BCR-ABL Ultra as an alternative to validated systems currently used by four highly specialized Italian laboratories affiliated with the Italian national laboratory network for CML. A total of 129 peripheral blood samples from CML patients at various disease stages, along with two external quality control materials, were analyzed. We assessed the test’s repeatability, specificity, and stability. Concordance of BCR::ABL1%IS values generated by the different methods was evaluated using EUTOS criteria and Bland–Altman analysis. Finally, MR value concordance was analyzed based on European LeukemiaNet recommendations or calculated using the formula 2 − log10(BCR::ABL1%IS). Xpert® BCR-ABL Ultra demonstrated high repeatability and stability. The BCR::ABL1%IS values obtained with this assay showed strong concordance with those generated by local reference methods, and MR classifications were consistent across platforms. These findings confirm the robustness, accuracy, and efficiency of the Xpert® BCR-ABL Ultra assay, supporting its use as a reliable alternative to currently validated systems for the routine clinical monitoring of CML patients.
Validation Study of Analytical Methods for Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Acute Myeloid Leukemia Martina Barone, Agnese Patuelli, Michele Dicataldo, Maria Irno Consalvo, Gabriella Chirumbolo, et al. International Journal of Molecular Sciences, 2025 The standardization of multiparameter flow cytometry-based measurable residual disease (MFC-MRD) assessment in acute myeloid leukemia (AML) lacks clear criteria to define leukemia-associated immunophenotypes (LAIPs). In addition, the most specific/sensitive aberrations used to define LAIPs are often partially expressed by the leukemic clone at diagnosis, raising questions about their reliability for accurate MRD quantification. To address this, we investigated whether the quantification of LAIP+ cells reflects residual disease in cases of partial LAIP expression. The following two MFC-MRD approaches were evaluated by comparing their results to RT-qPCR for NPM1 mutations: (1) the LAIP-method, wherein all cells within the patient-specific template created at diagnosis are counted without further gating; (2) the LAIP-based different-from-normal (DfN)-method, wherein cells+ for LAIP-specific aberrant markers are further selected. A total of 125 bone marrow samples from 25 NPM1-mutated AML patients were studied. Our data demonstrate that the LAIP-based DfN-method improves the MFC-MRD accuracy and comparability with molecular MRD. ROC analysis identified cut-offs of 0.034% and 0.095% to discriminate positive/negative results in patients receiving intensive chemotherapy and hypomethylating agents, respectively. We also found distinct accuracy degrees based on the LAIP-specific aberrant markers used for MRD assessment. These results refine the MFC-MRD method and highlight the importance of therapy-specific MRD cut-offs and LAIP classification based on specificity and sensitivity.
Upfront intensive treatment analysis of the Italian Cohort Study on FLT3-mutated AML patients (FLAM): The impact of a FLT3 inhibitor addition to standard chemotherapy in the real-life setting Jacopo Nanni, Irene Azzali, Cristina Papayannidis, Antonino Mulè, Ernesta Audisio, et al. Cancer, 2025 BackgroundThe addition of a FLT3 inhibitor (FLT3i) to standard chemotherapy to treat fit newly diagnosed (ND) patients with FLT3‐mutated acute myeloid leukemia (AML) represents the standard of care resulting from clinical trial results. However, evidence regarding FLT3i adoption in routine clinical practice is still scarce.MethodsClinical data are reported from 394 ND patients with FLT3‐mutated AML enrolled in the retrospective observational Italian Cohort Study on FLT3‐mutated patients with AML and treated with an upfront intensive regimen with (FLT3i group, n = 92) or without (CT group, n = 302) the addition of a FLT3i.ResultsWith a median follow‐up time of 34.5 months, an effectiveness benefit obtained by FLT3i incorporation both in terms of overall survival (median, 34.9 in the FLT3i vs 12.7 months in the CT group, p < .01) and relapse‐free survival (median, 18.9 in the FLT3i vs 7.6 months in the CT group, p = .01) was documented, with a higher composite complete remission rate (75.4% in the FLT3i vs 62.4% in the CT group, p = .052). FLT3i benefit seemed to be independent from the transplant rate.ConclusionsIn conclusion, the benefit of FLT3i addition to upfront intensive treatment in newly diagnosed FLT3‐mutated AML patients was confirmed in a large, real‐life cohort study.
