NPM1 Mutations Research Articles

Acute Myeloid Leukemia (AML) Risk Stratification Using the European Leukemianet 2022 Classification in a Real-Life Cohort of Patients from Sardinia, Italy

Introduction Acute Myeloid Leukemia (AML) identifies a clinically heterogeneous group of clonal disorders. Advances in Next Generation Sequencing (NGS) have increased the possibility to define the genetic lesions present in each patient, which has led to improved disease classification, risk stratification and identification of new therapeutic targets. The 2017 European LeukemiaNet (ELN) reccommendations for genetic risk stratification of AML have been widely adopted among physicians and investigators, even though patient responses remain heterogeneous. A new edition of reccomendations for diagnosis and management of AML in adults, ELN 2022, has been recently proposed to better define prognostic significance of genomic abnormalities. Aim of the study was to evaluate the usefulness of the new ELN 2022 genetic risk classification in a series of patients diagnosed with AML at two hematological institutions. Methods A retrospective analysis of 158 newly diagnosed AML patients according to WHO 2016 (100 de novo, 58 secondary) and treated from 2013 to 2021 in two hematological centers of Sardinia, Italy, was performed. Male patients were 96 (61%) and 63 out of 158 (40%) were >70 yrs old. One-hundred-five AML diagnosis samples were analyzed by targeted NGS on Ion Torrent platform using the DNA Oncomine Myeloid Research panel which explores 1256 hotspots in 33 key myeloid genes. Demographic and clinical data, disease characteristics at diagnosis, first-line treatment and clinical outcome data were available for all patients. Cytogenetic and molecular characteristics were used to classify patients into ELN 2017 and ELN 2022 risk groups. Results NGS detected 196 clinically revelant mutations in 87% AML patients with a limit of detection of 2%, while fusion genes were detected in 5% of patients (5/105). The most frequent genetic abnormalities were FLT3ITD/TKD and NPM1 mutations which represented the 29% and 28%, respectively. Co-occurrence of NPM1 and FLT3 driver gene mutations was found with a frequency of 16%. IDH1/2 mutations were observed in 20/105 pts (19%), RUNX1 and K/NRAS in 12/105 (11%), ASXL1 mutated in 11 pts (10%), STAG2 and BCOR mutations were present in 4 and 1 pts, respectively. TP53 mutations were observed with a frequency of 13% (14 patients). ELN 2017 classified 24 cases as favorable (23%), 40 as intermediate (38%), and 41 as adverse (39%) risk group. Sixteen out of 105 (15%) patients migrated to a more adverse risk group when ELN 2022 was applied: from favorable to intermediate (7) or adverse (2), 7 pts from intermediate to adverse. ELN 2022 re-classification assigned at favorable 15 pts (14%), 40 at intermediate ( 38%), and 50 at adverse risk groups (48%). In our cohort with the use of the new ELN 2022 classification no patient was reclassified with a better risk profile. Conclusions The recent ELN 2022 classification of AML with updated genetic integration has been proposed as a useful disease classification which impacts on the choice of treatment and outcome of individual patients. Our retrospective analysis confirms that the ELN 2022 is a robust tool of genetic risk classification and redefines a better genomic profile of AML patients prompting to individualized treatments. Re-classification according to ELN 2022 showed shifting from favorable to a worse category in the 10% of AML patients <60 years of age, who could have been eligible to a more intensive treatment. Furthermore, the implementation of the AML screening gene panel through NGS analysis allows to identify molecular abnormalities, such as IDH1/IDH2 mutations, susceptibles of target therapies.

Whole Genome Sequencing for Detection of Prognostically Relevant Genetic Abnormalities in Paediatric Acute Myeloid Leukaemia

Background Genetics is important in the diagnosis of paediatric acute myeloid leukaemia (AML). A number of chromosomal abnormalities and mutations have prognostic significance and timely genetic analysis is essential for risk stratification. The recent Myechild 01 clinical trial for children with AML required testing for >20 different genetic abnormalities to assign newly diagnosed patients into three prognostic genetic risk groups: good, intermediate and poor (Figure A). Throughout the trial a combination of techniques was employed to detect these abnormalities; including chromosomal analysis, FISH for cytogenetically cryptic abnormalities, notably the poor risk fusions: t(5;11)(q35;p15)/NUP98::NSD1, inv(16)(p13q24)/CBFA2T3::GLIS2, t(7;12)(q36;p13)/ETV6::MNX1, as well as molecular testing for NPM1, FLT3-ITD and CEBPA mutations. Here, we present a small retrospective study demonstrating that whole genome sequencing (WGS) has the potential to detect all prognostically significant abnormalities in childhood AML within a single test. Methods WGS was performed on matched diagnostic and remission samples from 