Given the heterogeneity of acute myeloid leukemia patients, it is necessary to identify patients considered fit for intensive therapy but who will perform poorly, and in whom alternative approaches deserve investigation. We analyzed 1034 fit adults ≤70 years intensively treated between 2012 and 2022 in the CETLAM group. Young adults ( ≤ 60 years) presented higher remission rates and improved survival than older adults above that age (CR 79% vs. 73%; p = 0.03 and 4-yr OS 53% vs. 33%; p < 0.001). Remission and survival outcomes varied among different genetic subsets. An especially adverse genetic group included complex, monosomal karyotype, TP53 alterations (deleted/mutated), and MECOMr. Transplant feasibility in this very adverse risk group was low, and OS and EFS at 4 years were 14% and 12%, in contrast to 70% and 57% in the favorable group and 38% and 32% in all other patients. We integrated clinical and genetic data into the Intensive Chemotherapy Score for AML (ICSA) with 6-risk categories with significantly different remission rates and OS, validated in another cohort of 581 AML patients from a previous CETLAM protocol. In summary, we identified groups of fit patients that benefit differently from an intensive approach which may be helpful in future treatment decisions.

Background Measurable residual disease (MRD) detection in acute myeloid leukemia (AML) patients in complete morphological remission (CR) is associated with an increased risk of relapse and mortality. While molecular methods such as qPCR offer high sensitivity, most patients lack a molecular marker and are monitored using flow cytometry (FC). Next-generation sequencing (NGS) and digital PCR (ddPCR) represent a potential alternative for MRD assessment in AML, given that they offer high sensitivity and broad applicability. In this prospective study, we aimed to compare the clinical performance of NGS and ddPCR for MRD assessment in a cohort of intensively treated AML patients. Methods Adult patients with newly diagnosed AML treated with intensive chemotherapy (IC) according to CETLAM 2022 protocol were included prospectively in this study. Bone marrow (BM) samples of patients in CR were collected for MRD analysis at different time points: after two courses of IC (all), at the end of IC (favorable patients), at the time of hematopoietic stem cell transplant (HSCT) and 1 month after (intermediate/adverse patients). A custom NGS panel for MRD detection (NGS-MRD) was designed including 30 genes frequently mutated in AML and a specific assay to detect FLT3-ITD. NGS-MRD was performed on DNA extracted from BM. ddPCR assays were selected personally based on the mutational profile of each patient and performed on the same DNA samples. Dilution studies were conducted to assess the level of detection of both techniques, establishing a positivity threshold of 0.2% VAF for NGS-MRD and 0.1% VAF for ddPCR. MRD status was defined according to the European LeukemiaNet (ELN) guidelines (Heuser M et al, Blood, 2021). Results Forty-nine BM samples in CR from 23 AML patients were included in this study. The median age at diagnosis was 61 (range 21-73), and 52.2% were male. Fifteen (65.2%) patients had HSCT indication based on ELN 2022 intermediate or adverse-risk, while 8 (34%) were classified as favorable-risk. Standard of care (SoC) methods for MRD evaluation were flow cytometry in 16 (69.5%) of them, NPM1-qPCR in 6 (21.8%), and CBF-qPCR in 1 (4.3%). With a median follow-up of 17.9 (range 3.7-24.9) months, there were 4 (17.4%) relapses and 2 (8.7%) deaths. All 49 samples were analyzed with NGS-MRD. A median of 3 mutations (range 1-8) could be tracked for each patient with a median read depth of 6336x (range 576-26402x). Twenty-five (51%) samples had a targetable mutation for ddPCR analysis including IDH1, IDH2, NRAS, and FLT3-TKD. In the 25 samples studied by NGS and ddPCR, we found a strong correlation (r=0.925) between the VAF values obtained by both methods. The median VAF for positive MRD samples was 4.2% (range 0.1-42.1%). All NGS-MRD-positive samples were also considered positive by ddPCR, while 5 NGS-MRD-negative had low-level detectable ddPCR-MRD (range 0.1-0.5% VAF). Next, we compared our results with standard methods. After two cycles of IC, 33.7% of patients had detectable MRD by SoC techniques, while 52.1% by NGS and 63.6% by ddPCR. All SoC-MRD+ were also positive by NGS and ddPCR, but we could identify MRD persistence in 50% of patients with undetectable MRD by FC or qPCR. Moreover, in our cohort, NGS-MRD status after the second induction significantly predicted RFS (22.2 vs not reached; p=0.025). ddPCR-MRD status showed a positive trend (15.2 vs NR; p=0.191) but did not reach statistical significance. At the end of chemotherapy, all favorable-risk patients had undetectable MRD by SoC tests, but we could detect 3 (37.5%) MRD+ cases by both NGS and ddPCR. Two of these cases relapsed within 6 months. Regarding HSCT recipients, pre-transplant MRD was detectable in 35.7% of patients by FC, 50% by NGS, and 75% by ddPCR. However, pre-HSCT MRD by any method did not show an impact on RFS. This could be due to the high MRD clearance rate after HSCT: all FC-MRD+ patients converted to MRD- and 92.9% of MRD+ patients with NGS or ddPCR. Conclusions NGS and ddPCR showed a high correlation as MRD assessment methods. While ddPCR offered higher sensitivity, NGS-MRD status was more predictive of relapse-free survival. Also, NGS allowed us to track multiple mutations and even new clones, while ddPCR was restricted to 1-2 targets per patient. Applying these techniques in a prospective AML cohort, we could detect MRD persistence in a substantial number of MRD-negative patients by standard methods.

