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Abstract Next-Generation Sequencing is needed for the accurate genetic risk stratification of acute myeloid leukemia according to European LeukemiaNet (ELN) guidelines. We validated and compared the 2022 ELN risk classification in a real-life cohort of 546 intensively and 379 non-intensively treated patients. Among fit patients, those aged ≥65 years old showed worse OS than younger regardless risk classification. Compared with the 2017 classification, 14.5% of fit patients changed the risk with the 2022 classification, increasing the high-risk group from 44.3% to 51.8%. 3.7% and 0.9% FLT3-ITD mutated patients were removed from the favorable and adverse 2017 categories respectively to 2022 intermediate risk group. We suggest that midostaurin therapy could be a predictor for 3 years OS (85.2% with vs. 54.8% without midostaurin, P = 0.04). Forty-seven (8.6%) patients from the 2017 intermediate group were assigned to the 2022 adverse-risk group as they harbored myelodysplasia (MDS)-related mutations. Patients with one MDS-related mutation did not reach median OS, while patients with ≥2 mutations had 13.6 months median OS (P = 0.002). Patients with TP53 ± complex karyotype or inv(3) had a dismal prognosis (7.1 months median OS). We validate the prognostic utility of the 2022 ELN classification in a real-life setting providing supportive evidences to improve risk stratification guidelines.

High dose-intensive or infusional intermediate-dose immunochemotherapy is highly effective treatment for Burkitt lymphoma irrespective of human immunodeficiency virus (HIV) infection. However, toxicities of these regimens are relevant, especially in older adults and elderly patients. The prospective multicenter BURKIMAB14 trial included four to six blocks of immunochemotherapy according to stage (localized: 1 and 2 non-bulky; advanced: 2 bulky, 3, 4) and age, with dose reduction in patients >55 years old. Dose-intensity of chemotherapy was reduced in patients ≤55 years old after achieving complete metabolic response (CMR). Their outcomes were compared with those of similar patients included in the former BURKIMAB08 trial, in which there was no dose reduction. CMR was attained in 86 of 107 (80%) patients (17/19 in localized stages and 69/88 in advanced stages). Patients from the BURKIMAB14 trial ≤55 years old showed similar overall survival (OS), fewer infections and cytopenias than patients from the BURKIMAB08 trial. Patients >55 years old had a significantly higher treatment- related mortality despite dose reduction of chemotherapy. With a median follow-up of 3.61 years the 4-year OS probability was 73% (range, 63-81%). Age (≤55 vs. >55 years) and stage (localized vs. advanced) had prognostic significance. No significant differences in OS were observed in HIV-positive versus HIV-negative patients. The results of BURKIMAB14 are similar to those of other dose-intensive immunochemotherapy trials. Age >55 years and advanced stage, but not HIV infection, were associated with poor survival. Dose reduction of chemotherapy in young adults in CMR is safe and does not impact outcomes (clinicaltrials gov. Identifier: NCT05049473).

Abstract In a context of therapeutic revolution in older adults with AML, it is becoming increasingly important to select patients for the various treatment options by taking account of short-term efficacy and toxicity as well as long-term survival. Here, the data from three European registries for 1,199 AML patients aged 70 years or older treated with intensive chemotherapy were used to develop a prognostic scoring system. The median follow-up was 50.8 months. In the training set of 636 patients, age, performance status, secondary AML, leukocytosis, and cytogenetics, as well as NPM1 mutations (without FLT3-ITD), were all significantly associated with overall survival, albeit not to the same degree. These factors were used to develop a score that predicts long-term overall survival. Three risk-groups were identified: a lower, intermediate and higher-risk score with predicted 5-year overall survival (OS) probabilities of ≥12% (n = 283, 51%; median OS = 18 months), 3–12% (n = 226, 41%; median OS = 9 months) and