25 paediatric AML patients entered to the Myechild 01 treatment trial. Remission samples, minimal residual disease negative by flow cytometry and/or molecular testing, were used as germline controls. For validation, whole transcriptome sequencing (WTS) was performed on diagnostic RNA from each patient in the study. Results Leukaemia associated genetic abnormalities were detected in all 25 patients, including structural variants associated with gene fusions (n=14), single nucleotide variants (SNV) (n=20) and/or copy number abnormalities/aneuploidies (n=17). All gene fusions and mutations were confirmed by WTS. WGS reliably detected all risk stratifying abnormalities identified by standard techniques (Figure B), including 11 patients with gene fusions: RUNX1::RUNX1T1 (n=6), CBFB::MYH11 (n=3), KMT2A::MLLT3 (n=1) and KMT2A::MLLT10 (n=1) and 3 patients with copy number abnormalities: del(5q) (n=2) and del(12p) (n=1) and one patient with an NPM1 mutation. Among 10 patients with no risk stratifying abnormalities detected by standard techniques, WGS identified risk defining genetic features in 3 of them. Two patients could be reclassified as good risk due to the discovery of biallelic CEBPA mutations. Interestingly, one of the CEBPA positive patients presented with a homozygous mutation (VAF=0.97), which WGS revealed to be the consequence of copy number neutral loss of heterozygosity for the long arm of chromosome 19. A single patient could be reclassified as poor risk due to the finding of a rearrangement of MECOM with CDK6. Cytogenetics showed a deletion of 7q as the sole karyotypic abnormality, which was revealed to be an unbalanced der(7)t(3;7)(q21.2;q26.2) translocation by WGS. Over-expression of MECOM was confirmed by WTS. This patient also had a GATA2 mutation. A risk defining abnormality was not detected by either standard testing or WGS in 7 patients, however 5 of them presented with chromosomal abnormalities which were confirmed and further characterised by WGS, including aneuploidies (n=3) and/or structural rearrangements (n=4). Across the cohort, WGS detected a wide range of abnormalities not currently used in the risk stratification algorithm, including the fusions genes: NPM1::MLF1 (n=1) and PICALM::MLLT10 (n=2), which, although not stratifying in Myechild 01, have been reported to be associated with a good and a poor prognosis, respectively. SNV in genes commonly mutated in AML were observed in 19 patients (76%) including NRAS (n=6), KIT (n=3) EZH2 (n=3) and GATA2 (n=2). It has been suggested that the co-occurrence of mutations may be used to refine risk groups in childhood AML, notably the presence of KIT and NRAS mutations in RUNX1::RUNXT1 and CBFB::MYH11 positive leukaemia, respectively. Conclusion This small pilot study has demonstrated the ability of WGS to identify a wide range of genetic abnormalities that are currently used for risk stratification in childhood AML. The full spectrum of genomic events observed here highlights the potential of WGS to refine risk stratification and identify novel genetic changes as therapeutic targets in future trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Measurable residual disease detected by flow cytometry independently predicts prognoses of NPM1-mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) with NPM1 mutation is a distinct genetic entity with favorable outcomes. Nevertheless, emerging evidence suggests that NPM1-mutated AML is still a highly heterogeneous disorder. In this study, 266 patients with AML with NPM1 mutations were retrospectively analyzed to evaluate the associations between variant allele frequency (VAF) of NPM1 mutations, co-mutated genes, measurable residual disease (MRD), and patient outcomes. Multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (RT-PCR) were used for monitoring MRD. Ultimately, 106 patients were included in the long-term follow-up period. Patients with high NPM1 VAF (≥ 42.43%) had poorer 2-year relapse-free survival (RFS) (55.7% vs. 70.2%, P = 0.017) and overall survival (OS) (63.7% vs. 82.0%, P = 0.027) than those with low VAF. DNMT3A mutations negatively influenced the outcomes of patients with NPM1 mutations. Patients with high DNMT3A VAF or NPM1/DNMT3A/FLT3-ITD triple mutations had shorter RFS and significantly lower OS than that in controls. After two cycles of chemotherapy, patients with positive MFC MRD results had lower RFS (MRD+ vs. MRD-:44.9% vs. 67.6%, P = 0.007) and OS (61.5% vs. 76.6%, P = 0.011) than those without positive MFC MRD results. In multivariate analysis, high NPM1 VAF (hazard ratio [HR] = 2.045; P = 0.034) and positive MRD after two cycles of chemotherapy (HR = 3.289; P = 0.003) were independent risk factors for RFS; MRD positivity after two cycles of chemotherapy (HR = 3.293; P = 0.008) independently predicted the OS of the patients. These results indicate that VAF of both NPM1 gene itself or certain co-occurring gene pre-treatment and MRD post-treatment are potential markers for restratifying the prognoses of patients AML having NPM1 mutations.