Background Iadademstat is a potent, selective, oral inhibitor of both the enzymatic and scaffolding activities of the transcriptional repressor lysine-specific demethylase 1 (LSD1; also known as KDM1A) that showed promising early activity and safety in a phase 1 trial and strong preclinical synergy with azacitidine in acute myeloid leukaemia cell lines. Therefore, we aimed to investigate the combination of iadademstat and azacitidine for the treatment of adult patients with newly diagnosed acute myeloid leukaemia. Methods The open-label, phase 2a, dose-finding ALICE study was conducted at six hospitals in Spain and enrolled patients aged 18 years or older with newly diagnosed acute myeloid leukaemia not eligible for intensive chemotherapy and an ECOG performance status of 0-2. In the dose escalation portion of the trial, patients received a starting dose of iadademstat at 90 mu g/m 2 per day (with de-escalation to 60 mu g/m 2 per day and escalation up to 140 mu g/m 2 per day) orally, for 5 days on, 2 days off weekly, with azacitidine 75 mg/m 2 subcutaneously, for seven of 28 days. The primary objectives were safety (analysed in the safety analysis set; all patients who received at least one dose of study treatment) and establishing the recommended phase 2 dose; secondary objectives included response rates in the efficacy analysis set (all patients who had at least one efficacy assessment). This study is registered on EudraCT (EudraCT 2018-000482-36) and has been completed. Findings Between Nov 12, 2018, and Sept 30, 2021, 36 patients with newly diagnosed acute myeloid leukaemia were enrolled; the median age was 76 (IQR 74-79) years, all patients were White, 18 (50%) were male, and 18 (50%) were female, and all had intermediate-risk or adverse-risk acute myeloid leukaemia. The median follow-up was 22 (IQR 16-31) months. The most frequent (>= 10%) adverse events considered to be related to treatment were decreases in platelet (25 [69%]) and neutrophil (22 [61%]) counts (all grade 3-4) and anaemia (15 [42%]; of which ten [28%] were grade 3-4). Three patients had treatment-related serious adverse events (one fatal grade 5 intracranial haemorrhage, one grade 3 differentiation syndrome, and one grade 3 febrile neutropenia). Based on safety, pharmacokinetic and pharmacodynamic data, and efficacy, the recommended phase 2 dose of iadademstat was 90 mu g/m 2 per day with azacitidine. 22 (82%; 95% CI 62-94) of 27 patients in the efficacy analysis set had an objective response. 14 (52%) of 27 patients had complete remission or complete remission with incomplete haematological recovery; of these, ten of 11 evaluable for measurable residual disease achieved negativity. In the safety analysis set, 22 (61%) of 36 patients had an objective response. Interpretation The combination of iadademstat and azacitidine has a manageable safety profile and shows promising responses in patients with newly diagnosed acute myeloid leukaemia, including those with high-risk prognostic factors.

Background/Objectives: Patients with relapsed/refractory (R/R) AML with FLT3 mutation (FLT3mut) have a dismal prognosis. FLT3mut offers a target for therapy in these patients. Gilteritinib (gilter) and quizartinib (quizar) have demonstrated efficacy as single agents in two phase 3 clinical trials. Methods: We retrospectively analyzed the characteristics, treatments, and outcomes of 50 patients with R/R FLT3mut AML who received gilter or quizar as monotherapy in 27 Spanish centers before their commercial availability. Forty-four patients were treated with gilter and six with quizar. Results: The median age was 62.5 years, and 52% were women. Most patients presented with FLT3-ITD mutations (80%); 46% had refractory disease and 54% had relapsed disease at treatment initiation. First-line treatment was chemotherapy in 80% of patients, with 40% of these also receiving midostaurin. Twenty-five patients (50%) had previously received FLT3 inhibitor, and twenty-eight (56%) had received more than one line treatment before starting gilter/quizar. The rates of complete remission (CR), CR without hematological recovery (CRi), and partial remission were 22%, 18%, and 16%, respectively. The median overall survival (OS) and disease-free survival were 4.74 months and 2.99 months, respectively. We observed a significant improvement in OS in patients who had received only one prior line of therapy compared to those who had received two or more therapies (10.77 months vs. 4.24 months, p = 0.016). Multivariate analysis identified failure to achieve CR/CRi, receiving more than one prior line of therapy, age, and white blood cells count as independent prognostic factors for OS. The most common toxicities were febrile neutropenia, liver function abnormalities, and QT interval prolongation. Conclusions: Gilter/quizar monotherapy are effective and tolerable options for patients with R/R FLT3mut AML in a real-world setting. Response and toxicity rates are similar to those reported in the phase 3 trials, despite the more heterogeneous nature of the study population.

INTRODUCTION Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia defined by a reciprocal translocation t(15;17)(q24.1;q21.2), giving rise to a PML::RARA fusion gene product (Arber et al.,2016). Arsenic trioxide (ATO) or chemotherapy plus all-trans retinoic acid (ATRA) frontline treatment regimens are the current standard of care (SOC) for patients with newly diagnosed APL (Sanz et al., 2019). Additional cytogenetic abnormalities (ACA) are frequently found in APL and more rarely complex karyotype (CK) (Arber et al.,2016). Different groups have tried to establish the prognostic impact of CK, but their low prevalence and the variability within the alterations do not make it an easy task (Epstein-Peterson et al., 2022). This study aims to analyze our cohort's overall survival (OS), event free survival (EFS) and to determine the prognostic impact of complex karyotype in this setting of patients. METHODS We included all APL patients diagnosed at 4 oncology centers associated with the Catalan Institute of Oncology (ICO-Institut Català d'Oncologia“) between 2005 and 2024 analyzing those with the presence of t(15;17) demonstrated by karyotype.CK was defined as the presence of ≥ 2 cytogenetic alterations in addition to the t(15;17). OS and EFS were determined by Kaplan-Meyer method. A multivariable Cox proportional hazards model was used to correlate OS and EFS with clinical and biological characteristics. RESULTS One hundred eighty-six patients were included and a total of 148 were analyzed. Thirteen patients were excluded due to lack of karyotype growth, 12 patients for presenting a normal karyotype which made impossible to distinguish between the normal clone selection or a cryptic karyotype, 11 patients for not being treated with SOC and received ATRA alone and 2 patients due to loss to follow-up. Median age at diagnosis was 51 years (range:16-85 years). Sixty-nine (46.6%) were male. At diagnosis: median hemoglobin 89 g/L (range:4.4-161g/L), platelets 28 x109/L (range: 3-256 x109/L), total leucocytes count 3.4 x109/L (range: 0.3-169.87 x109/L) and 103 patients (69.6%) presented with associated coagulopathy. One hundred and twenty-two (82.4%) patients had a karyotype with 46 chromosomes and the t(15;17) (n=122, 82.4%) as unique alteration. Four (2.7%) presented with ACA and 22 (14.9%) with CK. All patients received treatment, 93 (62.8%) with chemotherapy (idarubicin 12mg/m 2) plus ATRA and 55 (37.2%) with ATO plus ATRA. Median follow-up among surviving patients was 52 months (range: 0- 297 months) Thirty-five patients (23.6%) died (32 died from disease related complications and 3 non-disease related) and 15 (10%) relapsed during follow-up. OS was significantly worse in the CK-group as compared with non-CK (71.6 months vs not reached (NR); p=0.002). Moreover, CK patients had a significantly lower median EFS compared to non-CK patients (46.8 months vs NR; p<0.001). We also observed worse OS and EFS in the CK group regardless of the treatment received (ATRA plus chemotherapy vs ATRA plus ATO, p=0.012). Multivariate analysis performed including gender, age, total leucocyte count, karyotype at diagnosis and treatment received, showed that older age (>60years) was associated to a worse OS, independently of treatment, total leukocyte count karyotype and gender (p <0.001; HR 9.4). Regarding EFS, multivariate analysis showed that CK was associated with poor EFS, independently of treatment, leukocyte counts at diagnosis, age and gender (p=0.015; HR 4.1). CONCLUSIONS Our cohort results confirm the negative impact on OS and EFS of CK. As therapeutical decisions are based on the initial prognostic risk assessment, the inclusion of CK as a variable in this assessment may provide more precise prognostic information and may indicate a need for better treatment strategies to overcome this adverse effect and improve outcomes in this subgroup of patients. Larger prospective analyses are required to confirm our data.

