ABSTRACT: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.

ABSTRACT:Secondary myelofibrosis (SMF) represents a late stage of polycythemia vera (PV) and essential thrombocythemia (ET), with overall survival (OS) currently defined by the myelofibrosis secondary to PV and ET prognostic model (MYSEC-PM). To identify additional myeloid neoplasm-associated cancer gene variants (CGVs) associated with SMF outcome, we evaluated next-generation sequencing panel testing in 644 patients within the MYSEC cohort. Overall, 429 (66.6%) patients reported at least 1 CGV, with ASXL1, TET2, and DNMT3A being the most frequently involved. Specific molecular profiles affected OS (P< .001): U2AF1, TP53, or SRSF2 variants (UTS; 9.3%; median OS, 4.1 years) and ASXL1 without UTS (25.3%; median OS, 8.4 years). By integrating these genetic signatures within the MYSEC-PM through penalized Cox regressions, we identified the following independent predictors (P< .0001 to .02): hemoglobin level <11 g/dL (1 point), circulating blasts ≥3% (2 points), platelet count <150 × 109/L (2 points), age (0.21 points/y), ASXL1 without UTS mutations (1 point), and any UTS mutations (3 points). Finally, we developed the MYSEC-molecular prognostic model (MYSEC-mPM) allocating 582 patients with SMF into 4 categories with different OS (P < .001): low (median OS, 18.0 years; score <14), intermediate-1 (8.8. years; score, 14-16), intermediate-2 (4.6 years; score, 17-18), and high risk (1.9 years; score ≥19). Additionally, in 381 patients with SMF and available cytogenetics, the MYSEC-mPM was implemented with complex/monosomal karyotype, generating the karyotype-enhanced MYSEC-kmPM. Our study shows that genomic and cytogenetic profiling improves survival prediction in SMF, outperforming the MYSEC-PM.

The clinical management of myeloid neoplasms increasingly relies on the accurate detection and longitudinal monitoring of disease-defining genetic alterations. Many clinically relevant mutations are often present at very low variant allele frequencies, below the detection limits of conventional approaches routinely used in diagnostic workflows. In recent years, a growing number of ultra-sensitive molecular technologies have been developed to overcome these limitations, enabling the detection of rare variants with unprecedented precision, offering complementary strengths in terms of sensitivity, quantification, throughput, and clinical applicability. This review provides a comprehensive overview of established and emerging ultra-sensitive technologies for the diagnosis and molecular monitoring of myeloid neoplasms, discussing their technical principles, advantages, and limitations.

Abstract Ruxolitinib is widely used in polycythemia vera (PV) following hydroxyurea failure. However, validated response criteria to predict long-term outcomes in ruxolitinib-treated patients are lacking, complicating clinical decision-making. We investigated predictors of event-free survival ([EFS]; including progression to post-PV myelofibrosis, thrombosis, hemorrhages, or death) after 6 months of ruxolitinib in 178 patients with PV enrolled in the observational PV-ARC study. After a median follow-up from ruxolitinib start of 3.50 years, 15 patients died, 7 had a thrombosis, 9 had a hemorrhage, and 21 progressed to myelofibrosis. Overall, 5-year EFS was 70.4%. Leukocytosis, thrombocytosis, phlebotomy need, lack of spleen length reduction ≥50% (SR50), and ruxolitinib dose <10 mg twice daily at 3 time points (baseline and months 3 and 6), were tested for association with EFS. Multivariable analysis identified 3 independent risk factors: (1) ruxolitinib dose <10 mg twice daily at ≥1 time point (hazard ratio [HR], 1.94; P = .047), (2) no SR50 at months 3 and 6 (HR, 2.65; P = .009), (3) phlebotomy requirement at ≥2 time points (HR, 2.11; P = .039). Points were assigned as follows: 1 to phlebotomies at 1 time point; 2 to phlebotomies at ≥2 time points and to ruxolitinib <10 mg twice daily at ≥1 time point; and 2.5 to lack of SR50. Based on cumulative scores, we developed the PV–response to ruxolitinib after 6 months (PV-RR6) prognostic model, identifying 3 risk categories: low (score 0: 5-year EFS, 89.4% [n = 63]), intermediate (score 1-2.5: EFS, 71.0% [n = 82]), and high (score >2.5: EFS, 38.9% [n = 33]). PV-RR6 enables early identification of patients at risk of poor outcomes, supporting timely treatment optimization in ruxolitinib-treated patients. This trial was registered at www.clinicaltrials.gov as NCT06134102.

