The aim of our study was to analyze the incidence, co-mutation pattern, and prognostic impact of CEBPA gene mutations in a large multicenter consecutive series of 1367 adult patients AML patients treated with non-IT modalities. A total of 83 patients (6.1%) had mutations in CEBPA gene. Among these, 34 (2.5%) harbored mutations located in bZIP domain (bZIP in-frame N = 6) and 49 (3.6%) in other regions of the gene (other CEBPAmut). Genes most frequently co-mutated in these CEBPAmut patients were TET2 (45.8%, N = 38), SRSF2 (42.2%, N = 35), and ASXL1 (40.9%, N = 34). Using the Bradley-Terry model we identified that mutations in MDS-related genes, in TP53, and in epigenetic regulators appear to occur earlier. In contrast, genes involved in activating cell signaling appeared to occur later than CEBPAmut. Overall Survival (OS) of non-IT AML patients was analyzed according to the type of CEBPAmut. Median OS was 11.6 months in CEBPA-bZIP patients compared to 9.0 and 6.9 for patients with other CEBPAmut or CEBPAwt, respectively. When selecting 1129 AML patients treated with HMA or HMA-based combinations, CEBPA-bZIP patients had a median survival time of 11.6 months (range 9.6-NR) and 2.5 years survival probability of 20.1% which were outcomes comparable to remaining ELN2024 favorable patients. We concluded that our series of non-IT AML patients within the PETHEMA registry confirm a low percentage of CEBPA mutated cases which are frequently co-mutated with MDS related genes. Those CEBPA-bZIP cases harbor a similar median OS than those belonging to favorable ELN2024 risk category.

Background/Objectives: This PETHEMA PCR-LMA study aimed to evaluate whether mutations detected by NGS (VAF cut-off of ≥5%) correlate with NPM1, FLT3-ITD, FLT3-TKD, IDH1, and IDH2 mutations detected using conventional PCR (analytical sensitivity 3%) in a nationwide network of seven reference laboratories. Methods: Between 2019 and 2021, 1685 adult AML patients with at least one centralized sample (NGS or PCR) at primary diagnosis or relapse/refractory episode were included. Results: During this period, 1288 paired NGS/PCR samples (1094 at diagnosis, 103 at relapse and 88 at refractoriness) were analyzed. Considering PCR the gold-standard, for NPM1 NGS sensitivity was 98.5% and specificity 98.9%, for FLT3-ITD 73.8% and 99.6%, for FLT3-TKD 84.5% and 99.3%, for IDH1 98.7% and 98.7%, and for IDH2 99.1% and 97.7%, respectively. Overall concordance rate of positive results between NGS (and PCR was 95% (262/276) for NPM1, 72% (149/206) for FLT3-ITD, 74% (49/66) for FLT3-TKD, 87% (77/89) for IDH1 and 84% (107/127) for IDH2. Overall, median days from sample reception until report were 7 for PCR and 28 for NGS. Conclusions: This study shows high concordance between NPM1 and IDH results using PCR and NGS. However, sensible important discrepancies are observed for FLT3 mutations. In our context, rapid screening for these druggable mutations should be performed by conventional PCR.

The rapid and accurate laboratory identification of targetable therapeutic genes, together with the implementation of measurable residual disease (MRD) based decision is essential for optimal clinical management in acute myeloid leukemia (AML). The PETHEMA (Programa Español de Tratamientos en Hematología) cooperative group comprises nine laboratories that perform centralized molecular studies by conventional PCR and Real-time quantitative PCR (RT-qPCR) for AML patients. With the aim to validate laboratories performances, we conducted three rounds of interlaboratory cross validation (ICV) for the quantification of CBFB::MYH11, RUNX1::RUNX1T1 and NPM1 and one round to determine the mutational status of NPM1, FLT3 (ITD and TKD2) IDH1 and IDH2. For RT-qPCR a total of 19 samples were tested and only 3 (15.8 %) failed to achieve 100 % concordance, corresponding to samples with low target gene mean ratios (ICV1-S4, ICV2-S1, ICV2-S6). For mutation detection in a total of 227 returned results, concordance rate ranged from 95 % to 100 %. FLT3-ITD and NPM1 mutation detection had 100 % concordant results. One false negative was reported for IDH2- R140 mutation and 4 false positives were detected: 2 FLT3-TKD2 and 2 IDH1-R132, likely reflecting differences in assay sensitivity. Overall, the results were highly satisfactory, particularly regarding MRD assessment, and highlighted key points for improvement, especially in baseline detection of FLT3-TKD2 and IDH1 mutations. This study represents the first collaborative initiative to evaluate performance of AML molecular targets within a laboratory network and underscores the importance of regular exercises to monitor performance, identify and resolve technical or interpretive discrepancies to ultimately ensure accurate clinical decision-making.