Emergence and Cytogenetic Clonal Evolution of Chromosome 7 Abnormalities in Myeloid Malignancies: Investigating the Role of Telomere Dysfunction Carmen Baldazzi, Lorenza Bandini, Valentina Robustelli, Agnese Patuelli, Claudia Venturi, et al. International Journal of Molecular Sciences, 2025 Monosomy 7 and deletion 7q are common chromosomal abnormalities in myeloid malignancies, and they are associated with a poor prognosis. The mechanism underlying their acquisition remains elusive. We identified a cohort of 24 patients exhibiting clones with different chromosome 7 abnormalities, such as deletion 7q, unstable derivatives (ring chromosomes or ‘naked’ centromeres), and monosomy 7. We designated this group as having cytogenetic clonal evolution of chromosome 7 abnormalities (CCE7). In some cases, CCE7 correlated with disease progression, suggesting that deletions or other derivatives involving the q-arm of chromosome 7 may arise early in the disease course. These abnormalities may be transient but can potentially evolve into monosomy 7. Within the CCE7 group, telomere loss or shortening may contribute to chromosomal instability and the emergence of unstable derivatives, as the chromosome 7 derivatives displayed loss or rearrangement of subtelomeric regions. Moreover, we identified variants in genes implicated in telomere biology disorders and observed specific genetic mutation profiles associated with different chromosome 7 abnormalities. These findings shed light on a potential mechanism leading to monosomy 7 through the evolution of chromosome 7q abnormalities. Identifying patients at risk of developing monosomy 7, based on the presence of unstable derivatives with telomere loss or a specific mutation profile, could potentially enhance patient management and guide the development of novel therapeutic strategies.
Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets Samantha Bruno, Enrica Borsi, Agnese Patuelli, Lorenza Bandini, Manuela Mancini, et al. International Journal of Molecular Sciences, 2024 Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.
Real-life use of ropeg-interferon α2b in polycythemia vera: patient selection and clinical outcomes Francesca Palandri, F. Branzanti, M. Venturi, A. Dedola, G. Fontana, et al. Annals of Hematology, 2024 Ropeginterferon-alfa2b (ropegIFNα2b) is a long-acting IFN formulation with broad FDA/EMA approval as a therapy of polycythemia vera (PV) with no symptomatic splenomegaly. There is currently lack of information on the real-world patient selection, including the impact of local reimbursement policies, and drug management, particularly: type/timing of screening and follow-up tests; absolute/relative contraindications to therapy; ropegIFNα2b dose and combinations with hydroxyurea. As a sub-analysis of the PV-ARC retrospective study (NCT06134102), we here report our monocenter experience with ropegIFNα2b in the period from January 2021, corresponding to drug availability outside clinical trial, and December 2023. Among the 149 patients with EMA/FDA indication, only 55 (36.9%) met the local reimbursement criteria and 18 (12.1%) received ropegIFNα2b. Thanks to appropriate screening, relative/absolute contraindications to ropegIFNα2b were detected and managed in a multidisciplinary manner. Efficacy and safety of ropegIFNα2b was confirmed, with 3 cases of early molecular response. General use of low ropegIFNα2b dose, with frequent need for hydroxyurea combinations, was noted. This real-world experience suggests a significant impact of local regulations on drug prescription and the need for greater real-world data collection on ropegIFNα2b in PV patients. Also, it describes appropriate multidisciplinary screening and monitoring procedures during ropegIFNα2b therapy.