Treatment options for patients with secondary acute myeloid leukemia (sAML) and AML with myeloid-related changes (AMLMRC) aged 60 to 75 years are scarce and unsuitable. A pivotal trial showed that CPX-351 improved complete remission with/without incomplete recovery (CR/CRi) and overall survival (OS) as compared with standard "3+7" regimens. We retrospectively analyze outcomes of 765 patients with sAML and AML-MRC aged 60 to 75 years treated with intensive chemotherapy, reported to the PETHEMA registry before CPX-351 became available. The CR/CRi rate was 48%, median OS was 7.6 months (95% confidence interval [CI]: 6.7-8.5) and event-free survival (EFS) 2.7 months (95% CI: 2-3.3), without differences between intensive chemotherapy regimens and AML type. Multivariate analyses identified age ≥70 years, Eastern Cooperative Oncology Group performance status ≥1 as independent adverse prognostic factors for CR/CRi and OS, while favorable/intermediate cytogenetic risk and NPM1 were favorable prognostic factors. Patients receiving allogeneic stem cell transplant (HSCT), autologous HSCT, and those who completed more consolidation cycles showed improved OS. This large study suggests that classical intensive chemotherapy could lead to similar CR/CRi rates with slightly shorter median OS than CPX-351.

Acute myeloid leukemia has a poor prognosis in older adults, and its management is often unclear due to its underrepresentation in clinical trials. Both overall survival (OS) and health-related quality-of-life (HRQoL) are key outcomes in this population, and patient-reported outcomes may contribute to patient stratification and treatment assignment. This prospective study included 138 consecutive patients treated in daily practice with the currently available non-targeted therapies (intensive chemotherapy [IC], attenuated chemotherapy [AC], hypomethylating agents [HMA], or palliative care [PC]). We evaluated patients’ condition at diagnosis (Life expectancy [Lee Index for Older Adults], Geriatric Assessment in Hematology [GAH scale], HRQoL [EQ-5D-5L questionnaire], and fatigue [fatigue items of the QLQ-C30 scale]), OS, early death (ED), treatment tolerability (TT) and change in HRQoL over 12 months follow-up. The median OS was 7.1 months (IC not reached, AC 5.9, HMA 8.8, and PC 1.0). Poor risk AML category and receiving just palliative care, as well as a higher Lee index score in the patients receiving active therapy, independently predicted a shorter OS. The Lee Index and GAH scale were not useful for predicting TT. The white blood cell count was a valid predictor for ED. Patients’ HRQoL remained stable during follow-up.

Abstract FLT3-ITD mutations are detected in approximately 25% of newly diagnosed adult acute myeloid leukemia (AML) patients and confer an adverse prognosis. The FLT3-ITD allelic ratio has clear prognostic value. Nevertheless, there are numerous manuscripts with contradictory results regarding the prognostic relevance of the length and insertion site (IS) of the FLT3-ITD fragment. We aimed to assess the prognostic impact of these variables on the complete remission (CR) rates, overall survival (OS) and relapse-free survival (RFS) of AML patients with FLT3-ITDmutations. We studied the FLT3-ITD length of 362 adult AML patients included in the PETHEMA AML registry. We tried to validate the thresholds of ITD length previously published (i.e., 39 bp and 70 bp) in intensively treated AML patients (n = 161). We also analyzed the mutational profile of 118 FLT3-ITD AML patients with an NGS panel of 39 genes and correlated mutational status with the length and IS of ITD. The AUC of the ROC curve of the ITD length for OS prediction was 0.504, and no differences were found when applying any of the thresholds for OS, RFS or CR rate. Only four out of 106 patients had ITD IS in the TKD1 domain. Our results, alongside previous publications, confirm that FLT3-ITD length lacks prognostic value and clinical applicability.

Acute myeloid leukemia (AML) in the elderly remains a clinical challenge, with a five-year overall survival rate below 10%. The current ELN 2017 genetic risk classification considers cytogenetic and mutational characteristics to stratify fit AML patients into different prognostic groups. However, this classification is not validated for elderly patients treated with a non-intensive approach, and its performance may be suboptimal in this context. Indeed, the transcriptomic landscape of AML in the elderly has been less explored and it might help stratify this group of patients. In the current study, we analyzed the transcriptome of 224 AML patients > 65 years-old at diagnosis treated in the Spanish PETHEMA-FLUGAZA clinical trial in order to identify new prognostic biomarkers in this population. We identified a specific transcriptomic signature for high-risk patients with mutated TP53 or complex karyotype, revealing that low expression of B7H3 gene with high expression of BANP gene identifies a subset of high-risk AML patients surviving more than 12 months. This result was further validated in the BEAT AML cohort. This unique signature highlights the potential of transcriptomics to identify prognostic biomarkers in in elderly AML.