Background: Inhibition of FLT3 and other kinases through oral targeted agents could improve standard chemotherapy outcomes for fit AML FLT3-ITD negative patients. The randomized SORAML trial from the SAL group, including all genetic subtypes, showed that the addition of type II inhibitor sorafenib improved leukemia-free, but not overall survival (OS) among newly diagnosed fit AML patients. Quizartinib (Quiz) is a potent type II inhibitor showing about 30% of complete remissions (CR) and CR with incomplete recovery (CRi) as monotherapy for relapsed/refractory FLT3-ITD negative AML. With this rationale, the PETHEMA group designed a randomized, double-blind, placebo (PBO)-controlled phase II QUIWI trial (NCT04107727), with interim preplanned analyses (data cut-off March 2023) reporting 2-years overall survival (OS) 63.5% with Quiz vs. 47% with PBO (p=0.004). Here, we report the final analyses of the QUIWI trial with significantly prolonged follow-up (data cut-off July 26th, 2024) and full data-base cleaning. Methods: Multicenter, randomized, PBO-controlled, double-blinded phase II clinical trial. Patients with newly diagnosed FLT3-ITD negative AML, aged 18 to 70 years, and fit for intensive chemotherapy were centrally screened for FLT3-ITD prior to randomization. The trial was conducted in two phases: an open-label safety run-in phase exploring Cytarabine 200 mg/m2 (days 1-7), Idarubicin 12 mg/m2 (days 1-3), and Quiz 60 mg/d x 14 days to establish the dose for the randomized phase. The double-blinded phase 2:1 used randomization stratified by age (<60 vs. ≥60 years old) at diagnosis. A second identical induction cycle was allowed in case of failure to achieve CR/CRi after the first cycle. Consolidation (up to 4 cycles) consisted of high dose Cytarabine on Days 1, 3, and 5 plus Quiz or PBO for 14 days. Patients with high genetic risk or intermediate with MRD positivity were recommended for allo-SCT. A 12 cycles maintenance phase with 60 mg Quiz or PBO started after the consolidation or after allo-SCT. MRD monitoring, was performed through the PETHEMA centralized platform (PLATAFO-LMA). NPM1 and CBF patients was assessed for MRD using RT-qPCR standardized techniques, and the remaining subgroups by standardized multiparametric flow cytometry. The primary objective of QUIWI trial was to compare the event-free survival (EFS) (failure to achieve CR/CRi after 1 or 2 cycles, death in CR/CRi, or relapse, whichever occurs the first) between Quiz and PBO arms. OS was a key secondary endpoint. A blinded independent review committee (IRC) revised response assessment and European Leukemia Net (ELN) risk classification (2017 and 2022), among other critical parameters. Analyses were performed on an intent-to-treat basis. Results: From September 2019 to November 2021, 284 Pts were enrolled in 45 Spanish PETHEMA centers, 11 of them were included in the safety run-in phase establishing 60 mg/day of Quiz for the randomized phase. 273 Pts were randomized to Quiz (n=180) or PBO (n=93). The median age was 57 y [IQR, 48 - 64 y]. Baseline pts and disease characteristics were balanced between the 2 arms (ELN 2022 risk distribution for Quiz and PBO was low 29.4% vs. 25.8%, intermediate 12.8% vs. 13.9%, and high 57.8% vs. 60.2%, p=0.8). The median follow-up was 39.4 months. Median EFS was 18.8 mo with Quiz vs. 9.9 mo with PBO (hazard ratio [HR], 0.732; 95% CI, 0.533-1.005; 2-sided P=0.053). Regarding OS, 71 out of 180 patients died in the Quiz arm, and 51 out of 93 in the PBO. Median OS was not reached with Quiz vs 29.3 mo with PBO (HR, 0.625; 95% CI, 0.436-0.897; P=0.009), and the 3-years OS was 61% with Quiz vs. 46% with PBO. Quiz benefit was observed among both <60 vs. ≥60 years old pts (HR, 0.63, P=0.067; and HR, 0.63, P=0.085, respectively), and among allo-SCT and no allo-SCT pts (HR, 0.59; P=0.16; and HR, 0.64; P=0.03, respectively). CR/CRi rate after 2 cycles was 77.2% in the Quiz arm and 76.3% in the PBO. Death during first induction cycle was 7 (4%) for Quiz and 5 (5%) for PBO. Overall, 86 (31.5%) pts received an allo-SCT after first CR/CRi, 58 (32.2%) in Quiz and 28 (30.1%) in PBO arm. Maintenance therapy started in 102 (37%) pts, 72 (40%) in Quiz and 30 (32%) in PBO arm. No new safety signals were observed among Quiz and PBO arms. Conclusion: Our study strongly suggests that the addition of Quiz to 3+7 may prolong OS in newly diagnosed FLT3-ITD negative AML. A large global phase III randomized trial (Quantum-WT) will aim to confirm the PETHEMA-QUIWI results.