ABSTRACT:Ineffective erythropoiesis and the resulting anemia are the main characteristics of myelodysplastic syndromes (MDS). Drugs designed to promote erythropoiesis in patients with MDS include erythropoiesis-stimulating agents, such as recombinant human erythropoietin, and transforming growth factor β inhibitors, such as luspatercept, which is approved for the treatment of anemia associated with MDS or β-thalassemia. However, these types of drugs are ineffective in some patients and fail to elicit durable responses in others, underscoring the need for additional therapeutic targets. Here, we sought to define the role of ubiquitin-conjugating enzyme E2O (UBE2O), which remodels the proteome during late erythroid differentiation, in the pathogenesis of ineffective erythropoiesis in the setting of MDS and investigate its potential as a therapeutic target for improving erythropoiesis. UBE2O expression was analyzed in K562 leukemic cells and bone marrow samples from patients with MDS before and after treatment with erythropoietin and luspatercept. Bioinformatic analysis identified a GATA1 binding motif on the UBE2O promoter region, and chromatin immunoprecipitation validated the interaction. Our findings demonstrate that GATA1 binds to the UBE2O promoter, thereby regulating UBE2O transcription and expression. Although further studies are needed to explore the implications of UBE2O in MDS treatment, our work provides potential strategies for novel therapeutic approaches in MDS.

BACKGROUND:Cytopenia is a common complication in patients with myelofibrosis and may worsen during treatment with ruxolitinib. METHODS:The RUX-MF multicenter study evaluated 879 patients treated with ruxolitinib for at least 6 months, categorizing them into four groups based on the evolution of cytopenia: never cytopenic, treatment-emergent cytopenia, persistent cytopenia, and improved anemia. RESULTS:At baseline, 40.6% of patients presented with cytopenia, increasing to 57.8% after 6 months. Baseline cytopenia was associated with significantly reduced median overall survival (OS) compared to noncytopenic patients (3.7 vs. 6.7 years). Prognosis varied notably across groups: patients who remained noncytopenic had the median best OS (8.1 years), whereas those with persistent cytopenia had the worst (3.7 years). Treatment-emergent cytopenia was linked to intermediate outcomes (5.1 years), with isolated thrombocytopenia showing the poorest prognosis (4.3 years) and anemia a slightly better one (6.1 years). Patients with improved anemia had better survival than those with persistent anemia (5.2 vs. 3.5 years). Symptom response mirrored survival trends, with the best outcomes in noncytopenic and improved anemia groups. CONCLUSIONS:These findings highlight the prognostic significance of cytopenia dynamics during ruxolitinib therapy and support the use of cytopenia trajectory monitoring as a valuable tool for risk stratification and treatment optimization in myelofibrosis.

Objectives: Venetoclax (VEN) is approved for acute myeloid leukemia (AML) in association with azacitidine, in a 28-day schedule at a fixed dosage, which requires reduction if azoles are co-administered. The present study aims to evaluate VEN therapeutic drug monitoring (TDM) in a real-word setting, where the VEN schedule is frequently reduced, investigating: (i) the posaconazole impact, and (ii) whether VEN exposure correlates with safety and efficacy. Methods: We analyzed data from 43 AML patients treated with different VEN-containing regimens, for whom a near-trough VEN plasma concentration (Cmin) was determined at different timepoints (days 5-8-11-15-22-29) across different cycles (163 cycles, 290 determinations). The posaconazole impact was explored in the whole study population, while safety and efficacy were investigated only in patients treated with azacitidine-VEN, respectively in the safety (35 patients) and in the efficacy subset (29 patients at their first cycle). VEN exposure was expressed through multiple parameters, taking into account both VEN concentrations and the days of VEN administration. Results: Posaconazole was used in 40.5% of cycles and, despite dose adjustment, was associated with: (i) greater interpatient variability, (ii) higher VEN concentrations, (iii) delayed elimination, (iv) accumulation along the cycle, and (v) the need for VEN-dosage change. In the safety subset, VEN exposure correlated with neutropenia and its duration, Granulocyte Colony-Stimulating Factor requirement, platelet transfusions, cycle duration, and infections. Finally, no correlation was found between VEN exposure and response in the efficacy subset. Conclusion: VEN TDM appears valuable in clinical practice to reduce toxicity, especially in patients receiving posaconazole, where VEN exposure remains highly unpredictable.