Optimizing the Supportive Care of Intensive Chemotherapy in AML : Impact of Vitamin C and D Supplementation in Patients with NPM1 Mutation. a Dataml Registry Study

Background Vitamin (vit) deficiencies are frequently observed in patients (pts) with newly diagnosed AML (ND-AML). A lower level of vit D is associated with a worse prognosis in pts treated with intensive chemotherapy (IC) (Lee HJ, Cancer 2014). Similarly, a pretransplant vit D deficiency is associated with a higher risk of relapse (Radujkovic JCO, 2017). Moreover, preclinical studies demonstrated the antileukemic activity of vit D through the modulation of cell cycle, differenciation and apoptosis even though the clinical translation has not been successful (Cao H, Exp Hematol 2017 ; Paubelle E, Cell Rep 2020). More recent data have highlighted the role of vit C in AML. Vit C is a cofactor and increases the activity of TET2 enzyme, inducing DNA demethylation, stem cell differentiation and antileukemic activity (Agathocleous M, Nature 2017; Cimmino L, Cell 2017). Cases of remission following vit C supplementation have been reported (Foster M, Antioxidants, 2018 ; Das A, Blood Cancer J, 2019) and a small study showed that the addition of vit C to decitabine/AraC/aclarubicin/G-CSF improved outcome compared to DCAG (Zhao H, Leuk Res 2018). Based on these data, we modified our practice by adding vit C and D to the supportive care (SC) of IC. Methods The study population included all pts with ND-AML treated by IC and registered in Toulouse DATAML from 01/2015 to 05/2020. As of March 2018, as part of the SC for AML pts treated by IC and in the absence of contra indication (renal lithiasis, renal insufficiency, G6PD deficiency, hypercalcemia), vit C (1g x 2/d, three times a week ; IV) and vit D (100,000 IU/week ; PO) have been administered from d10 until hematologic recovery of induction and consolidation. The principal objective of this retrospective study was to assess the impact of the vit supplementation on EFS. Secondary objectives were response to induction, early death, OS, RFS, incidence of relapse, vitamin levels during treatment and safety. Results 431 pts were included, 262 without (from 01/2015 to 02/2018 ; control group) and 169 with vit C/D supplementation (03/2018-05/2020 ; vitC/D group). Data were updated on 03/03/2022. The median FU was 45.6 months. Pt characteristics were well balanced except for age, PS and creatinine (Table 1). In the vitC/D group, the median plasma level of vit C was 3.20 mg/L (range, 0.47-15.42) at diagnosis and 4.62 mg/L (range, 0.18-18.1) at time of hematologic recovery from induction (P=0.18). The level of vit D at diagnosis was 16 ng/mL (range, 4-56) and 18 ng/mL (range, 4-42) in the control and vitC/D groups, respectively. Upon recovery from induction, vit D levels significantly increased compared to diagnosis in the vitC/D group (median, 39 ng/ml, range, 5-93 ; P<0.0001). CR/CRi rate was 84.4% (control) and 84% (vitC/D) (P=0.93). Day-30 death rate was 4.2% (control) and 2.4% (vitC/D) (P=0.31). During induction, the rates of grade 3-4 bacterial infections (vitC/D, 27.2% vs control, 35.1%, P=0.086), fungal infections (10.1 vs 18.3%, P=0.019), hemorrhages (1.8 vs 5.7%, P=0.045) or macrophage activation syndrome (1.8 vs 8.8%, P=0.002) were lower in the vitC/D group. Median level of day-21 calcemia was higher in the vitC/D group (2.48 mmol/L vs 2.44, P=0.003). Grade 2 hypercalcemia was observed in only 1 patient of the control group. Allo-SCT was performed in 37 (26.1%) and 75 (33.9%) pts of the vitC/D and control groups, respectively. Median pretransplant vit D levels were significantly higher in the vitC/D group (33 vs 19 ng/mL, P<0.0001). There was no significant difference in EFS (vitC/D, median 13.9 vs 17.4 months, P=0.26), RFS (15.5 vs 23.5, P=0.24) and OS (30.8 vs 34.5, P=0.80) between both groups. However, multivariate analysis for OS showed a significant interaction between vitC/D and NPM1 mutation meaning that vitC/D supplementation was significantly and independently associated with better OS in pts with NPM1 mutations (HR, 0.52 ; 95%CI: 0.30-0.90, P=0.019) but not in NPM1 wildtype pts (HR, 1.01 ; 95%CI: 0.68-1.51, P=0.95). MV analysis for EFS, RFS and CIR did not find any significant interactions. Conclusion Vit C and D supplementation is feasible and safe in AML pts during IC. Normal Vit D levels can be restored before transplant, which may be important. The potential impact of vit C/D supplementation in AML with NPM1 mutations needs further studies and pave the way for exploratory analyses to explain how the VDR pathway and/or vit C activity may interact with the biology of NPM1 mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1b Dose Escalation and Expansion Study of SNDX-5613, Azacitidine (AZA) and Venetoclax (VEN) in Newly Diagnosed, Patients ≥ 60 Years with Untreated NPM1-Mutated/ FLT3-Wild Type AML or KMT2A-Rearranged Acute Myeloid Leukemia (AML)