The pathogenesis of COVID‐19 warrants unravelling. Genetic polymorphism analysis may help answer the variability in disease outcome. To determine the role of KIR and HLA polymorphisms in susceptibility, progression, and severity of SARS‐CoV‐2 infection, 458 patients and 667 controls enrolled in this retrospective observational study from April to December 2020. Mild/moderate and severe/death study groups were established. HLA‐A , ‐B , ‐C , and KIR genotyping were performed using the Lifecodes® HLA‐SSO and KIR‐SSO kits on the Luminex® 200™ xMAP fluoroanalyser. A probability score using multivariate binary logistic regression analysis was calculated to estimate the likelihood of severe COVID‐19. ROC analysis was used to calculate the best cut‐off point for predicting a worse clinical outcome with high sensitivity and specificity. A p ≤ 0.05 was considered statistically significant. KIR AA genotype protected positively against severity/death from COVID‐19. Furthermore, KIR3DL1 , KIR2DL3 and KIR2DS4 genes protected patients from severe forms of COVID‐19. KIR Bx genotype, as well as KIR2DL2 , KIR2DS2 , KIR2DS3 and KIR3DS1 were identified as biomarkers of severe COVID‐19. Our logistic regression model, which included clinical and KIR/HLA variables, categorised our cohort of patients as high/low risk for severe COVID‐19 disease with high sensitivity and specificity (Se = 94.29%, 95% CI [80.84–99.30]; Sp = 84.55%, 95% CI [79.26–88.94]; OR = 47.58, 95%CI [11.73–193.12], p < 0.0001). These results illustrate an association between KIR/HLA ligand polymorphism and different COVID‐19 outcomes and remarks the possibility of use them as a surrogate biomarkers to detect severe patients in possible future infectious outbreaks.

The advent of tyrosine kinase inhibitors (TKIs) has changed the natural history of chronic myeloid leukemia (CML), and the transformation from the chronic phase to the blast phase (BP) is currently an uncommon situation. However, it is one of the major remaining challenges in the management of this disease, as it is associated with dismal outcomes. We report the case of a 63-year-old woman with a history of CML with poor response to imatinib who progressed to myeloid BP-CML, driven by the acquisition of t(8;21)(q22;q22)/RUNX1::RUNX1T1. The patient received intensive chemotherapy and dasatinib, followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, she suffered an early relapse after allo-HSCT with the acquisition of the T315I mutation in ABL1. Ponatinib and azacitidine were started as salvage treatment, allowing for the achievement of complete remission with deep molecular response after five cycles. Advances in the knowledge of disease biology and clonal evolution are crucial for optimal treatment selection, which ultimately translates into better patient outcomes.