Diagnosis and treatment of hematological cancers is usually provided in many healthcare facilities including large but also middle size centers.1 Providing cancer care in local institutions might be advantageous for patients and caregivers in terms of financial burden and quality of life. However, it might carry potential risks derived of the limited experience of smaller centers and differences in accessibility to complex therapies including allogeneic hematopoietic cell transplantation (allo-HCT) and chimeric antigen receptor (CAR) T-cells. These risks might be especially relevant in infrequent cancers as adult acute lymphoblastic leukemia (ALL). In most European countries, ALL treatment protocols are based on pediatric-inspired regimens which include a large number of immune-chemotherapeutic agents and several key decision points to allocate patients to distinctive treatment arms based on genetics and treatment response.3,4 Several patient and disease characteristics have been identified as prognostic factors for outcomes including age, white blood cell count (WBC) at diagnosis, central nervous system (CNS) infiltration, clearance of measurable residual disease (MRD),4 and disease genetics.5–7 The outcome of patients with ALL may also depend on external factors including center experience, access to cellular therapies and economic variables.8–10 The impact of these center-related and macroeconomic variables on the outcome of patients has been scarcely studied. Thus, the aim of this study was to analyze the potential impact of center-related and macroeconomic variables on the outcome of newly diagnosed adult ALL patients included in 4 consecutive trials of the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) Group. Patients with Philadelphia chromosome (Ph) positive or negative ALL enrolled in one of the 4 consecutive protocols of the PETHEMA group by Spanish institutions adhered to the public health system from 2003 to 2018 were included in this study. The 4 protocols have been closed and reported elsewhere.4,11–13 Centralized analysis of MRD was performed in 5 centers in the ALL-AR-03 and in a single institution in the ALL-HR-11 trial. The PH-08 protocol included adults with newly diagnosed Ph-positive ALL up to the age of 60 years. Patients received an induction therapy with daunorubicin, vincristine, and steroids in combination with imatinib 600 mg/d followed by a 12-week consolidation chemotherapy based on alternated cycles of high-dose methotrexate and cytarabine in combination with imatinib. Allo-HCT was offered to all fit patients with a suitable donor. Treatment protocols used in the ALL-RE-2008, ALL-AR-03, and ALL-HR-11 trials were pediatric-inspired.4,11–13 The ALL-RE-2008 trial included intermediate risk patients based on age (<30 years), WBC count (<25,000 cells/μL), and cytogenetics. The ALL-AR-03 and ALL-HR-11 protocols included high-risk patients up to the age of 60 years diagnosed from 2003 to 2011 and 2011 to 2019, respectively. In both trials, bone marrow MRD assessment by flow cytometry was performed at the end of induction (week 5) and at the end of the third consolidation cycle (weeks 16–18). Only patients with slow clearance of MRD in both trials were allocated to allo-HCT, while patients with good MRD clearance continued with chemotherapy for up to 2 years. Clinical variables analyzed in this study included age, gender, ECOG performance status, WBC, CNS infiltration, precursor lineage (B or T) presence or absence of Ph and treatment period (2003–2010 versus 2011–2018). The Allo-HCT center was defined as centers having authorization by the Spanish government to perform allo-HCT in the same institution where the patient was treated for the ALL. The Allo-HCT center in the same province was defined as having a designated allo-HCT center in the same province where the patient was treated. Reported ALL referred to the number of ALL patients reported to the PETHEMA database by a particular center and served as a surrogate marker of center experience in treating ALL. Nine centers reporting at least 30 ALL patients each and around half of the patients together in this data set were considered as “experienced centers.” Protocol deviation center referred to centers with identified protocol deviations in key treatment decisions (allo-HCT versus chemotherapy allocation or autologous HCT instead of allo-HCT when not indicated in the protocol) in at least 5% of the patients. Other demographic and macroeconomic variables are self-explanatory and listed in Table 1. Demographic and economic variables were obtained from the Spanish Government (See footnote in Table 1). Table 1 - Patients, Disease, Center, and Macroeconomic Characteristics Characteristic N = 816 Age, y, median (range) 35.53 (15; 60) Gender, n (%) Male 475 (58) Female 341 (42) ECOG PS, n (%) 0–1 648/772 (84) 2–3 124/772 (16) WBC, ×109/L, median (range) 16 (0; 842) CNS infiltration, n (%) No 707/769 (92) Yes 62/769 (8) Ph+ ALL, n (%) No 688 (84) Yes 128 (16) Precursor phenotype, n (%) B 603/803 (75) T 200/803 (25) Treatment period, n (%) 2003–2010 341 (42) 2011–2018 475 (58) Protocol, n (%) ALL-AR-03 323 (40) ALL-RE-08 86 (10) PH-08 128 (16) ALL-HR-11 279 (34) Allo-HCT center, n (%) No 292 (36) Yes 524 (64) Allo-HCT center in the same province, n (%) No 168/292 (57) Yes 124/292 (43) ALL cases reported to PETHEMA, median (range) 30 (1; 74) Protocol deviation in ≥5% of reported patients from this center, n (%) No 445 (55) Yes 371 (45) Beds in the hospitala, median (range) 832 (248; 1525) City populationa, median (range) 409,661 [29,288; 3,223,334] AC populationa, median (range) 6,578,079 [580,229; 8,384,408] Relative Health investment GDPa (%), median (range) 6.4 [3.9; 9.5] Health investment/inhabitant €a, median (range) 1,312 [1,090; 1,631] GDP capita/AC, median (range)a 22,700 [17,554; 33,824] aSource: Ministry of Health and Social Services (https://www.sanidad.gob.es/en/estadEstudios/estadisticas/sisInfSanSNS/tablasEstadisticas/InfAnSNS.htm) and Spanish National Statistics Institute (https://www.ine.es/dyngs/INEbase/en/categoria.htm?c=Estadistica_P&cid=1254734710984).AC = autonomous community; Allo-HCT = allogeneic hematopoietic cell transplantation; CNS = central neurological system; ECOG PS = Eastern Cooperative Oncology Group Performance Status; GDP = growth domestic product; Ph = Philadelphia chromosome; WBC = white blood cell count. Clinical endpoints included overall survival (OS), disease-free survival (DFS), cumulative incidence of relapse (CIR), and nonrelapse mortality (NRM) were defined as previously described.11 Infection-related mortality (IRM) was considered an exploratory endpoint and was defined as patients after the first CR dying of infectious causes during ALL treatment or after allo-HCT without previous ALL relapse. All numerical variables were summarized and categorized by medians. Spearman’s rank coefficient or median test was used to analyze correlations between factors. OS and DFS curves were plotted by the Kaplan–Meier method and compared by the log-rank test. CIR, NRM, and IRM were estimated using cumulative incidence functions by competing risks analysis, and