Acute myeloid leukemia (AML) is a heterogeneous disease classified into three risk categories (favorable, intermediate and adverse) with significant differences in outcomes. Definitions of risk categories evolve overtime, incorporating advances in molecular knowledge of AML. In this study, we analyzed the impacts of evolving risk classifications in 130 consecutive AML patients in a single-center real-life experience. Complete cytogenetic and molecular data were collected using conventional qPCR and targeted Next Generation Sequencing (NGS). Five-year OS probabilities were consistent among all classification models (roughly 50–72%, 26–32% and 16–20% for favorable, intermediate and adverse risk groups, respectively). In the same way, the medians of survival months and prediction power were similar in all models. In each update, around 20% of patients were re-classified. The adverse category consistently increased over time (31% in MRC, 34% in ELN2010, 50% in ELN2017), reaching up to 56% in the recent ELN2022. Noteworthily, in multivariate models, only age and the presence of TP53 mutations remained statistically significant. With updates in risk-classification models, the percentage of patients assigned to the adverse group is increasing, and so will the indications for allogeneic stem cell transplantation.

Next–Generation Sequencing (NGS) implementation to perform accurate diagnosis in acute myeloid leukemia (AML) represents a major challenge for molecular laboratories in terms of specialization, standardization, costs and logistical support. In this context, the PETHEMA cooperative group has established the first nationwide diagnostic network of seven reference laboratories to provide standardized NGS studies for AML patients. Cross–validation (CV) rounds are regularly performed to ensure the quality of NGS studies and to keep updated clinically relevant genes recommended for NGS study. The molecular characterization of 2856 samples (1631 derived from the NGS–AML project; NCT03311815) with standardized NGS of consensus genes (ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1) showed 97% of patients having at least one mutation. The mutational profile was highly variable according to moment of disease, age and sex, and several co–occurring and exclusion relations were detected. Molecular testing based on NGS allowed accurate diagnosis and reliable prognosis stratification of 954 AML patients according to new genomic classification proposed by Tazi et al. Novel molecular subgroups, such as mutated WT1 and mutations in at least two myelodysplasia–related genes, have been associated with an adverse prognosis in our cohort. In this way, the PETHEMA cooperative group efficiently provides an extensive molecular characterization for AML diagnosis and risk stratification, ensuring technical quality and equity in access to NGS studies.

FLT3–ITD results in a poor prognosis in terms of overall survival (OS) and relapse-free survival (RFS) in acute myeloid leukemia (AML). However, the prognostic usefulness of the allelic ratio (AR) to select post-remission therapy remains controversial. Our study focuses on the prognostic impact of FLT3–ITD and its ratio in a series of 2901 adult patients treated intensively in the pre-FLT3 inhibitor era and reported in the PETHEMA registry. A total of 579 of these patients (20%) harbored FLT3–ITD mutations. In multivariate analyses, patients with an FLT3–ITD allele ratio (AR) of >0.5 showed a lower complete remission (CR rate) and OS (HR 1.47, p = 0.009), while AR > 0.8 was associated with poorer RFS (HR 2.1; p < 0.001). Among NPM1/FLT3–ITD-mutated patients, median OS gradually decreased according to FLT3–ITD status and ratio (34.3 months FLT3–ITD-negative, 25.3 months up to 0.25, 14.5 months up to 0.5, and 10 months ≥ 0.5, p < 0.001). Post-remission allogeneic transplant (allo-HSCT) resulted in better OS and RFS as compared to auto-HSCT in NPM1/FLT3–ITD-mutated AML regardless of pre-established AR cutoff (≤0.5 vs. >0.5). Using the maximally selected log-rank statistics, we established an optimal cutoff of FLT3–ITD AR of 0.44 for OS, and 0.8 for RFS. We analyzed the OS and RFS according to FLT3–ITD status in all patients, and we found that the group of FLT3–ITD-positive patients with AR < 0.44 had similar 5-year OS after allo-HSCT or auto-HSCT (52% and 41%, respectively, p = 0.86), but worse RFS after auto-HSCT (p = 0.01). Among patients with FLT3–ITD AR > 0.44, allo-HSCT was superior to auto-HSCT in terms of OS and RFS. This study provides more evidence for a better characterization of patients with AML harboring FLT3–ITD mutations.