Restoring apoptosis in malignant cells represents a central goal of anticancer therapy. Tumour cells often escape cell death by overexpressing anti-apoptotic members of the BCL-2 protein family, particularly BCL-2, BCL-xL, and MCL1. These proteins inhibit the intrinsic mitochondrial apoptotic pathway through intricate interactions with pro-apoptotic partners and direct modulation of the mitochondrial outer membrane. Their pivotal role in cell survival has established them as attractive therapeutic targets. Over the past two decades, significant efforts have been devoted to developing selective small-molecule inhibitors capable of neutralising these proteins and reactivating apoptosis. A first milestone was the discovery of ABT-263 (navitoclax), a dual BCL-2/BCL-xL inhibitor. Building on this achievement, the development of venetoclax, a highly selective BCL-2 inhibitor, marked a major breakthrough, demonstrating potent pro-apoptotic activity and clinical efficacy in several leukaemia subtypes. Despite these advances, the design of inhibitors of BCL-2 family members remains challenging, largely due to the structural characteristics of the BH3-binding groove, which is both shallow and hydrophobic, complicating the identification of molecules with optimal binding affinity and selectivity. PROTACs targeting BCL-xL may represent a promising future strategy, potentially overcoming the intrinsic limitations of small molecule inhibitors.

The FLT3-ITD mutation is a critical prognostic marker in acute myeloid leukemia (AML) and recent clinical trials demonstrate that FLT3-based measurable residual disease (MRD) is both prognostic and predictive, guiding therapeutic interventions in intensive and post-transplant settings. Conventional detection methods lack the sensitivity required for effective MRD monitoring. We developed a patient-specific droplet digital PCR (ddPCR) approach achieving analytical sensitivity of 10-5 (0.001%) for FLT3-ITD quantification. In our cohort, ddPCR enabled longitudinal monitoring of clonal dynamics, allowing the detection of re-emerging FLT3-ITD clones months before hematologic relapse and earlier than standard capillary electrophoresis. Notably, 25% of patients who relapsed as FLT3-ITD positive despite being classified as FLT3-negative at diagnosis harbored detectable microclones when retrospectively analyzed by ddPCR, suggesting that FLT3-ITD-positive relapse frequently originates from pre-existing subclones below conventional detection thresholds. These findings challenge current diagnostic classification and may influence risk stratification and treatment decisions, particularly regarding FLT3 inhibitor eligibility. While ddPCR is limited to tracking known dominant clones, it represents a practical, cost-effective solution for high-sensitivity MRD surveillance. In the era of targeted FLT3 therapies, integrating sensitive molecular monitoring into routine AML management may enable timely therapeutic adjustments and improve patient outcomes.

BACKGROUND:The 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. METHODS:Clinical 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. RESULTS:With 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. CONCLUSIONS:In 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.

Background:Approximately one-third of chronic myeloid leukemia (CML) patients may develop resistance and/or intolerance to the current therapies and need to switch to later lines of treatment. However, how to choose a later line of therapy is still a matter of discussion. Methods:A survey was performed by the Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) to understand how the scenario has changed after the introduction of the first allosteric inhibitor, asciminib, in later lines. Results:The GIMEMA survey aimed to reassess the Italian approach to third-line or later-line treatments in CML. In the whole cohort of 1,637 patients, to treat resistance, ponatinib was used with a mean of 41% [standard deviation (SD) = 29] and a median of 50% (0-100), while asciminib was used with a mean of 27% (SD = 23) and a median of 25% (0-100). Indeed, to treat intolerance, asciminib was the most used with a mean of 32% (SD = 30) and a median of 30 (0-100), followed by bosutinib with a mean of 25% (SD = 25) and a median of 20 (0-90). Several possible treatment sequences were analyzed, and asciminib emerged as the best third-line treatment. Conclusions:The survey attempted to understand the major reasons for treatment switch, how tyrosine kinase inhibitors (TKIs) were selected, and which drug was preferred based on patient and disease characteristics. The current algorithm of treatment seems to have changed in both resistant and intolerant CML patients in later lines. The reduction of TKI dose is a current practice to maintain efficacy while reducing the occurrence of side effects.