Background: SNDX-5613 is a potent, orally available, small molecule inhibitor of menin, which binds to MLL1, encoded by the KMT2A gene, and MLL1 fusion proteins. Menin expression leads to aberrant gene transcription through its activation of Homeobox-related genes, leading to maturation arrest and leukemogenesis in KMT2A-rearranged and NPM1-mutated leukemias. Inhibition of menin activity significantly blocks the transcriptional signature and subsequently the proliferative capacity of leukemia cells harboring NPM1 mutations or KMT2A rearrangements. SNDX-5613 and its analogs have demonstrated robust single-agent anti-proliferative activity in NPM1-mutated AML cells and AML patient-derived xenograft models. Currently, SNDX-5613 is being explored as a single agent in a phase 1/2 study in patients with NPM1-mutated or KMT2A-rearranged relapsed/refractory AML, mixed phenotypic acute leukemia and acute lymphoblastic leukemia. The primary objective of this study is to determine the safety and recommended dose of SNDX-5613 combined with AZA and VEN in untreated AML patients age ≥ 60 years who are not candidates for intensive chemotherapy. Study Design and Methods: This study is a sub-study of the BeatAML Master Trial (NCT03013998) in which untreated AML patients age ≥ 60 are assigned an investigational therapy based on cytogenetic and central genomic analysis. Patients must specifically harbor an identifiable NPM1 mutation without a concomitant FLT3 mutation or have a KMT2A rearrangement to be eligible. In this phase 1b study, the primary objective is to determine the safety and recommended dose using a standard 3+3 design based on dose-limiting toxicities with 3 dose levels (DL) of SNDX-5613 to be evaluated in combination with VEN/AZA and an anti-fungal (required during induction): 75mg (DL-1), 113mg (DL1), and 163mg (DL2). In continuation phase with anti-fungals not required, SNDX-5613 will be dosed at 113mg (DL-1), 163mg (DL1), or 226mg (DL2) with appropriate dose reductions added for both SNDX-5613 and VEN if continuing on anti-fungals. The secondary objectives are to assess the overall response rate, measurable residual disease (MRD) status, overall survival, transplant rate and duration of remission. During induction cycles, VEN (D1-28) and AZA (D1-7) are dosed per standard of care every cycle. SNDX-5613 is dosed continuously orally twice daily with dose levels listed above. In patients who achieve a morphologic remission (<5% blasts), SNDX-5613 will be held if absolute neutrophil count (ANC) is < 0.5x109/L and/or platelets (PLT) < 50x109/L. Patients can receive up to 3 induction cycles to achieve a composite complete remission (CRc: CR, CRi, CRh). Patients can receive continuation therapy with AZA, VEN and SNDX-5613 once they achieve CRc and after hematologic recovery. Following the determination of a safe and recommended dose in phase 1b, there will be a dose expansion cohort of 12-20 patients following the same schedule as described above (Figure 1) to further evaluate safety, tolerability and correlative biomarkers. Correlative objectives include assessment of SNDX-5613 pharmacokinetics and its correlation with response and toxicities. Additionally, the study will evaluate the impact of MRD, as measured by central flow cytometry and next-generation sequencing of NPM1 mutations. Additional evaluations and biomarkers of potential interest include menin- KMT2A-specific transcriptomic changes, myeloid differentiation/cell subset changes, mechanisms of resistance, and discovery of baseline features (methylation, mutations, etc.) that can impact outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The FLT3-ITD Allelic Ratio and NPM1 Mutation Do Not Impact Outcomes in AML Patients with FLT3-ITD after Allo-HSCT: A Retrospective Propensity-Score Matching Study

Introduction: FLT3-ITD occurred in about 25% of adult patients with acute myeloid leukemia (AML), which has been correlated with poor outcomes mainly in cytogenetically normal AML, thus being one of the most frequently affected genes in AML. Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eliminate the detrimental effects of FLT3-ITD mutation in AML patients remain debatable. Studies from our group and others showed that allo-HSCT may overcome the adverse effect of FLT3-ITD on survival outcomes, comparable outcomes were not confirmed in other studies. Additionally, high allelic ratio was correlated with inferior outcomes while NPM1 mutation was associated with favorable outcomes in several studies, but their association with prognosis was not observed in other studies. Previous study from our group showed comparable outcomes between FLT3-ITDmut and FLT3-ITDwt patients, however, the influence of NPM1 mutation and allelic ratio on FLT3-ITDmut patients remains unclear in our database. Hence, we designed a retrospective matched-pair study to compare the survival outcomes between FLT3-ITDmut and FLT3-ITDwt patients who underwent allo-HSCT and further analyzed the impact of NPM1 and allelic ratio on outcomes. Methods: 118 FLT3-ITDmut patients and 497 FLT3-ITDwt patients undergoing allo-HSCT were observed. Each FLT3-ITDmut patients was matched with three FLT3-ITDwt patients using the nearest neighbor-matching method with a caliper size of 0.2. Age and sex of patients, disease status, cytogenetics, pre-MRD, and follow-up days were included in the propensity score model. Results: 116 FLT3-ITDmut patients and 314 FLT3-ITDwt patients were included with a median (range) age of 35 years (1-64). FLT3-ITDmut patients showed higher WBC counts at diagnosis (P<0.001) and NPM1 mutation rate (P<0.001). CIR and LFS of FLT3-ITDmut patients were similar to FLT3-ITDwt patients (CIR:16.9% vs 14.2%, P= 0.266; LFS: 75.1% vs 77.7%, P= 0.399). 