Ex vivo characterization of acute myeloid leukemia patients undergoing hypomethylating agents and venetoclax regimen reveals a venetoclax-specific effect on non-suppressive regulatory T cells and bona fide PD-1+TIM3+ exhausted CD8+ T cells Giulia Corradi, Dorian Forte, Gianluca Cristiano, Andrea Polimeno, Marilena Ciciarello, et al. Frontiers in Immunology, 2024 Acute myeloid leukemia (AML) is an aggressive heterogeneous disease characterized by several alterations of the immune system prompting disease progression and treatment response. The therapies available for AML can affect lymphocyte function, limiting the efficacy of immunotherapy while hindering leukemia-specific immune reactions. Recently, the treatment based on Venetoclax (VEN), a specific B-cell lymphoma 2 (BCL-2) inhibitor, in combination with hypomethylating agents (HMAs) or low-dose cytarabine, has emerged as a promising clinical strategy in AML. To better understand the immunological effect of VEN treatment, we characterized the phenotype and immune checkpoint (IC) receptors’ expression on CD4+ and CD8+ T cells from AML patients after the first and second cycle of HMA in combination with VEN. HMA and VEN treatment significantly increased the percentage of naïve CD8+ T cells and TIM-3+ CD4+ and CD8+ T cells and reduced cytokine-secreting non-suppressive T regulatory cells (Tregs). Of note, a comparison between AML patients treated with HMA only and HMA in combination with VEN revealed the specific contribution of VEN in modulating the immune cell repertoire. Indeed, the reduction of cytokine-secreting non-suppressive Tregs, the increased TIM-3 expression on CD8+ T cells, and the reduced co-expression of PD-1 and TIM-3 on both CD4+ and CD8+ T cells are all VEN-specific. Collectively, our study shed light on immune modulation induced by VEN treatment, providing the rationale for a novel therapeutic combination of VEN and IC inhibitors in AML patients.
A venetoclax and azacitidine bridge-to-transplant strategy for NPM1-mutated acute myeloid leukaemia in molecular failure C. Sartor, L. Brunetti, E. Audisio, A. Cignetti, L. Zannoni, et al. British Journal of Haematology, 2023 NPM1-mutated acute myeloid leukaemia (NPM1mut AML) represents a mostly favourable/intermediate risk disease that benefits from allogeneic haematopoietic stem cell transplantation (HSCT) in case of measurable residual disease (MRD) relapse or persistence after induction chemotherapy. Although the negative prognostic role of pre-HSCT MRD is established, no recommendations are available for the management of peri-transplant molecular failure (MF). Based on the efficacy data of venetoclax (VEN)-based treatment in NPM1mut AML older patients, we retrospectively analysed the off-label combination of VEN plus azacitidine (AZA) as bridge-to-transplant strategy in 11 NPM1mut MRD-positive fit AML patients. Patients were in MRD-positive complete remission (CRMRDpos ) at the time of treatment: nine in molecular relapse and two in molecular persistence. After a median number of two cycles (range 1-4) of VEN-AZA, 9/11 (81.8%) achieved CRMRD -negative (CRMRDneg ). All 11 patients proceeded to HSCT. With a median follow-up from treatment start of 26 months, and a median post-HSCT follow-up of 19 months, 10/11 patients are alive (1 died from non-relapse mortality), and 9/10 patients are in MRDneg status. This patient series highlights the efficacy and safety of VEN-AZA to prevent overt relapse, achieve deep responses and preserve patient fitness before HSCT, in patients with NPM1mut AML in MF.
Prognosis in Chronic Myeloid Leukemia: Baseline Factors, Dynamic Risk Assessment and Novel Insights Miriam Iezza, Sofia Cortesi, Emanuela Ottaviani, Manuela Mancini, Claudia Venturi, et al. Cells, 2023 The introduction of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of chronic myeloid leukemia (CML), leading to a dramatic improvement of the outcome of CML patients, who now have a nearly normal life expectancy and, in some selected cases, the possibility of aiming for the more ambitious goal of treatment-free remission (TFR). However, the minority of patients who fail treatment and progress from chronic phase (CP) to accelerated phase (AP) and blast phase (BP) still have a relatively poor prognosis. The identification of predictive elements enabling a prompt recognition of patients at higher risk of progression still remains among the priorities in the field of CML management. Currently, the baseline risk is assessed using simple clinical and hematologic parameters, other than evaluating the presence of additional chromosomal abnormalities (ACAs), especially those at “high-risk”. Beyond the onset, a re-evaluation of the risk status is mandatory, monitoring the response to TKI treatment. Moreover, novel critical insights are emerging into the role of genomic factors, present at diagnosis or evolving on therapy. This review presents the current knowledge regarding prognostic factors in CML and their potential role for an improved risk classification and a subsequent enhancement of therapeutic decisions and disease management.