Background: Waldenström macroglobulinemia (WM) is a rare, indolent B-cell lymphoproliferative disorder, often preceded by a history of IgM monoclonal gammopathy of undetermined significance (IgM-MGUS). WM progression mechanisms are not fully understood given the complex integration of clinical and molecular features. Aims: To determine the impact of clinical, molecular and flow cytometry factors on overall survival (OS) and time to first treatment (TTFT) in a large, real life series of patients with IgM gammopathy. Methods: In this retrospective multicenter study, we collected real-life data on 577 patients with IgM gammopathy from 22 Spanish Centers. Moreover, 166 additional patients, treated at the University Hospital of Torino, Italy, were used as validation series. Multiparameter Flow Cytometry (MFC) and MYD88L265P evaluation (either by ddPCR or qPCR) were performed on baseline bone marrow (BM) samples in Salamanca and Torino laboratories respectively. Results: Overall median OS of the Spanish series was 126.7 months: 85.0 for symptomatic (sWM), 143.6 for asymptomatic WM (aWM), and 180.6 months for IgM-MGUS (p<0.001) respectively, while median TTFT for asymptomatic patients (aWM+MGUS) was 228.5 months, with 89% of pts remaining off treatment at 5 years. By multivariate analysis, significant clinical prognostic factors for OS included age > 65 years, male gender, diagnosis of sWM and beta-2-microglobulin >3, while age > 65 years, BM infiltration, hemoglobin <11.5 g/dl and platelets <100.000/mmc were associated with shorter TTFT. Data from the Spanish and the Torino cohorts were pooled and divided into two groups based on high vs low baseline values of MYD88L265P Mut/WT ratio (cut-off point 0.162) and high vs low baseline MFC clonal B cell marrow infiltration (cut-off point 4.39%). By univariate analysis, the MYD88low group showed better OS (p=0.005, HR=0.44) and TTFT (p=0.024, HR=0.33) compared to the MYD88high group. Similarly, the MFClow group had increased OS (p=0.033, HR=0.65) and TTFT (p=0.008, HR=0.37) compared to the MFChigh group. Moreover, by combining MFC and MYD88 baseline levels, patients were stratified into low, intermediate and high risk classes (LR: MFClow/MYD88low, IR: either MFClow/MYD88high or MFChigh/MYD88low; HR: MFChigh/MYD88high). The high risk group significantly differed from the others in terms of OS (n= 156, p=0.005, HR=3.28, figure 1) and TTFT (n= 92, p=0.003, HR= 9.61), while the intermediate-risk group (p=0.015, HR=4.76) was also significantly different from the low risk group for TTFT. Multivariable analysis confirmed a significant impact of MYD88 mutation and MFC baseline levels on both OS and TTFT. Finally, competing risk analysis showed, in the MFChigh/MYD88high group, a statistically significant increment in disease-related rather than unrelated deaths at 5 years (2.2% vs 19%, p=0.002, for MFClow/MYD88low vs MFChigh/MYD88high respectively). Conclusions: Our retrospective study shows that MYD88L265P, by PCR quantitative analysis, and marrow infiltration, by MFC, play a significant prognostic role in OS and in the treatment indications in patients with IgM gammopathy. Prospective validation studies may greatly help to define prognostic tools for IgM gammopathy and to better define patients with high risk WM.

Background: Quizartinib (Quiz) is a potent type II inhibitor showing clinical activity in FLT3-ITD negative acute myeloid (AML) patients (pts), as suggested by the interim analyses of the PETHEMA randomized, double-blind, placebo (PBO)-controlled phase II QUIWI trial (NCT04107727). Here, we describe the frequency of baseline gene mutations among FLT3-ITD negative (allelic ratio by PCR <0.03) randomized subjects and evaluate their impact on OS in QUIWI final dataset. Methods: Eligible pts were 18-70 years old, with newly diagnosed FLT3-ITD negative AML (FLT3-ITD allelic ratio <0.03, centralized analyses). Pts were randomized 2:1 to receive 3+7 induction chemotherapy with PBO or Quiz (60 mg x 14 days). A second identical induction cycle was allowed in case of failure to achieve CR/CRi after the first cycle. Consolidation (up to 4 cycles) consisted of high dose Cytarabine on Days 1, 3, and 5 plus PBO or Quiz for 14 days. Patients with high genetic risk or intermediate with MRD positivity were recommended for allo-SCT. A maintenance phase with 60 mg Quiz or PBO started after the consolidation or after allo-SCT. A blinded independent review committee revised response assessment and European Leukemia Net (ELN) risk classification (2017 and 2022). Analyses were performed on an intent-to-treat basis. Baseline mutational statuses for 26 genes relevant to AML were analyzed in bone marrow via next-generation sequencing panels in the PETHEMA central lab plataform (PLATAFO-LMA) (Quiz, n=180; placebo, n=93 pts). A gene