32 patients and 41 patients showed NPM1 mutation in FLT3-ITDmut and FLT3-ITDwt patients, respectively. We divided them into four groups based on the status of FLT3-ITD and NPM1 status, no significant difference in CIR and LFS was observed between FLT3-ITDwt NPM1wt, FLT3-ITDwt NPM1mut, FLT3-ITDmut NPM1wt and FLT3-ITDmut NPM1mut patients (FLT3-ITDwt NPM1wt vs FLT3-ITDwt NPM1mut vs FLT3-ITDmut NPM1wt vs FLT3-ITDmut NPM1mut: CIR: 14.8% vs 9.4% vs 16.4% vs 17.8%, P= 0.570; LFS: 76.9% vs 84.4% vs 79.5% vs 67.1%, P= 0.176). 49 patients with available allelic ratio were divided into low allelic ratio (n=25) and high allelic ratio groups (n=24) according to a cut-off 0.50. High allelic ratio was not correlated with inferior outcomes in this study (high allelic ratio vs low allelic ratio: CIR: 21.1% vs 18.1%, P= 0.617; LFS: 70.6% vs 73.7%, P= 0.563). To further analyze the differences between different donor type, we compared the outcomes of 318 haploidentical transplantation and 98 sibling-matched transplantation patients, respectively. FLT3-ITDmut patients had higher CIR and lower LFS than FLT3-ITDwt patients among sibling-matched transplantation (CIR:24.7% vs 12.0%, P= 0.064; LFS:68.2% vs 83.5%, P= 0.0502), but no difference was observed in haploidentical transplantation (CIR: 13.7% vs 15.0%, P= 0.996; LFS:77.8% vs 75.9%, P= 0.885). FLT3-ITD mutation didn't become a prognostic factor in survival outcomes when all patients received transplantation. Prognostic factors with P< 0.1 in univariate analysis were included in multivariate analysis. Apart from pre-MRD, and disease status influencing OS and CIR, another factor affecting OS was the gender of patients. Age played a prognostic role in NRM while pre-MRD and disease status also became the significant factors influencing LFS. Conclusion: Our results showed that allo-HSCT, especially haploidentical HSCT, may overcome the adverse effect of FLT3-ITD mutation, regardless of the NPM1 status or allelic ratio. Allo-HSCT could be an ideal option for AML patients with FLT3-ITD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Improved Post-Transplant Outcomes in Recent Years for AML Patients with Intermediate Karyotype, FLT3-ITD and Wild Type NPM1: A Report from the EBMT Acute Leukemia Working Party

Rationale: Allogeneic hematopoietic cell transplantation (allo-HCT) is recommended in first complete remission (CR1) in acute myeloid leukemia (AML) patients harboring FLT3 internal tandem duplication (FLT3-ITD). However, historically, long-term survival has been poor due to early relapse, particularly for patients with wild-type NPM1. Recently, FLT3 inhibitors in combination with chemotherapy before allo-HCT, or as post-transplant maintenance, have significantly reduced the risk of relapse and improved survival. Here, we assessed real-world changes over time in transplant characteristics and post-transplant outcomes in young AML patients with FLT3 ITD and either NPM-1 mutated or wild type, using a large dataset from the European Society of Blood and Marrow Transplantation (EBMT) registry. Patients and Methods: We identified 1827 adult AML patients (54% female; median age 49 years, range 18-60) with FLT3 ITD, intermediate karyotype and available NPM1 mutation status, allografted between 2012 and 2021 in CR1 from a matched related (32%), matched unrelated (56%) or haploidentical donor (12%). NPM1 was mutated in 72% of patients. At transplant, 834 patients were measurable residual disease (MRD) positive, 464 MRD negative, and 529 were not evaluated or missing. Comorbidity index (CI) was zero in 59% of patients with available data. Conditioning was myeloablative (MAC) in 64% of patients. In vivo T cell depletion (TCD) graft was given to 62% of patients, post-transplant cyclophosphamide (PTCy) to 17%, and 87% received peripheral blood stem cells (PBSC). Most patients (66%) and donors (54%) were cytomegalovirus (CMV) positive. Median follow-up of alive patients was 30 months (IQR 28-32). Results: We compared changes in patient and transplant characteristics over time in 688 (38%) patients transplanted between 2012 and 2016 and 1139 (62%) patients transplanted between 2017 and 2021. Patients transplanted in recent years had a shorter follow up, were more likely to have NPM1 mutation, to be MRD positive, to have a higher CI, to receive a transplant from a haploidentical donor, a CMV positive donor, and to receive PBSC and PTCy. For patients with wild-type NPM1, day 100 acute graft-versus-host disease (GVHD) grade II-IV and grade III-IV were encountered in 29% and 11% of patients transplanted in 2021-2016, respectively, and 21% and 7% for patients transplanted in 2017-2021, respectively. The 2-year cumulative incidence of chronic and extensive chronic GVHD were 32% and 16%, respectively for 2012-2016 and 36% and 20%, respectively for 2017-2021. Over time, the 2-year cumulative incidence of relapse (CIR) significantly decreased from 43% to 31% between the two time periods (p<0.04) and non-relapse mortality (NRM) significantly decreased from 14% to 8% (p<0.05). The 2-year leukemia free survival (LFS) and overall survival (OS) significantly improved over time from 54% to 64% (p<0.02) and from 63% to 