Innate immune cellular effectors are actively consumed during systemic inflammation, but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here, we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF-κB independent, Traf6/IκB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.

This retrospective study investigated outcomes of 404 patients with relapsed/refractory (R/R) FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) enrolled in the PETHEMA registry, pre-approval of tyrosine kinase inhibitors. Most patients (63%) had received first-line intensive therapy with 3 + 7. Subsequently, patients received salvage with intensive therapy (n = 261), non-intensive therapy (n = 63) or supportive care only (n = 80). Active salvage therapy (i.e., intensive or non-intensive therapy) resulted in a complete remission (CR) or CR without hematological recovery (CRi) rate of 42%. More patients achieved a CR/CRi with intensive (48%) compared with non-intensive (19%) salvage therapy (p < 0.001). In the overall population, median overall survival (OS) was 5.5 months; 1- and 5-year OS rates were 25% and 7%. OS was significantly (p < 0.001) prolonged with intensive or non-intensive salvage therapy compared with supportive therapy, and in those achieving CR/CRi versus no responders. Of 280 evaluable patients, 61 (22%) had an allogeneic stem-cell transplant after they had achieved CR/CRi. In conclusion, in this large cohort study, salvage treatment approaches for patients with FLT3-ITD mutated R/R AML were heterogeneous. Median OS was poor with both non-intensive and intensive salvage therapy, with best long-term outcomes obtained in patients who achieved CR/CRi and subsequently underwent allogeneic stem-cell transplant.

The hypomethylating agents, decitabine (DEC) and azacitidine (AZA), allowed more elderly acute myeloid leukemia (AML) patients to be treated. However, there are little direct comparative data on AZA and DEC. This multicenter retrospective study compared the outcomes of AZA and DEC in terms of response and overall survival (OS). Potential predictors associated with response and OS were also evaluated. A total of 626 AML patients were included (487 treated with AZA and 139 with DEC). Response rates were similar in both groups: CR was 18% with AZA vs. 23% with DEC (p = 0.20), CR/CRi was 20.5% vs. 25% (p = 0.27) and ORR was 32% vs. 39.5% (p = 0.12), respectively. Patients with leukocytes < 10 × 109/L, bone marrow blasts < 50% and ECOG ≥ 2 had higher ORR with DEC than with AZA. OS was similar in both groups: 10.4 months (95% CI: 9.2–11.7) vs. 8.8 months (95% CI: 6.7–11.0, p = 0.455), for AZA and DEC, respectively. Age (≥80 years), leukocytes (≥ 10 × 109/L), platelet count (9/L) and eGFR (≥45 mL/min/1.73 m2) were associated with higher OS with AZA compared to DEC. In conclusion, we found no differences in response and OS rates in AML patients treated with AZA or DEC.

Next-generation sequencing (NGS) has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia (AML). However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. In order to overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for NGS panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse AML. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized NGS analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for AML patients of the PETHEMA group (clinicaltrials gov. Identifier: NCT03311815).