was considered mutated if it exhibited at least one somatic mutation with a VAF of ≥ 5%. For FLT3-TKD and NPM1 mutations, conventional PCR was also used for positivity diagnosis (sensitivity 3%). The impact of Quiz according to ELN 2017 and 2022 risk categories on OS was assessed. Based on their prevalence and prognostic significance, the effect of NPM1, DNMT3A, IDH1, IDH2, FLT3-TKD, and TP53 mutations on OS was explored. Hazard ratios (HRs) comparing Quiz vs. PBO were calculated using unstratified Cox proportional hazards models. These analyses were exploratory in nature and not powered for formal hypothesis testing. Results: At baseline, gene mutations were detected in 256/273 (93.8%) analyzed pts (all pts had available NGS results). The most common mutations were: DNMT3A (24 %), NPM1 (21 %), TET2 (19 %), IDH2 (18 %), RUNX1 (16 %), SRSF2 (13 %), TP53 (12 %), ASXL1 (10 %), FLT3-TKD (10 %), NRAS (9 %), IDH1 (9 %), CEBPa (8 %), KRAS (7 %), PTPN11 (7 %), SF3B1 (6 %), EZH2 (5 %), U2AF1 (5 %), WT1 (5 %), GATA2 (3.3 %), and SETBP1 (3.3 %). ELN 2017 risk distribution for Quiz and PBO arms was low 28.3% vs. 25.8%, intermediate 27.2% vs. 25.8%, and high 44.4% vs. 48.4%, p=0.82; and ELN 2022 risk distribution for Quiz and PBO was low 29.4% vs. 25.8%, intermediate 12.8% vs. 13.9%, and high 57.8% vs. 60.2%, p=0.8). OS benefit with Quiz vs PBO was observed among ELN 2017 low-risk (3-years OS 90% and 71%, respectively; HR, 0.3 [0.095, 0.946]) and intermediate-risk (3-years OS 67% and 44%, respectively; HR, 0.466 [0.227, 0.956]); with no detrimental OS effect among high-risk group (HR,0.8 [0.5-1.3]). OS benefits with Quiz vs PBO was observed among ELN 2022 low-risk (3-years OS 92% and 71%, respectively; HR, 0.23 [0.067, 0.786]) and intermediate-risk (3-years OS 68% and 53%, respectively; HR, 0.53 [0.185, 1.513]); with no detrimental OS effect among high-risk group (HR, 0.7 [0.5-1.13]). OS benefits with Quiz vs. PBO was observed across most of the assessed subgroups, including NPM1mut (3-years OS 92% and 53%, respectively; HR, 0.123 [0.033, 0.467]) and DNMT3Amut (3-years OS 71% and 48%, respectively; HR, 0.458 [0.205, 1.024]); FLT3-TKDmut (3-years OS 95% and 33%, respectively; HR, 0.057 [0.006, 0.514]); IDH2mut (3-years OS 72% and 47%, respectively; HR, 0.503 [0.204, 1.24]). No detrimental OS effect was observed in IDH1mut (3-years OS 65% and 62%, respectively; HR, 0.88 [0.22, 3.52]), and TP53mut (3-years OS 16% and 0%; HR, 0.989 [0.473, 2.071]). Conclusions: The addition of Quiz to intensive chemotherapy was associated with significant OS advantage among ELN low-intermediate risk pts. NPM1mut and FLT3-TKDmut appeared to have special sensitivity to Quiz, resulting in high survival rates. The addition of Quiz vs. PBO was not associated with improved or worsened survival among ELN adverse-risk patients. These results suggest that baseline mutational status of FLT3-ITD negative pts could be relevant to predict benefit from Quiz treatment.

Background: There are no studies assessing the evolution and patterns of genetic studies performed at diagnosis in acute myeloid leukemia (AML) patients. Such studies could help to identify potential gaps in our present diagnostic practices, especially in the context of increasingly complex procedures and classifications. Methods: The REALMOL study (NCT05541224) evaluated the evolution, patterns, and clinical impact of performing main genetic and molecular studies performed at diagnosis in 7285 adult AML patients included in the PETHEMA AML registry (NCT02607059) between 2000 and 2021. Results: Screening rates increased for all tests across different time periods (2000-2007, 2008-2016, and 2017-2021) and was the most influential factor for NPM1, FLT3-ITD, and next-generation sequencing (NGS) determinations: NPM1 testing increased from 28.9% to 72.8% and 95.2% (p < .001), whereas FLT3-ITD testing increased from 38.1% to 74.1% and 95.9% (p < .0001). NGS testing was not performed between 2000-2007 and only reached 3.5% in 2008-2016, but significantly increased to 72% in 2017-2021 (p < .001). Treatment decision was the most influential factor to perform karyotype (odds ratio [OR], 6.057; 95% confidence interval [CI], 4.702-7.802), and fluorescence in situ hybridation (OR, 2.273; 95% CI, 1.901-2.719) studies. Patients >= 70 years old or with an Eastern Cooperative Oncology Group >= 2 were less likely to undergo these diagnostic procedures. Performing genetic studies were associated with a favorable impact on overall survival, especially in patients who received intensive chemotherapy. Conclusions: This unique study provides relevant information about the evolving landscape of genetic and molecular diagnosis for adult AML patients in real-world setting, highlighting the increased complexity of genetic diagnosis over the past 2 decades.