the Gray test was used for comparisons.14 The effects of center-related and macroeconomic indicators on OS and DFS were analyzed using Cox proportional hazards regression models, and the Fine and Gray model was used for analyzing these effects on CIR and NRM.15 For each main effect, a separate multivariable model was built, adjusted for other potential risk factors (listed in table 1). All statistical analyses were performed using SPSS (v.24) and R (v.4.1.0). Two-sided values of P < 0.05 were considered statistically significant. Eight hundred sixteen patients were included in the study. Main characteristics at diagnosis appear in Table 1. Median follow-up for patients alive was 3 years (range 0.1–9.5). Two-hundred twenty-four (38%) of the evaluable patients received allo-HCT in the first CR. In line with protocol recommendations, allo-HCT in CR1 ranged from 94% of the patients in the PH-08 trial to 6% patients in the ALL-RE-08 trial. The remaining patients were allocated to further chemotherapy consolidation and maintenance regimens according to each protocol. Fifty-five centers in 40 different cities located in 15 Spanish Autonomous Communities included at least 1 patient in one of the 4 protocols. Twenty-three (42%) centers were accredited for allo-HCT and 47% of nontransplant institution had an allo-HCT center in the same province. Median number of ALL reported by center to PETHEMA database during the study period was 10 (interquatile range 4–20). Approximately half of the patients (n=424, 52%) were reported by ten centers which were considered experienced centers for this study. Patient characteristics were similar between experienced and less-experienced centers, except for treatment period (Suppl.Table S1). Twenty centers (36%) had at least a major deviation in more than 5% of their reported patients. Details on center-related and macroeconomic variables are summarized in Table 1. As expected, there were some associations among center-related and macroeconomic variables. Hence, being a transplant center was correlated with the number of ALL cases reported (P = 0.002), the number of beds in the hospital (P < 0.001), and with major protocol deviations (P = 0.039). Health investment relative to GDP was inversely correlated with the number of inhabitants in the Autonomous Community (P = 0.032) and directly associated with its GDP per capita (P = 0.001). Other explored associations are listed in Suppl. Table S2. Probability of OS at 5 years for the whole cohort was 46% (95% confidence interval [CI] 42-50). Variables associated with lower OS in the univariable analysis were older age, higher ECOG PS score, higher WBC at diagnosis, Ph-negative ALL and diagnosis in the earlier period (Suppl. Figure S1). Probability of DFS at 5 years for the whole cohort was 43% (95% CI: 38-47). In the univariable analysis, ECOG PS > 1, higher WBC at diagnosis, Ph-negative ALL, and earlier diagnosis were associated with worse DFS (Suppl. Table S3). In the multivariable model adjusted for macroeconomic variables, none of the center-, region- and economic-related variables was associated with lower OS or DFS (Table 2; Suppl. Figure S2). Table 2 - Multivariable Adjusted Model for Center-related and Macroeconomic Variables for Patients Outcomes Factor N OS, HR (IC 95%) P N DFS, HR (IC 95%) P N CIR, HR (IC 95%) P N NRM, HR (IC 95%) P Allo-HCT center No 285 1 0.803 257 1 0.475 274 1 0.450 259 1 0.610 Yes 488 1.029 (0.822;1.288) 442 1.087 (0.864; 1.368) 462 1.111 (0.847; 1.456) 449 1.114 (0.736; 1.688) ALL reported cases ≤30 407 1 0.543 370 1 0.623 394 1 0.320 374 1 0.930 >30 366 1.069 (0.862; 1.326) 329 1.056 (0.850; 1.313) 342 1.137 (0.882; 1.465) 334 0.983 (0.664; 1.457) Number of beds in the hospital >824 384 1 0.707 346 1 0.504 359 1 0.670 353 1 0.640 ≤824 389 1.042 (0.839; 1.295) 353 1.078 (0.865; 1.342) 377 1.057 (0.818; 1.365) 355 0.909 (0.612; 1.352) Region population ≤6,578,079 442 1 0.302 406 1 0.450 429 1 0.210 410 1 0.930 >6,578,079 331 1.122 (0.901; 1.398) 293 1.090 (0.872; 1.363) 307 1.177 (0.913; 1.517) 298 1.019 (0.681; 1.524) City population ≤409,661 396 1 0.446 354 1 0.176 368 1 0.010 360 1 0.240 >409,661 377 1.087 (0.878: 1.345) 345 1.162 (0.935; 1.444) 368 1.399 (1.084; 1.804) 348 0.792 (0.537; 1.169) Region GDP per capita (€) >22,700 380 1 0.690 341 1 0.660 355 1 0.310 344 1 0.930 ≤22,700 393 1.045 (0.842; 1.297) 358 1.050 (0.844; 1.306) 381 1.143 (0.884; 1.477) 364 0.982 (0.660; 1.462) Health investment (%) ≥6.4% 419 1 0.919 380 1 0.859 403 1 0.660 386 1 0.850 <6.4% 354 1.011 (0.814; 1.256) 319 1.020 (0.819; 1.270) 333 1.060 (0.820; 1.371) 322 1.038 (0.670; 1.547) Health investment (per capita) ≥1312 393 1 0.766 350 1 0.963 362 1 0.460 356 1 0.440 <1312 380 0.968 (0.779; 1.202) 349 0.995 (0.799; 1.238) 374 1.100 (0.582; 1.418) 352 0.855 (0.576; 1.270) AC = autonomous community; Allo-HCT = allogeneic hematopoietic cell transplantation; CNS = central neurological system; CIR = cumulative incidence of relapse; DFS = disease-free survival; ECOG PS = Eastern Cooperative Oncology Group Performance Status; GDP = growth domestic product; HR = hazard ratio; NRM = nonrelapse mortality; OS = overall survival; Ph = Philadelphia chromosome; WBC = white blood cell count. Of the 816 patients, 738 (90%) achieved CR after one or two induction cycles. Cumulative incidence of relapse among them was 41% (95% CI: 36-45). In the univariable analysis, factors associated with an increased risk of relapse were older age, higher WBC at diagnosis, Ph-negative ALL, and T-cell phenotype. The only center-, region- and economic-related variable associated with a higher CIR in the multivariable model was a higher number of inhabitants in the city where the treatment center was located hazard ratio (HR) 1.399 (95% CI: 1.084-1.804, P = 0.01) (Table 2). One hundred ten patients died of non-relapsing causes, 49 of them (45%) after allo-HCT. Cumulative incidence of NRM at 5 years was 17% (95% CI: 14-20). In the univariable analysis, older age, higher EOCG PS score at diagnosis, Ph-negative ALL, and diagnosis in the earlier period were associated with higher NRM. In the multivariable adjusted model, none of the center-, region- and economic-related variables were associated with higher NRM (Table 2). Finally, of the 738 patients achieving CR, 110 died without previous ALL relapse. Of them, causes of death could be identified in 104 (94.5%). Seventy-three patients (70%) died of infectious causes (44 during ALL treatment and 29 after allo-HCT) and were considered to have IRM. Factors associated with higher IRM in the univariable analysis were older age (HR = 1.031 [95% CI: 1.012-1.050], P = 0.001) and earlier treatment period (HR = 2.061 [95% CI: 1.289-3.297], P = 0.003). Again, none of the center-related and macroeconomic variables were associated with higher IRM (not shown). The objective of this study was to analyze the potential impact of center-related and macroeconomic variables on the outcome of adult patients with ALL treated within 4 consecutive PETHEMA protocols. These variables were considered along with patient and disease factors to adjust the impact of each of them. Our results indicate