Abstract: Although hypomethylating agents (HMAs) are frequently used in the frontline treatment of older acute myeloid leukemia (AML) patients, little is known about their effectiveness in relapsed or primary treatment–refractory (RR)-AML. Using an international multicenter retrospective database, we studied the effectiveness of HMAs in RR-AML and evaluated for predictors of response and overall survival (OS). A total of 655 patients from 12 centers received azacitidine (57%) or decitabine (43%), including 290 refractory (44%) and 365 relapsed (56%) patients. Median age at diagnosis was 65 years. Best response to HMAs was complete remission (CR; 11%) or CR with incomplete count recovery (CRi; 5.3%). Additionally, 8.5% experienced hematologic improvement. Median OS was 6.7 months (95% confidence interval, 6.1-7.3). As expected, OS differed significantly by best response, with patients achieving CR and CRi having a median OS of 25.3 and 14.6 months, respectively. In multivariate analysis, the presence of ≤5% circulating blasts and a 10-day schedule of decitabine were associated with improved response rates, whereas the presence of >5% circulating blasts and >20% bone marrow blasts were associated with decreased OS. A significant subset of RR-AML patients (16%) achieved CR/CRi with HMAs and experienced a median OS of 21 months. Outside of a clinical trial, HMAs represent a reasonable therapeutic option for some patients with RR-AML.

We sought to predict treatment responses and outcomes in older patients with newly diagnosed acute myeloid leukemia (AML) from our FLUGAZA phase III clinical trial (PETHEMA group) based on mutational status, comparing azacytidine (AZA) with fludarabine plus low-dose cytarabine (FLUGA). Mutational profiling using a custom 43-gene next-generation sequencing panel revealed differences in profiles between older and younger patients, and several prognostic markers that were useful in young patients were ineffective in older patients. We examined the associations between variables and overall responses at the end of the third cycle. Patients with mutated DNMT3A or EZH2 were shown to benefit from azacytidine in the treatment-adjusted subgroup analysis. An analysis of the associations with tumor burden using variant allele frequency (VAF) quantification showed that a higher overall response was associated with an increase in TET2 VAF (odds ratio (OR), 1.014; p = 0.030) and lower TP53 VAF (OR, 0.981; p = 0.003). In the treatment-adjusted multivariate survival analyses, only the NRAS (hazard ratio (HR), 1.9, p = 0.005) and TP53 (HR, 2.6, p = 9.8 × 10−7) variants were associated with shorter overall survival (OS), whereas only mutated BCOR (HR, 3.6, p = 0.0003) was associated with a shorter relapse-free survival (RFS). Subgroup analyses of OS according to biological and genomic characteristics showed that patients with low–intermediate cytogenetic risk (HR, 1.51, p = 0.045) and mutated NRAS (HR, 3.66, p = 0.047) benefited from azacytidine therapy. In the subgroup analyses, patients with mutated TP53 (HR, 4.71, p = 0.009) showed a better RFS in the azacytidine arm. In conclusion, differential mutational profiling might anticipate the outcomes of first-line treatment choices (AZA or FLUGA) in older patients with AML. The study is registered at ClinicalTrials.gov as NCT02319135.

BACKGROUND: Induction schedules in acute myeloid leukemia (AML) are based on combinations of cytarabine and anthracyclines. The choice of the anthracycline employed has been widely studied in multiple clinical trials showing similar complete remission rates. MATERIALS AND METHODS: Using an ex vivo test we have analyzed if a subset of AML patients may respond differently to cytarabine combined with idarubicin, daunorubicin or mitoxantrone. Bone marrow (BM) samples of 198 AML patients were incubated for 48 hours in 96 well plates, each well containing different drugs or drug combinations at different concentrations. Ex vivo drug sensitivity analysis was made using the PharmaFlow platform maintaining the BM microenvironment. Drug response was evaluated as depletion of AML blast cells in each well after incubation. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis, and pharmacological responses were calculated using pharmacokinetic population models. RESULTS: Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC50 with idarubicin (p=1.462E-06), whereas the interpatient variability of either drug was large. To identify those cases of selective sensitivity to anthracyclines, potency was compared, in terms of area under the curve. Differences in anthracycline monotherapy potency greater than 30% from 3 pairwise comparisons were identified in 28.3% of samples. Furthermore, different sensitivity was detected in 8.2% of patients comparing combinations of cytarabine and anthracyclines. DISCUSSION: A third of the patients could benefit of the use of this test in the first line induction therapy selection, although it should be confirmed in a clinical trial specifically designed.

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P