BACKGROUND:The response to ruxolitinib after 6 months (RR6) model allows early identification of ruxolitinib-treated myelofibrosis (MF) patients with poorer overall survival (OS); however, it is less applicable to lower-risk patients. METHODS:To further explore this, the authors performed a subanalysis of the "RUX-MF" study (NCT06516406) with an aim to validate the RR6 and to develop a score specific for intermediate-1 DIPSS/MYSEC-PM risk patients. RESULTS:Among the 776 evaluable patients, 34.4%, 47.8%, and 17.8% were at low, intermediate, and high RR6 risk, with 5-year OS of 64.1%, 51.8%, and 44.5%, respectively (p < .001). In the 428 intermediate-1 patients, the RR6 model did not discriminate between intermediate and low-risk patients (5-year OS: 74.4% vs. 72.0%, p = .24). The intermediate-1 specific RR6 (iRR6) model was therefore developed by incorporating new variables: underdosed ruxolitinib with respect to platelet count at one or more time points (hazard ratio [HR], 3.91; p < .001), absence of palpable spleen reduction by ≥50% at 6 months (HR, 1.45; p = .02), and red blood cell transfusion requirement at all time points (HR, 1.85; p = .01). The iRR6 model stratified patients into three risk categories: low (score 0, 20.3%), intermediate (score 1-2, 45.8%), and high-risk (score >2, 33.9%), with 5-year OS of 84.8%, 76.4%, and 56.6%, respectively (p < .0001). The iRR6 model was validated in a cohort of 95 intermediate-1 risk patients from the Moffitt Cancer Center, yielding stratification into the same three risk categories, with 5-year OS of 83.3% (low-risk), 71.7% (intermediate-risk), and 54.5% (high-risk) (p = .01). CONCLUSIONS:The iRR6 model provides a more refined tool for the identification of intermediate-1 MF patients who may benefit from early therapy shift.

INTRODUCTION:In patients with myelofibrosis (MF), overall survival (OS) after ruxolitinib discontinuation is poor, with leukemic transformation, clonal evolution and thrombocytopenia as the main factors worsening prognosis. PATIENTS AND METHODS:To assess the impact of disease phenotype on outcome after ruxolitinib discontinuation in chronic phase patients, we performed a sub-analysis of the "RUX-MF" study (NCT06516406), which now includes 1055 MF patients who received ruxolitinib in a real-life context. RESULTS:After a median follow-up of 3.3 years, 397 patients discontinued ruxolitinib therapy while in chronic phase. At treatment end, 208 patients (52.4%) had a severely cytopenic phenotype (defined as platelets < 100 × 109/L and/or hemoglobin < 8 g/dL); among the remaining myeloproliferative 189 patients, 97 had no cytopenia (51.3%) and 92 (48.7%) had mild anemia only (hemoglobin between 8 and 10 g/dL). Overall, 175 patients (44.1%) had a large splenomegaly (palpable at ≥ 10 cm below costal margin). After ruxolitinib discontinuation, 3-year OS was 33.4% in severely cytopenic and 54.4% in myeloproliferative patients (P < .001); this was confirmed after adjustment for risk categories. Noncytopenic and mildly anemic patients had comparable OS (P = .73). Patients with large splenomegaly had significantly poorer OS compared to nonsplenomegalic patients (OS: 33.5% vs. 51.6% P = .01). Large splenomegaly confirmed its negative prognostic impact on OS of patients with myeloproliferative MF (60.7% vs. 44.5%, P = .05). In patients with severe cytopenia, the presence of a large splenomegaly did not influence OS (41.7% vs. 26.1%, P = .26). CONCLUSIONS:Cytopenic phenotype and large splenomegaly in myeloproliferative MF are key prognostic determinants of outcome after ruxolitinib discontinuation.