that detailed treatment protocols with standardized disease evaluations in reference center laboratories allowed adult patients with ALL to have similar outcomes irrespective of demographic, social and economic characteristics of the centers, cities, and regions where they were treated. These results support the work done by PETHEMA and other National Cooperative Groups in Europe as they assure quality of care and equity to all citizens. To the best of our knowledge, this is the first study focused on a wide variety of center-related and macroeconomic variables in adult ALL. Contrary to ours, other studies have either focused in one or very few center-related variables (eg, distance to ALL treating center) or have not included well known clinical and genetic factors in their analyses.16 Impact of insurance status has also been identified as a prognostic factor for adults with ALL,17 although this would not apply to most European public health systems where the great majority of ALL patients are treated in fully accessible public hospitals. Regarding efficacy, we did not observe differences across centers in terms of center-related and macroeconomic variables. The only variables associated with DFS and OS were those dependent on patient and disease characteristics including age, ECOG performance status and WBC at diagnosis, all of them being previously identified as prognostic factors for ALL patients.7,11,18 Noteworthy, none of the variables associated with the experience of the center or size of the city or region where the patient was treated associated with DFS or OS. Patients treated in larger cities had a higher CIR which did not translated into lower OS or DFS. Reasons for this finding are uncertain and might reflect early referrals of more complex cases at diagnosis to larger centers located in highly populated cities. Center experience has been identified as a prognostic factor in other high-risk procedures performed in hematological patients such as allo-HCT.19 However, we did not find any impact of center experience on the outcome of patients. Transplantation is probably a more complex and less well standardized procedure than first-line therapy for ALL, in which physician’s expertise might be more relevant on patient outcomes. Conversely, PETHEMA ALL protocols include very detailed information in terms of dosing and modification of chemotherapeutic agents and transplant indications which might have contributed to mitigate the potential impact of center experience and assure the equity and access to complex therapies to all the population. Limitations of our study include a local diagnosis of ALL in most patients, a relatively limited sample size and the fact that our results are based on data reported to our cooperative group database. Prospective reporting of data to a cooperative group might indicate certain commitment on scientific studies of these centers. While using data prospectively reported to a cooperative group might harbor a selection bias toward well structured and organized centers, the use of national cancer statistics including all patients diagnosed with ALL in our country would not have allowed us to include homogeneous population of patients treated under the same protocols and with centralized MRD determination, which constitutes a strength of our study. In summary, center-related and macroeconomic variables do not seem to have an impact on outcomes in newly diagnosed adult patients with ALL in the setting of a public health system with clearly defined and structured treatment protocols. Based on our results, these protocols can be safely administered to ALL patients in all hospitals adhered to a national cooperative ALL group. These data should be considered for nation-wide planning of healthcare infrastructures for cancer patients. AUTHOR CONTRIBUTIONS Conception and Design: PB, JMR. Collection of data: All authors. Data analysis and interpretation: PB, MM, JMR. Manuscript writing: All authors. Final approval of manuscript: All authors. DISCLOSURES PB declares having received honoraria from Allogene, Amgen, BMS, Gilead, Incyte, Jazz Pharmaceuticals, Miltenyi Biomedicine, Novartis and Roche. J.M.R. has received honoraria and grants from Amgen, Pfizer, Incyte, Servier, Takeda and Novartis. All the other authors have no conflicts of interest to disclose. SOURCES OF FUNDING PB received research funding from the Carlos III Health Institute (PI16/01433, PI21/0199), Asociación Española contra el Cáncer (Ideas Semilla 2019) and a PERIS 2018-2020 grant from the Generalitat de Catalunya (BDNS357800). J.M.R. has received grants from Asociacion Española contra el Cáncer (GC16173697BIGA) and HARMONY Alliance (HORIZON 2020, European Union).

Midostaurin added to intensive chemotherapy is the standard of care for acute myeloid leukemia (AML) with FLT3 mutations ( FLT3 mut). We analyzed the impact of midostaurin in 227 FLT3 mut-AML patients included in the AML-12 prospective trial for fit patients ≤70 years (#NCT04687098). Patients were divided into an early (2012–2015) and late (2016–2020) cohorts. They were uniformly treated except for the addition of midostaurin in 71% of late group patients. No differences were observed in response rates or the number of allotransplants between groups. Outcome was improved in the late period: 2-year relapse incidence decreased from 42% vs 29% in early vs late group ( p = 0.024) and 2-year overall survival (OS) improved from 47% vs 61% ( p = 0.042), respectively. The effect of midostaurin was evident in NPM1 mut patients ( n = 151), with 2-yr OS of 72% (exposed) vs 50% (naive) patients ( p = 0.011) and mitigated FLT3 -ITD allelic ratio prognostic value: 2-yr OS with midostaurin was 85% and 58% in low and high ratio patients ( p = 0.049) vs 67% and 39% in naive patients ( p = 0.005). In the wild-type NPM1 subset ( n = 75), we did not observe significant differences between both study periods. In conclusion, this study highlights the improved outcome of FLT3 mut AML fit patients with the incorporation of midostaurin.

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases without a care standard and show variability in treatment outcomes. This Spanish, observational, prospective study ERASME (CEL-SMD-2012-01) assessed the evolution of newly diagnosed and treatment-naive high-risk MDS patients (according to IPPS-R). 204 patients were included: median age 73.0 years, 54.4% males, 69.6% 0-1 ECOG, and 94.6% with comorbidities. Active treatment was the most common strategy (52.0%) vs. stem cell transplantation (25.5%) and supportive care/watchful-waiting (22.5%). Overall (median) event-free survival was 7.9 months (9.1, 8.3, and 5.3); progression-free survival: 10.1 months (12.9, 12.8, and 4.3); and overall survival: 13.8 months (15.4, 14.9; 8.4), respectively, with significant differences among groups. Adverse events (AEs) of >= 3 grade were reported in 72.6% of patients; serious AEs reported in 60.6%. 33.1% of patients died due to AEs. Three patients developed second primary malignant neoplasms (median: 8.2 months). Our study showed better outcomes in patients receiving active therapy early after diagnosis.