71% (p<0.05) respectively, whereas GVHD-free, relapse-free survival (GRFS) improved from 39% to 46% (p=0.08). In multivariate Cox analysis (MVA), transplantation in recent years significantly reduced the CIR (hazard ratio [HR]=0.65; p<0.01), and acute GVHD grade II-IV (HR=0.6; p<0.01) and significantly improved LFS (HR=0.67; p=0.02), OS (HR=0.66; p<0.03), and GRFS (HR=0.77; p<0.05). The use of unrelated donors increased the risk of acute GVHD but reduced the CIR whereas the use of haploidentical donors increased NRM. In vivo TCD reduced the risk of chronic GVHD whereas female donors to male recipients increased it. PTCy decreased the risk of NRM whereas MAC reduced the CIR. For patients with NPM1 mutation, no significant changes over time in post-transplant outcomes were noted except for decreased NRM from 15% for 217 patients transplanted in 2012-2016, to 10% for 288 patients transplanted in 2017-2021 (p<0.02). In MVA, transplantation in recent years did not significantly affect any of the post-transplant outcomes. Conclusion: In AML patients with FLT3 ITD and wild-type NPM1, we noticed a significant decrease over time in the CIR and a significant improvement of LFS, OS and GRFS, likely reflecting the efficacy of FLT-3 inhibitors, including when used as post-transplant maintenance, in this high-risk setting. On the contrary, no significant change over time was noticed in post-transplant outcomes of patients harboring NPM1 mutation.

Phase 1/2, Open-Label, Dose Escalation, Dose Expansion Study of Menin Inhibitor DSP-5336 in Adult Patients with Acute Leukemia with and without Mixed-Lineage Leukemia (MLL)-Rearrangement (R) or Nucleophosmin 1 (NPM1) Mutation (m)

Patients who relapse after initial acute leukemia (AL) treatment have median survivals <6 mo for AML and <1 y for ALL, especially AML or ALL with MLL rearrangements (MLLr) or relapsed AML with NPM1 mutations. Translocations of the MLL gene (5% to 10% of adult patients) produce an aggressive subtype, with a median overall survival (OS) of approximately 9 months. The NPM1 mutation (NPM1m) occurs in 30-35% of adult de novo AML and is associated with favorable prognosis. However, 40-50% of these patients have concurrent FLT3-internal tandem duplication mutations negating this favorability. DSP-5336, a novel oral small-molecule, was developed to prevent menin protein binding to MLL fusion proteins that drive leukemogenesis. Nonclinical studies of DSP-5336 showed selective growth inhibition against human AL cell lines with MLLr or NPM1m. Evidence of antitumor activity and survival advantage was demonstrated in 3 AML xenograft models with MLLr and/or NPM1m. Treatment with DSP-5336 may reduce leukemic cells and benefit patients with relapsed or refractory (R/R) MLLr or NPM1m AML or pts with R/R MLLr ALL. An open-label, single-arm, phase 1/2 study (NCT04988555) will evaluate the safety and efficacy of DSP-5336 and determine the recommended phase 2 dose (RP2D) in adults aged ≥18 y with relapsed/refractory AML or ALL after at least one line of therapy. Key inclusion criteria are ECOG performance status 0-2 and adequate organ function. Patients will be dosed twice daily in 28-day cycles. In phase 1, 21-30 pts will be enrolled into multiple dose cohorts (1-6 pts per dose level), with dose escalation determined using a two-parameter Bayesian logistic regression model. The RP2D will be informed by the maximum biologic effect or maximum tolerated dose, whichever is lower. The phase 2 study will include two arms (relapsed or refractory [R/R] AML with MLLr and R/R AML with NPM1m). Patients will be treated at the RP2D to evaluate clinical activity and safety, monitored using Bayesian posterior probability to optimize enrollment, with Bayesian stopping rules. Bayesian monitoring of responses begins after the first 10 enrolled patients are evaluable for efficacy. This study is recruiting in the United States and Japan. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NPM1 Mutation Quantitation Evaluated By a Commercially Available Assay and the Cepheid Prototype‡ Xpert® NPM1 Mutation Assay

Aims The objective of this study was to compare the performance of the novel Cepheid Prototype Xpert® NPM1 Mutation Assay to the Qiagen ipsogen® NPM1 mut A, B&D MutaQuant Kits using samples contrived using cell line-spiked peripheral blood. Our goal was to evaluate the comparative performance of a faster and easier to use assay than commercially available products for use in the monitoring of minimal residual disease (MRD) in NPM1-positive AML patients. Background A common mutation in AML (20-30% of cases) is a four base pair insertion in the NPM1 gene.¹ NPM1 plays a role in ribosome biogenesis, maintaining genomic stability, and modulating growth suppression by binding and stabilizing p53.² NPM1 mutant A consists of about 80% of NPM1 mutations, while mutants B or D are present in a sizeable minority of patients.¹² Monitoring of NPM1 mutations can provide prognostic value and can assist with choice of treatment.