Background/Objectives: Treatment with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has revolutionized disease management and has transformed CML from a life-threatening disease to a chronic condition for many patients. However, overcoming resistance, particularly related to leukemic stem cells (LSC) that can persist even when the bulk of the leukemic cells are eliminated, remains a significant challenge. Methods: K562 and KU812 cell lines were treated in vitro with the TKI Imatinib (IM). Gene expression, protein analysis, and metabolomic screening were conducted to investigate the ability of the drug to enhance stem cell (SC) features. Moreover, a gene ontology analysis was performed on different available datasets, to further consolidate our data. Results: 48 h of IM treatment can significantly increase the expression of genes related to SC self-renewal, particularly SOX2 and OCT 3/4. Interestingly, these modulations occur in cells that remain alive after drug treatment and that displayed features consistent with leukemia stem-like CML cells, suggesting that SC genes levels are crucial even in cell population survived upon TKI treatment. Moreover, after in silico analysis of available data, we observed an enrichment of SOX2/NANOG and OCT 3/4 signatures after TKI treatment, thus strengthening our results. Conclusions: Our results confirmed the relevance of LSC features after TKI treatment, highlighting the need for more effective and potentially curative strategies targeting LSCs to overcome resistance in CML.

Expectation of survival of patients receiving HMA + VEN is influenced by pre-treatment comorbidity burden.

Introduction: Acute myeloid leukemia (AML), a clonal neoplastic disease, mainly affects older adults, peaking at age 68, with two-thirds of cases diagnosed after age 55. Treatment is influenced by disease biology and patient-specific factors like comorbidities and performance status. The current risk stratification system, the European LeukemiaNet (ELN) 2022 risk model, was developed for fit patients receiving intensive therapy and is less effective for unfit patients. Therefore, two new models, ELN 2024 and Beat-AML 2024, have been proposed for patients who are candidates for non-intensive treatments. Our study aims to assess which model best predicts median Overall Survival (OS) in unfit AML patients in Italy, highlighting strengths and limitations and exploring strategies to identify patients suitable for further reduced-intensity therapies. Materials and Methods: We analyzed 171 unfit AML patients from 8 Italian centers. Molecular and cytogenetic data were used to apply ELN2022, ELN2024, and Beat-AML 2024 stratifications. Genetic alterations were assessed using myeloid NGS panels and conventional molecular analysis; cytogenetics followed ISCN guidelines. Patients received hypomethylating agents (HMA) with/without venetoclax. Risk distributions were analyzed with contingency tables; OS was assessed using Kaplan-Meier, log-rank tests, and Cox models; model performance was evaluated with the C-index. Results: The study population had a median age of 74.8 years. Among all patients, 132 received HMA-Ven, 31 received HMA alone and 8 received other low-intensity therapies. Median OS was 14 months (mos) (CI 10-17), similar to the one observed in de VIALE-A study (14,7 mos). Patients were stratified according to the ELN2022, ELN2024, and BEAT-AML2024 risk models. Based on ELN2022, the majority of patients (63%) were classified as adverse risk, while 17% and 20% were categorized as intermediate and favorable risk respectively. In contrast, using the ELN2024 model, 16% of patients fell into the adverse risk category, 22% into intermediate, and 62% into the favorable risk group. Finally according to BEAT-AML2024, 20%, 43% and 37% were observed for the same categories respectively. The curves based on the ELN2022 and the ELN2024 models demonstrated a statistically significant difference only between the adverse risk groups and the others but proved unable to discriminate between favorable and intermediate risks. The BEAT-AML2024 model, instead, showed the strongest statistical significance in differentiating between all risk groups (p-value <0.0001). The 2-years OS was 48.7% (CI 35.5-66.9) and median OS was 21 mos (CI 15-NA) for favorable risk, 25.6% (CI 15.8- 41.6) and 13 mos (CI 12-20) for intermediate risk, 10.33% (CI 2.8- 37.7) and 8 mos (CI 4-11) for adverse risk. In contrast ELN2024 showed a 2-year OS of 35.06% (CI 25.5-48.7) and median OS of 16 mos (CI 13-24) for favorable risk, 28.9%(CI 15.4-54.3) and 9 mos (CI 8-NA) for intermediate risk, 17.51% (CI 6.3-48.3) and 9 m (CI 5-16) for adverse risk. The C-index was highest for BEAT-AML24, confirming its better predictive performance (0.603, SE 0.031 vs 0.573, SE 0.028). Within the limits of our sample, we evaluated the influence of age, type of therapy, and mutation burden on survival, without obtaining statistical significant predictions. Conclusion: The BEAT-AML2024 model emerged as the best statistically significant model, demonstrating a superior C-index compared to the others. As shown by our data, BEAT-AML2024 better discriminates the 2-years OS and median OS compared to ELN2024. In our opinion, the more balanced distribution in the 3 risk classes of BEAT-AML2024 prevent the overestimation of favorable risk patients observed in ELN2024. Furthermore, patients stratified as adverse risk by BEAT-AML2024 show a lower OS compared to ELN2024-defined counterparts, suggesting a better discriminative capacity. However, the study has some limitations. Only two centers routinely analyze mutations in the DDX41, BCOR, and STAG2 genes. Moreover, these models rely on both karyotyping and NGS analysis, whose additional costs are not yet justified by the current therapeutic implications. Further research is needed to identify which patients might benefit from a further reduction in treatment intensity.