The project Precision Medicine Alliance in Hematology for myeloid malignancies supported by the Spanish Society of Hematology and Hemotherapy (SEHH) was carried out with the aim of providing a nationwide consensus of the processes involved in the molecular diagnosis of myeloid disorders using next-generation sequencing (NGS) techniques.One of the current needs is to improve harmonization across centers of genetic/molecular studies in the diagnosis and monitoring of patients with acute myeloid leukemia, myelodysplastic syndromes, and Philadelphia-chromosome negative myeloproliferative disorders.This would allow all patients, regardless of the center or region in which they are located, to have access to a correct diagnosis based on the use of these studies, in order to receive the optimal approach targeting the molecular characteristics of each neoplasm.Although it is not yet possible to achieve a total international standardization in which all the centers use the same panel and same technology, to fix the minimum studies that should be carried out.This should include the initial assessments and the NGS studies, as well as the minimum quality criteria required for diagnosis, which would warrant the necessary diagnostic uniformity for patients with myeloid malignancies.

Background The identification of predictive biomarkers is crucial for guiding treatment decisions in acute myeloid leukemia (AML). Previously, we identified a FLT3-like gene expression signature in FLT3 wild-type AML patients, which clustered a variable proportion of FLT3 wild-type patients with FLT3-ITD and TKD mutated patients. The QUIWI trial was a randomized, placebo-controlled, phase II study preliminary showing a significant increase in overall survival (OS) in wild-type FLT3 AML patients treated with the FLT3 inhibitor quizartinib (Quiza) plus standard chemotherapy. This preplanned correlative study was designed to assess the value of the FLT3-like signature to predict responses to Quiza. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced (161 from FLT3-ITD negative enrolled in QUIWI; and 55 from FLT3-ITD positive patients who were screen failure of the QUIWI trial by this reason). The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Gene expression quantification was performed using the Bioconductor workflow, and gene expression estimates (FPKM) were obtained. The gene expression estimates were log2 normalized. Finally, those genes mapping to the original FLT3-like signature (595 genes) were selected for downstream analysis. Clustering was based on the hierarchical method, using standard euclidean distance metrics. OS was defined as time from start of screening to death. Event-free survival (EFS) was defined as time from randomization to failure to achieve CR/CRi after 1 or 2 cycles, death in CR/CRi, or relapse (whichever occurred the first). Relapse-free survival was defined as time from randomization to disease relapse or death by any cause. Results Among the total 206 patients, a cluster of 54.37% (N=112) was enriched in FLT3-mutant cases (71.11% of cases, Figure 1A). This subgroup comprised 49.67% of wild-type FLT3 cases (N=80), hereafter FLT3-like patients. In the group of not FLT3-like patients, no differences were identified between the placebo and Quiza group in the total number of deaths (Fisher's p-value, 0.63), EFS (cox p-value, 0.83; HR 1.07 [0.56-2.06]), RFS (cox p-value 0.76; HR 0.88 [0.38-2.01]) and OS (cox p-value, 0.62; HR 1.22 [0.55-2.67]). Among FLT3-like patients, significant differences were identified in the total number of deaths (Fisher's p-value, 0.004), EFS (cox p-value, 0.009; HR 0.45 [0.25-0.82], RFS (cox p-value 0.01, HR 0.37 [0.18-0.79]) and OS (cox p-value 0.01, HR, 0.41 [0.20-0.84]) ( Figure 1B). No statistically significant association was observed between the FLT3-like pattern and the ELN-17 classification: 30.4% were low risk, 40.5% intermediate risk, 29.1% high risk. Conclusion The FLT3-like gene expression signature successfully identified a subset of patients who derived the most benefit from Quiz, while patients without the FLT3-like signature did not demonstrate a benefit compared with placebo. These findings support the use of the FLT3-like signature as a potential biomarker to identify those wild-type FLT3 AML patients who may benefit from Quiz, providing a valuable insight for personalized treatment in AML.