³ Methods Three NPM1-positive cell lines, OCI-AML3 MutA, OCI-AML3 MutB, and OCI-AML3 MutD, which express NPM1 MutA, NPM1 Mut B, and NPM1 Mut D, respectively, were grown and isolated. After processing the cells into cell lysate, the cell-lysates were spiked into NPM1-negative EDTA whole blood lysate and serially diluted to target 13 or 14 NPM1/ABL levels from approximately 2500% (log reduction (LR) 1.4) to approximately 0.01% (LR -4.0). A subset of each level underwent hands-off sample preparation, total RNA extraction, and RT-qPCR all within the Cepheid GeneXpert automated cartridge system, and another subset had total RNA extracted using the Zymo Direct-zol™ RNA Miniprep Plus Kit. RNA was qualified and quantified using the Thermo Scientific NanoDrop™ 2000 Spectrophotometer before being tested per the comparator assay vendor's recommendations. The Cepheid Prototype assay utilized 4.5mL of lysate, whereas the Qiagen kits used 100 ng of RNA per test. Specimens spiked with cell lines expressing different NPM1 mutations were tested using the respective ipsogen kit designed for that mutation. The Cepheid Prototype assay was evaluated against the comparator assay for each subtype using Deming regressions. Bland-Altman plots were utilized to evaluate bias between assay quantitation outputs. Results Deming regressions and associated Bland-Altman plots showed a high degree of correlation and agreement between comparator assays and the Cepheid Prototype assay (Figure 1). Conclusions The novel Cepheid Prototype assay showed a high level of agreement with Qiagen kits in this study using whole blood EDTA lysate samples spiked with three NPM1-positive cell-lysates. Agreement tended to decline at low input levels, and a small but acceptable degree of nonlinearity was present in MutA and MutD. Total time to result (TTR) was minimized in the Cepheid Prototype assay to under 170 minutes, while the comparator assay had a TTR of approximately six hours plus RNA extraction. While RNA extraction is performed within Cepheid Prototype assay cartridge and is included in this 170-minute TTR, the Qiagen kit requires additional expensive and labor-intensive extraction steps that can last one-hour to multiple days, which are not included in the six-hour TTR estimate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Menin Inhibitor DS-1594b Drives Differentiation and Induces Synergistic Lethality in Combination with Venetoclax in AML Cells with MLL-Rearranged and NPM1 Mutation

Introduction: Mixed-lineage-leukemia (MLL) is one of the most frequently translocated genes in hematologic malignancies, which produces aggressive leukemias that are refractory to conventional treatment resulting in a 5-year survival rate of only 35%. MLL translocations lead to fusion of the N-terminal fragment of MLL with one of over sixty protein partners, producing MLL fusion proteins that enhance proliferation, block hematopoietic differentiation, and drive leukemia development. Acute myeloid Leukemia (AML) is a heterogeneous and rapidly progressing blood cancer with a poor prognosis, especially in elderly patients where it is more common. Mutations in the nucleophosmin (NPM1) gene are one of the most common causes of AML. Interestingly, the protein-protein interaction between Menin, MLL and MLL fusion proteins plays a critical role in the pathogenesis of acute myeloid leukemias, both with MLLr as well as in leukemias carrying mutations in NPM1. Inhibition of the MLL-Menin interaction with novel Menin inhibitors has shown efficacy in both AML subsets, in pre- clinical and clinical studies. However, the responses are short-lived and hence it is imperative to identify therapeutic approaches that may synergize with Menin inhibition. Our recent work showed that MLLr t(4;11) Acute Lymphoblastic leukemia () patient cells express high levels of the anti-apoptotic BCL-2 protein and are highly sensitive to treatment with the BCL-2 selective antagonist, venetoclax (Benito, J.M., et al , Cell Reports, 2015). Here, we report experiments to establish the efficacy of the novel Menin-MLL inhibitor DS-1594b which was developed by Daiichi Sankyo co., jp, in combination with the BCL-2 antagonist venetoclax (VEN) in MLLr and NPM1-mutated acute leukemias. Methods: The efficacy of DS-1594b/VEN combination in MLLr and NPM1mut AML cell lines and primary leukemia samples was evaluated by examining the cell viability in response to serial dilutions of tested compounds as single agents or in combination using Cell Titer Glo (CTG) assay at 3, 5 and 7 days of treatment. Induction of apoptosis was assessed by Annexin V/ staining. Since down-regulation of MLL fusion target genes leads to differentiation in MLL leukemic cells, we monitored cell differentiation by flow cytometry in AML cell lines (MLLr: OCI-AML2, MOLM13, MOLM14, MV411. NPM1 Mut: OCI-AML3 . Leukemia cell lines with no MLLr or NPM1 mutations: HL60, U937) and primary leukemia samples ( blood from relapsed MLLr and NPM1mut AML) using differentiation markers CD11b, CD14, CD15. MLLr cell line MOLM13 resistant to venetoclax was also included in these studies. Results: 7 days treatment with DS-1594b reduced viability in MLLr and NPM1mut cell lines at low nM concentrations but did not affect growth of negative control U937 and HL60 . DS-1594b induced differentiation in OCI-AML3 and Ven resistant cell after 5 days and in other lines after 10 days (figure 1A). DS-1594b did not induce apoptosis when used as a single agent in any AML cell line tested indicating that differentiation is a key mechanism of Menin inhibition. Five days of treatment with single