Background and aims: The addition of Venetoclax (VEN) to the treatment of myeloid neoplasms, particularly newly diagnosed and relapse/refractory (R/R) Acute Myeloid Leukemia (AML), has shown improved responses compared to hypomethylating agents (HMA) in unfit patients. Nevertheless, side effects derived from deep cytopenias (infections) could potentially be a caved of such strategy. The use of antibacterial prophylaxis in this setting is controversial and its employment heterogeneous across centers, making difficult to draw conclusions from real-world studies. In this study we aimed to ascertain whether the use of antibacterial prophylaxis has an impact on the incidence of bacterial infections, hospitalization rates, infection severity, and mortality due to the infection. Finally, we also aimed to identify potential risk factors for bacterial infections. Methods: Retrospective data collection of patients with de novo, R/R or secondary AML, High Risk Myelodisplastic Syndrome and accelerated phases of Myeloproliferative Neoplasms treated with HMA or low dose cytarabine + VEN between January 2018 and May 2023 in 10 Spanish centers. Baseline characteristics and infectious episodes [presented as Microbiologically Documented Bacterial Infections (MDBI) or Clinically Documented infections without Isolation (CDI)], or febrile neutropenia without isolation or focus (FN) were recorded. Confirmed or suspected infections of parasites/fungi/viruses were excluded in this study. For each event, antibacterial prophylaxis, days of hospitalization, severity, disease status and dead due to infectious events/FN were collected. Results: In total, 165 patients have been included (clinical characteristics summarized in Table 1). VEN was initially used as 28 days/cycle and it had to be adjusted in 78 (48.75%) patients, mainly due to cytopenias and infections. The univariate analysis showed that infectious events and FN occurred more frequently in patients with high ELN risk than in those with Intermediate/favorable (OR; 2.71, 95% CI; 1.03-7.11, p = 0.041). In our cohort, 175 infections (MDBI/CDI) or FN occurred in 113 patients (68.5%) with an incidence of 26.1/100 cycles. These episodes correspond to 70 (40%) cases of MBI, 59 cases of CDI (33.7%) and 46 (26.3%) cases FN, with a median of 1 episode per patient (range 1-5). Most events occurred in the first (85; 48.9%) and second cycle (43; 24.7%) with a median of cycles at the moment of the event of 2 (p25-p75: 1-3) and a median time from VEN start to episode of 44 days (p25-p75: 18-105) because infections/FN usually led to delays on subsequent cycles. Considering the total number of events in patients with at least first disease evaluation available at the time of infection, only 48 (31%) had cytologic response (<5% blasts). Table 1 shows type of episode/isolation/focus. Antibacterial prophylaxis was used in 48 patients (29.5%), primarily with quinolones (levofloxacin 74%, ciprofloxacin 23%) according to physician decision. Incidence of infections (MDBI/CDI) or FN was lower in patients with antibacterial prophylaxis (OR; 0.37, 95% CI; 0.18-0.74, p = 0.05). Additionally, the median number of infections was lower in prophylaxis group (0.77 vs 1.18; p= 0.004). However, no differences were observed between both groups regarding hospitalization incidence or infection severity (defined as need of vasoactive drugs, high-flow oxygen therapy or deaths caused by the infectious episode). Microbiological isolations were significantly higher in non-prophylaxis group with infections (45.6% vs 23.7%, p 0.015). Incidence of multidrug-resistant organisms was 10.2% among all patients, with no differences between groups neither in Clostridioides difficile incidence (3.6%). In the competitive risk survival analysis, cumulative incidence of death from MBI/CDI or FN cause showed no differences between patients receiving or not antibacterial prophylaxis ( p= 0.98) (Figure 1). Conclusions: Our data suggest that avoiding antibacterial prophylaxis in non-intensive VEN-based treatments may result in a higher incidence of FN and infectious events, but it does not lead to impaired outcomes in terms of severity and/or mortality related to infections. Having a greater availability of microbiological isolations could enable more targeted antibiotic therapy, avoiding drug resistance. Additional data from the prophylaxis cohort will be included in the presentation.

Introduction The association of polymorphisms in molecules involved in the immune response (checkpoint inhibitors) with the clinical outcome after allogeneic transplantation (alloHSCT) has been described. Lymphocyte Activation 3 (LAG3) is a surface protein that plays a regulatory role in immunity as an inhibitory immune checkpoint molecule. Methods To determine its role in the alloHSCT setting, we analyzed 797 patients transplanted from HLA-identical sibling donors. The LAG3 rs870849 C>T polymorphism was genotyped in donors. Results We detected a higher incidence of severe acute GVHD in patients transplanted from donors with TT genotype (p: 0.047, HR 1.64; 95% CI 1.01 – 2.67). Overall survival (OS) was worse for patients transplanted from donors with the rs870849 CT/TT genotype (0.020; HR, 1.44; 95% CI 1.06 – 1.96), as well as disease-free survival (DFS) (p: 0.002; HR 1.58, 95%CI: 1.18 – 2.14) and transplant-related mortality (TRM) (p< 0.001; HR: 1.88, 95% CI 1.29 – 2.74). When combining the LAG3 rs870849 and the PDCD1 rs36084323 genotypes of the donor, three genetic groups were well defined, allowing a good stratification of the risk of acute GVHD, TRM, OS and DFS. Discussion We conclude that the LAG3 genotype of the donor may be considered in donors’ selection. As this selection may be limited in the HLA-identical sibling donor scenario, further studies exploring the impact of LAG3 genotype of the donor in unrelated transplantation are warranted.