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: The identification of FLT3 mutation in acute myeloid leukemia (AML) has become a key issue since the incorporation of FLT3 inhibitors in front-line therapy. Aims: To compare the identification of FLT3 mutations by the classical PCR technique vs. NGS with the two main platforms (capture-based Illumina and amplicon-based ThermoFisher). Methods: Data was collected from the 7 NGS reference laboratories of PETHEMA AML diagnostic network. A total of 354 FLT3-ITD positive AML samples at diagnosis or relapse were analyzed by PCR (followed by capillary electrophoresis) and NGS. A ratio ≥0.03 was established for a positive PCR result. ThermoFisher (amplicon-based technology) and Illumina (capture-based technology) platforms were used to characterize FLT3-ITD mutations in 196 and 158 samples respectively. NGS mutation characterization was performed with the commercial and custom myeloid panels and software used in routine diagnosis at each reference laboratory. Results: Using the ThermoFisher NGS platform, 14/196 samples (7.1%) were detected by PCR but not NGS. Of them, 6 samples had a PCR ratio between 0.03–0.19 and 7 samples presented an ITD of 129–200bp length. Meanwhile, all NGS FLT3-ITD positive samples were also identified by PCR. With Illumina platform, 12/158 (7.6%) were not characterized with PCR (but all of them were NGS positive). Ten out of the 12 samples presented a VAF between 1-2.8%, and 1 sample had an ITD mutation at position c.2503, thus, outside the PCR amplicon. We also compared concordance of PCR-ratio and VAF-ratio (mutated VAF/wild-type VAF) for each NGS platform. Twenty six samples with high ITD length (> 110bp) were excluded from the analyses as they showed highly dissimilar results. As shown in Figure 1, Illumina platform showed higher concordance (mean PCR-ratio: 0.902, mean VAF-ratio: 0.937, R of lineal regression=0.875, N=145) compared to ThermoFisher (mean PCR-ratio: 0.856, mean VAF-ratio: 0.399, R of lineal regression=0.655, N=182). Considering the whole series, we identified 12 (12/158, 7.6%) highly discordant cases with the Illumina platform, with a difference between PCR-ratio and VAF-ratio of at least two units. Half of these cases showed an ITD insert between 114–237bp which corresponded with overestimated VAF values between 74–99.4%. We also found 11 highly discordant cases (11/196, 5.6%) with the ThermoFisher platform, although only 2 cases had a high length insert (112 and 180bp) which conversely corresponded to infraestimated or negative NGS VAF values. Summary/Conclusion: Most of the samples were characterized by both PCR and NGS methods and the Illumina capture-based technique showed higher sensitivity and concordance. High-length ITD mutations are problematic as they are either undetected by amplicon-ThermoFisher technology or overestimated with the capture-Illumina workflow. These may be relevant considerations in the accurate identification of FLT3-ITD mutations as well as for NGS-MRD monitoring. Figure1. Lineal regression of PCR-ratio vs. VAF-ratio in each NGS platformKeywords: Acute myeloid leukemia, Flt3-ITD, PCR

Introduction Wild-type FLT3 AML presents significant therapeutic challenges due to limited treatment options. The QUIWI trial investigated quizartinib (Quiz) combined with standard chemotherapy, showing promising results. To understand drug response and refractoriness, we conducted a correlative analysis, exploring differential expression patterns among distinct patient groups. We reasoned that the molecular fingerprints of good responders to Quiz plus chemotherapy would be different with respect to patients who respond only to standard chemotherapy, helping to elucidate critical molecular mechanisms associated with Quiz response. Our study provides a basis for personalized therapeutic strategies in FLT3-ITD negative AML. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced, out of which 55 cases were FLT3-ITD and were discarded for this analysis. For this analysis, only samples with a minimum of 20% blasts were considered. The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Differential expression was assessed with the DESeq2 algorithm, using Blast proportion to adjust for disparities among the samples. P-values were adjusted for multiple testing using the FDR method. OS was defined as time from start of screening to death. Gene ontology and pathways analysis were performed using the WebGestalt portal. Results In this study, we compared the gene expression profiles of Quiz versus placebo-treated patients categorized as good responders, defined arbitrarily as those who continue alive and have a minimum follow-up of 6 months after diagnosis. Out of 82 eligible patients, we identified 100 (q-value < 0.05) and 172 (q-value < 0.1) differentially expressed genes between the Quiz and Placebo groups. Notably, 132 genes were overexpressed in the Quiz group, while 40 genes showed overexpression in the placebo group. Moreover, we observed a highly significant overexpression of ribosomal genes in the Quiz group (17 overlaps, KEGG pathways q-value < 2.2 x 10 -16), and a significant overexpression of genes related to the attenuation of the heat shock protein transcriptional response, which is involved in protein folding, in the placebo group (Reactome q-value, 0.008; gene ids: HSPA1A, HSPA1B and DNAJB1). Additionally, the Quiz group exhibited a significant overexpression of transmembrane receptor protein tyrosine phosphatase-related genes (Gene Ontology Molecular Function q-value, 0.02; gene ids: PTPRB, PTPRG and PTPRU). Conclusion This correlative analysis of the Quiwi trial revealed critical molecular mechanisms associated with Quiz response. The overexpression of ribosomal and transmembrane receptor protein tyrosine phosphatase-related genes in good responders within the Quiz group, along with the overexpression of the heat shock pathway genes in the placebo group, aligns with existing knowledge about the FLT3 pathway biology and the molecular determinants to tyrosine-kinase inhibitors. These findings offer potential biomarkers for personalized therapeutic approaches to improve clinical outcomes in this challenging AML population.