The BCR::ABL1 transcript is a defining characteristic of Chronic Myeloid Leukemia (CML) and European LeukemiaNet (ELN) guidelines emphasize monitoring its levels to evaluate treatment efficacy. While tyrosine kinase inhibitor (TKI) therapy has significantly improved outcomes for patients with chronic phase CML (CP-CML), a subset of patients still exhibits suboptimal responses. Qualitative differences in BCR::ABL1 transcript subtypes appear to affect response rates. Previous studies, primarily focusing on imatinib (IM) as frontline therapy, indicated that CML patients carrying the e14a2 transcript achieve more frequently optimal molecular responses by RT-QPCR if compared with e13a2. However, similar data in patients receiving second-generation TKIs (2G-TKIs) as frontline treatment is limited. Hence, we evaluated in the real-life scenario of the GIMEMA LabNet CML network the impact of the most frequent BCR::ABL1 transcript types (e14a2, e13a2, double isoform e14a2/e13a2) molecular response rate of patients with newly diagnosed CP-CML at 6, 12, 24 months after frontline treatment. Findings were stratified as IM-treated versus 2G-TKIs. Out of 9,699 patients, 1471 met study criteria with evaluable samples at the above timepoints. BCR::ABL1 transcript subtype carriers were: 675 (46%) with e14a2, 375 (25%) with e13a2, 290 (20%) with e14a2/e13a2, others 131 (9%). Current analysis focused on 1340 patients carrying the most frequent transcripts. Median age at diagnosis was 59 years (range 18-91), males were 766 (57%) and females 574 (43%). Frontline CML therapy was IM in 721, 2G-TKIs in 619 (230 with DAS, 386 with NIL, 3 with BOS). Within the IM cohort, at 6, 12 and 24 months, e14a2 carriers achieved > MR2 responses (38%, 60%, 80%;) if compared with e13a2 patients (21%, 39%, 68%, p=0.001, p<0.001, p=0.037 respectively). At the same time-points double isoform carriers performed better than e13a2 (35%, 62%, 85%; p=0.012, p<0.001, p=0.009). Among the 2G-TKIs cohort, e14a2 patients obtained better > MR2 responses at 6 and 12 months (67%, 75%) than e13a2 (50%, 62%; p=0.003, p=0.02 respectively) but there was no statistical difference at 24 months (85% vs 76%). No differences were documented when e14a2 was compared to the double isoform. Double isoforms showed better > MR2 rates than e13a2 at 6 and 12 months (67% vs 50%, 77% vs 62%; p=0.024, p=0.042) while at 24 months the rates of > MR2 were comparable (79% vs 76%). Notably, when e13a2 carriers were treated with 2G-TKIs achieved better and faster responses at 6 and 12 months (p<0.001) than with IM. Our data confirms that the kinetic behavior of BCR::ABL1 molecular reduction on frontline TKIs treatment shows different trajectories when evaluated by transcript types. Even if a limitation of our analysis might be the lack of risk distribution by Sokal or ELTS, these results, obtained in a high number of analyzed patients, preserve a notable meaning. These kinetic differences are also evident in 2G-TKIs treated patients. However, to what extent these differences are related to RTQ-PCR technical limitations (not relevant with digital PCR) or to real differences intrinsically related to the transcript type is yet to be clarified.