Background Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by the activation of several kinase pathways that promote treatment resistance leading to poor survival of patients at all ages. Despite the use of tyrosine kinase inhibitors in the Ph-like ABL-class subtype (15% Ph-like ALL), there is not a consensus approach for treatment of most patients (JAK-class especially), and the role of allogeneic stem cell transplantation (alloSCT) has scarcely been explored so far (i.e., mandatory vs. MRD-oriented). Objective To assess the outcome of Ph-like patients enrolled in the ongoing ALL19 trial from the Programa Español de Tratamientos en Hematología (PETHEMA) for adults (18-60y) with Ph-negative ALL, and to evaluate the effectiveness of alloSCT in first complete remission (CR). Methods In the ALL19 trial, patients are allocated to alloSCT according both to the end of induction (EOI) measurable residual disease (MRD) level and to high-risk genetic markers such as low-hypodiploidy, KMT2A rearrangement, TP53 biallelic alteration and concomitant deletion of IKZF1 and CDKN2A/B. Therefore, BCR::ABL1-like subtype was not a criteria for early transplantation unless meeting either poor MRD clearance at EOI or concomitant deletion of IKZF1+CDKN2A/B. Patients with MRD≥0.01% on day+35 (EOI) and/or high-risk genetics were assigned to early alloSCT (preceded by 1 cycle of consolidation chemotherapy) while those with good MRD clearance and standard risk genetics received early and delayed consolidation (3 cycles each) and maintenance therapy. Bone marrow or peripheral blood samples were analyzed by G-banding+FISH, SNP array (750K Affymetrix, Thermo Fisher) and next generation sequencing (NGS, with a custom DNA gene panel, Illumina) at diagnosis in 4 reference laboratories. MRD was centrally assessed by next generation flow cytometry with a sensitivity up to 2x10 -6. Results Twenty-four Ph-like out of 248 adult BCP ALL (10%) were detected. Baseline characteristics were similar between Ph-like and the remaining BCP-ALL. Most Ph-like ALL patients showed the JAK-class profile (20/24 [83%]: 17/24 CRLF2 rearrangement[r], 1/24 JAK2r, 1 BLNK::DNTT and 1 JAK2 R683 mutation), 3/24 belonged to the ABL-class subtype (2 ABL2r, 1 NUP214::ABL1) and 1 patient was identified by RNAseq (gene expression profiling similar to BCR::ABL1 ALL) without identifying any rearrangement. Regarding secondary genetic alterations, 17/24 (71%) patients showed the IKZF1 plus profile, 10/23 (43%) harbored JAK2 R683 mutation, 4/23 N/KRAS mutations, 2/23 CRLF2 mutation and 1/23 showed mutations in several genes such as IKZF1, NR3C1 or FLT3, among others. There were no significant differences in the probability of achieving CR between Ph-like and the remaining BCP ALL (15/20 [75%] vs. 137/162 [85%], p=0.334). However, EOI MRD level of Ph-like individuals was significantly poorer (MRD<0.01% Ph-like 4/15 [27%] vs. other BCP-ALL 85/136 [63%], p=0.007). By intention to treat, most Ph-like patients (17/19, 89%) were allocated to alloSCT (vs. 91/153 [59%] in the other BCP-ALL, p=0.011) due to either EOI MRD≥0.01% (9/18), IKZF1+CDKN2A/B concomitant deletions (3/18) or both (6/18). The 2-y overall survival (OS) in both groups was not significantly different (57% [95% CI, 27%-78%] vs. 67% [55%-77%, p=0.304]) (Figure 1). Interestingly, most Ph-like patients died due to treatment-related toxicity (2 transplant-related mortality, 1 CART-related, 1 during induction, 1 in CR and 1 by COVID-19 infection). The 2-y cumulative incidence of relapse (CIR) was also similar (Ph-like 27% [8%-50%] vs. other BCP-ALL 35% [25%-45%], p=0.946) (Figure 1). Conclusions Despite the short follow up of this series, our results show that Ph-like ALL patients have poorer EOI MRD clearance and higher rate of alloSCT realization than the remaining BCP ALL patients, without significant differences in outcome. This suggests that early alloSCT in first CR might overcome the poor prognosis of Ph-like ALL patients. Funding Funded in part by: PI10/01417 y FIS PI19/01183 Ministerio de Salud Carlos III, JCyL_SA118P20, FMM21/002_AP176752021, “La Caixa” Foundation, Pfizer Spain, Fundación PETHEMA. This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No IMI001-07. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA.

Introduction RAS mutations are subclonal events associated with both good and bad prognosis AML categories (Papaemmanuil E et al.N Engl J Med 2016; 374). An adverse prognostic meaning of RAS mutations in treated AML with myelodysplastic-related changes has also been reported.In solid tumors, it has been shown that patients with KRASQ61K do not confer resistance to therapy, unlike other mutations: KRASQ61K introduces a cryptic splice donor site and causes a short inactive isoform (Kobayashi Y et al. Nature 2022 ;603). However, most KRASQ61K patients acquired the silent mutation G60G, which removes the new splice donor site and increases therapy resistance. This splicing mechanism was not present in cases with NRASQ61K and HRASQ61K. We investigated the frequency of RAS mutations, their molecular associations, and the prevalence of splicing mechanisms involving KRASQ61K and NRASQ61K in AML. Methods We included a consecutive series of adult de novo AML enrolled in the CETLAM trials, a total of 620 patients who were diagnosed between 2017 to 2022. DNA and RNA were extracted from bone marrow or peripheral blood. Next Generation Sequencing (NGS) was carried out with three panels containing at least 42 AML-related genes, including KRAS and NRAS. Only variants with a VAF (variant allele frequency) >4% and ranked as pathogenic or likely pathogenic were used for the analysis. Conventional karyotype was routinely assessed. cDNA was obtained from 7 patients with NRASQ61K, and was amplified through a conventional PCR. cDNA products were observed in gel electrophoresis and sequenced by the Sanger method. IBM SPSS Statistics was used to perform Chi-Square tests. In statistical tests regarding molecular associations, patients with co-mutated NRAS and KRAS are excluded. Results Our study found RAS mutations in 16.9% (105 patients) of adult AML cases. 11.9% of AML patients had mutated NRAS, while 6.1% mutated KRAS (74 and 38 patients, respectively). In our study, Q61 was the third most common mutation in NRAS with an incidence of 26%, while it was the fourth most common in KRAS with a frequency of 12%. The distribution of mutations in KRAS and NRAS is shown in figure 1. Regarding the Q61 codon, the distribution of each amino acid change in our cohort is shown in Figure 1b. Nine NRASQ61K were found, and cDNA could be obtained from 7 patients. NRASQ61K and non-mutated NRAS samples presented the standard large transcript in the gel electrophoresis. These findings ruled out the presence of exon skipping in AML with NRASQ61K mutations. No single case of KRASQ61K was observed in this extensive series of AML, so we could not identify alternative transcripts described in solid tumors nor compensatory KRASG60G mutations.1.1% of patients (7 cases) presented co-mutated NRAS and KRAS. In 9 subjects, more than one NRAS mutation was found, whereas, in 3 patients we detected two KRAS mutations. Lastly, we focused on molecular associations of RAS mutated patients (Figure 2). Of the total RAS-mutated cohort (105 patients), DNMT3A and NPM1 had the highest mutation frequency. Notably, 40.3% of NRAS-mutated patients had NPM1 mutations, significantly higher (p=0.018) than KRAS (16.1%). Another interesting finding was that KRASK117N mutations were mutually exclusive with DNMT3A and NPM1. All KRASK117N (6 cases) were associated with TET2 and or ASXL1 mutations. Cytogenetics information was available for 87 patients with RAS mutations (59 NRAS and 35 KRAS). NRAS-mutated patients showed a higher frequency of core binding factor (CBF) alterations than KRAS (18.1% vs. 3.6%, respectively) (p=0.063). Complex and miscellaneous karyotypes had higher rates of KRAS mutations than NRAS mutations (57.1% vs. 32.7%, respectively, p=0.032).In 26 cases, RAS mutations were the single mutation or the one with the highest VAF; notably, 42.3% had an NRASQ61 mutation. We had cytogenetic information of 20 patients, and twelve had inv(16) (60%). Conclusions In this cohort of adult AML cases, RAS mutations were present in 16.9%. Mutations at codon G12 were the most prevalent lesions in AML. NRAS mutations were more frequent than those of KRAS. Up to 17% of RAS mutated AML cases showed more than one mutation. CBF chromosomal abnormalities were commonly associated with NRAS mutations. Complex and miscellaneous karyotypes showed KRAS mutations more often. KRASK117N mutations were consistently associated with TET2 and ASXL1 mutations and mutually exclusive with NPM1 and DNMT3A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The 2017 European LeukemiaNet (ELN 2017) guidelines for the diagnosis and management of acute myeloid leukemia (AML) have become fundamental guidelines to assess the prognosis and postremission therapy of patients. However, they have been retrospectively validated in few studies with patients included in different treatment protocols. We analyzed 861 patients included in the Cooperativo Para el Estudio y Tratamiento de las Leucemias Agudas y Mielodisplasias-12 risk-adapted protocol, which indicates cytarabine-based consolidation for patients allocated to the ELN 2017 favorable-risk group, whereas it recommends allogeneic stem cell transplantation (alloSCT) as a postremission strategy for the ELN 2017 intermediate- and adverse-risk groups. We retrospectively classified patients according to the ELN 2017, with 327 (48%), 109 (16%), and 245 (36%) patients allocated to the favorable-, intermediate-, and adverse-risk group, respectively. The 2- and 5-year overall survival (OS) rates were 77% and 70% for favorable-risk patients, 52% and 46% for intermediate-risk patients, and 33% and 23% for adverse-risk patients, respectively. Furthermore, we identified a subgroup of patients within the adverse group (inv(3)/t(3;3), complex karyotype, and/or TP53 mutation/17p abnormality) with a particularly poor outcome, with a 2-year OS of 15%. Our study validates the ELN 2017 risk stratification in a large cohort of patients treated with an ELN-2017 risk-adapted protocol based on alloSCT after remission for nonfavorable ELN subgroups and identifies a genetic subset with a very poor outcome that warrants investigation of novel strategies.