Introduction:Despite recent advances in AML treatment, patients continue to relapse and become chemotherapy resistant, leading to poor long-term overall survival. The identification of this measurable residual disease (MRD), defined as the post-therapy presence of leukemic cells, currently stands as one of the most well-established risk factors. The abnormal phenotypic and molecular characteristics of AML cells offer an opportunity for disease monitoring using techniques like multiparameter flow cytometry (MFC) and qPCR, which are currently considered the gold standard for MRD detection. However, these techniques have limitations. The aim of this study was to validate a different approach using single-cell DNA sequencing (scDNAseq) technology to detect MRD in patients achieving complete response (CR) and to characterize the genomic landscape of MRD clones and the potential identification of clonal evolution. Methods:We selected 24 cryopreserved bone marrow samples from 15 AML patients who participated in the QUIWI-PETHEMA clinical trial (NCT04107727) and achieved CR after induction and consolidation (n=20). 4 diagnosis samples were analyzed to determine genomic differences between the MRD clone and the initial leukemic population. All patients had bulk targeted NGS data available at diagnosis. Selection of CD34+ and/or CD117+ cells was performed using magnetic beads for enrichment of blasts and Mission Bio multiome single cell DNA+protein was performed using an AML-related 469 amplicon panel and 19 surface antibody mix. Multiplex of 3 independent samples was performed in each library preparation. Analysis was done according to manufacturer's instructions. MRD was also analyzed using MFC (n=14) EuroFlow panel with a limit of detection of 0.1% aberrant cells. RNA qRT-PCR was used in cases with NPM1 mutations (n=6). Quantification of MRD by scDNAseq was calculated according to the enrichment performance obtained from the manufacturer and the percentage of mutant cells. Results: The concordance between gold standard techniques for MRD and scDNAseq was 75% (15/20) (Table 1). Concordance between MFC and scDNAseq was 78% (11/14). The 3 discordant cases, positives by scDNAseq, MRD levels ranged between 0.04-0.09%, below the consensus cutoff of 0.1% to define MRD+. These results suggest that scDNAseq may complement MFC in the detection of very low levels of MRD. Concordance with qRT-PCR was 66% (4/6), but we only detected one patient with a persistent NPM1 clone. Taking advantage of the single cell approach, we were able to assess the genomic landscape of MRD clones and the clonal evolution in sequential samples. The number of clones/subclones and the number of variants per clone varied between patients with positive MRD as shown in Table 1. Interestingly, we analyzed 4 samples at diagnosis by scDNAseq and observed 2 cases in which MRD mutation was already present at diagnosis but was not informed as VAF was < 1%. In 4 patients, we detected small clones (around 1%) that remained unchanged in size despite treatment, suggesting that they likely represent clonal hematopoiesis. In 6 cases, consecutive samples were obtained showing clearance of some clones with other ones remaining stable (UPN2), progressive clearance of clones (UPN3, UPN4), acquisition of new clones (UPN9) and clearance of some clones and acquisition of new ones (UPN15) (Figure 1). Integration of scDNA and cell surface protein expression in the same cell allowed us to perform mutation-clone specific immunophenotypic analysis. Some cases showed a clear pattern in which one of the mutant clones had a significantly higher expression of some markers that was correlated with previous flow cytometry data (e.g. UPN1). Conclusions: Our study suggests that the use of scDNA technology is a feasible approach for detection of MRD in AML patients. Moreover, the use of this approach in sequential samples may allow deciphering clonal evolution, the co-occurrence of different mutations including potential clonal hematopoiesis mutations and identifying winner clones potentially responsible for disease persistence and relapse. Finally, the integration of mutations and surface antibody markers in the same cell provides a means for identifying the presence of mutations in different cell populations. Validation of these results in larger series of patients and correlation with clinical outcome are the next steps for validation of this technology.

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.