AbstractThe negative prognostic impact of internal tandem duplication of FLT3 (FLT3-ITD) in patients with acute myeloid leukemia with mutated NPM1 (AML-NPM1) is restricted to those with a higher FLT3-ITD allelic ratio (FLT3high; ≥0.5) and considered negligible in those with a wild-type (FLT3WT)/low ITD ratio (FLT3low). Because the comutation of DNMT3A (DNMT3Amut) has been suggested to negatively influence prognosis in AML-NPM1, we analyzed the impact of DNMT3Amut in FLT3-ITD subsets (absent, low, and high ratios). A total of 164 patients diagnosed with AML-NPM1 included in 2 consecutive CETLAM protocols and with DNMT3A and FLT3 status available were studied. Overall, DNMT3Amut status did not have a prognostic impact, with comparable overall survival (P = .2). Prognostic stratification established by FLT3-ITD (FLT3WT = FLT3low > FLT3high) was independent of DNMT3Amut status. Measurable residual disease (MRD) based on NPM1 quantitative polymerase chain reaction was available for 94 patients. DNMT3Amut was associated with a higher number of mutated NPM1 transcripts after induction (P = .012) and first consolidation (C1; P < .001). All DNMT3Amut patients were MRD+ after C1 (P < .001) and exhibited significant MRD persistence after C2 and C3 (MRD+ vs MRD−; P = .027 and P = .001, respectively). Finally, DNMT3Amut patients exhibited a trend toward greater risk of molecular relapse (P = .054). In conclusion, DNMT3Amut did not modify the overall prognosis exerted by FLT3-ITD in AML-NPM1 despite delayed MRD clearance, possibly because of MRD-driven preemptive intervention.

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

INTRODUCTION: Venetoclax (VEN), a BCL-2 specific inhibitor, used in combination with azacitidine (AZA) in patients with newly-diagnosed acute myeloid leukemia (AML) who were ineligible for intensive chemotherapy treatment, has shown high response rate and overall survival compared with AZA monotherapy (Di Nardo et al. N Engl J Med 2020; 383:617-629). For patients failing frontline, VEN-based salvage therapy prognosis appears very poor, although a subgroup of patients might benefit. Moreover, recently, had been published the European LeukemiaNet 2022 recommendations and classification in AML (Döhner et al. Blood 2022). AIMS: We performed a retrospective study of patients with relapsed/refractory (R/R) AML receiving salvage treatment with VEN combinations in the Catalan Institute of Oncology (ICO) in order to determine the efficacy and safety of the combination. We also explored the probability of response according to the ELN 2022 risk classification by genetics. METHODS: We analyze 60 patients with R/R AML at 4 hospitals belonging to ICO in Spain, treated with VEN (400mg/24h or 600mg/24h; with recommended 3-day ramp-up to target dose) in combination with hypomethylating (HMA) agents (AZA 75mg/m2 7/28 days or Decitabine (DEC) 20mg/m2 5/28 days) or low-dose cytarabine (LDAC) (20mg/m2 10/28 days) from May 2019 until December 2021. Event was defined as death, refractoriness to treatment or progressive disease. RESULTS: Characteristics of our cohort are described in table 1. Thirty-seven (62%) patients had next-generation sequencing (NGS) panel study at diagnosis and/or at the time of R/R disease. 31 (52%) patients had high-risk AML according to ELN 2017 classification, and 38 of 46 (83%) patients had high-risk AML according to ELN 2022 classification. Six of 37 patients (15%) had TP53 mutation at a variant allele fraction (VAF) of at least 10%. Seven (19%) patients were reclassified as high-risk AML according to ELN 2022 classification. NGS panel study didn't show high risk mutations in 10 patients. According to ELN 2022 classification, 8, 11 and 8 patients, presented 1, 2 or ≥ 3 high risk gene mutations, respectively. Fourteen (23%) patients received VEN + DEC, 34 (57%) VEN + AZA, and 12 (20%) VEN + LDAC. The median number of cycles received was 3 (range 1-28), with a median of one cycle to achieve the best response (range: 1-4). Early mortality in the first 30 days was 15% (9 patients), 5 due to progression disease, 2 due to infection and 2 due to clinical worsening. Overall response rate after first cycle [Complete response (CR) + complete response without hematological recovery (CRi) and partial response (PR)] was 58%. Three patients were treated in a molecular relapse (MRD positive status), and all of them achieved molecular response. Six of 19 fit young patients (32%) achieved CR and received an allogeneic stem cell transplantation (alloSCT). The median event-free survival (EFS) and overall survival (OS) was 4.03 months and 7.63 months, respectively. No significant differences of EFS and OS were found according to ELN 2022 risk classification, mutational profile or TP53 status. Response to treatment after 3-4 cycles, discriminate two groups of patients with an OS of 17.26 months in those patients who achieved CR or PR vs 2.4 months in non-responders (p=0.000). Patients relapsed after alloSCT (7 patients) presented a poor outcome (median OS was 1.6 months). SUMMARY/CONCLUSIONS: Our study showed that real-world experience of treating patients with R/R AML with VEN + HMA or LDAC is feasible as salvage treatment in patients relapsed to HMA and as bridge therapy to alloSCT with a rapid response rate. No significant differences of response were found according to ELN 2022 risk classification. Rapid responses shown with the combination, allow us to identify those patients who may benefit from this approach. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.