agent showed a downregulation of Menin in all cell lines and downregulation of BCL2 in OCI-AML3 and OCI-AML2 in Western blot analysis. Remarkably, combination with Venetoclax induced synergistic lethality and growth inhibition in all cell lines. Most importantly, was observed in primary leukemia samples (Figure 1B), with differentiation effects after DS-1594b single agent (bottom). Conclusions: Here we show in vitro efficacy of DS-1594b, a selective small molecule menin inhibitor, as single agent or in combination with Venetoclax in MLLr and NPM1 mutated cell lines and primary leukemia samples. Preliminary data demonstrate differentiation effects of DS-1594b as single agent in both cell lines and primary AML leukemia samples. Combination effects show synthetic lethality in almost all cell lines except OCI-AML3. The combination elicited profound apoptosis in primary leukemia samples. In vivo studies using MLLr and NPM1 PDXs models are ongoing and will be presented. Overall, the preliminary findings presented here pave the way for further studies aimed at understanding mechanisms of and further development and testing of this combination in AML with MLLr and/or NMP1 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of Patient-Level Data from 3 Cooperative Group Trials Confirms a Survival Advantage for NPM1m Patients Achieving MRD-Negative CR after Intensive Induction

Background:NPM1 mutations (NPM1m), predominantly tetra-nucleotide insertions in exon 12, are clonal driver events in acute myeloid leukemia (AML) and are found exclusively in leukemic cells, making them ideal for sensitive detection of measurable residual disease (MRD) using quantitative polymerase chain reaction (qPCR)- or next generation sequencing (NGS)-based methods. Over 50 peer-reviewed publications across multiple cooperative group trials have shown that NPM1m AML patients in complete remission (CR), CR with incomplete count recovery (CRi), or CR with partial hematologic recovery (CRh) have significantly better survival if they are MRD negative compared to MRD positive. Based on this body of evidence, the European LeukemiaNet recommends monitoring molecular MRD in NPM1m AML patients during treatment to help guide treatment decisions. Purpose: To provide further evidence for the value of NPM1 MRD as a surrogate endpoint for prospective, randomized trials in patients receiving novel agents in combination with intensive chemotherapy by evaluating the relationship between MRD negativity in patients with CR after 2 cycles of chemotherapy and event-free survival (EFS) and overall survival (OS) in a pooled analysis of patient-level data from 3 large cooperative group trials. MRD in all 3 studies was assessed using reverse transcriptase-mediated qPCR (RT-qPCR) of NPM1m transcripts normalized to ABL transcripts. Methods: Deidentified patient data for a total of 1,128 patients who achieved CR, CRi, or CRh, and who had MRD data after 2 cycles of chemotherapy, were provided by the UK National Cancer Research Institute AML Study Group (SG) for the AML17 (N = 539), the AMLSG for the AMLSG 09-09 (N = 497), and the Study Alliance Leukemia (SAL) for the AML2003 (N = 105) trials and standardized using Standard Data Tabulation Models. Data included demographic information, disease history, induction and consolidation treatment received, morphologic response, MRD data after 2 cycles of treatment, relapse, and survival. Cutoffs for MRD negativity were defined according to the definitions used in the individual studies (Ivey A, et al. N Engl J Med. 2016; Kapp-Schwoerer S, et al. Blood. 2020; Shayegi N, et al. Blood. 2013). In addition, sensitivity analyses were conducted using a range of cutoff values. CR was defined as <5% blasts in the bone marrow (BM) with count recovery, occurring within 42 days of the start of the last induction cycle; only these patients were included in the MRD analysis. EFS and OS were estimated using the Kaplan-Meier method and defined according to FDA criteria. Briefly, EFS was measured from day 1 of randomization to the date of treatment failure, hematologic relapse from CR or death from any cause; treatment failure was defined as not achieving CR after two cycles of chemotherapy. OS was measured from day 1 to the date of death from any cause. The absolute risk difference among MRD response status groups in EFS/OS at the 2-month, 1-year, 2-year, 3-year, and 5-year landmarks and their corresponding 95% confidence intervals (CI) were calculated using complementary log-log transformation. Hazard ratios (HR) were fitted using a Cox regression model with EFS/OS as the dependent variable and with MRD-response status and study as independent variables. Results: Among the 515 patients who achieved a CR within 42 days at the start of chemotherapy Cycle 2, MRD negativity confers a substantial survival advantage with EFS HR (95% CI) = 1.6 (1.1-2.3) when measured in BM and 1.8 (1.4-2.4) in peripheral blood (PB) using the study-defined cutoffs for positive and negative. This advantage is also observed using cutoff definitions of MRD negativity ranging from <10 to <1,000 NPM1m/104ABL transcripts. A survival advantage for OS was also observed with HR (95% CI) = 1.5 (1.0-2.4) and 1.7 (1.2-2.3) in BM and PB, respectively. Analysis of the predictive value of MRD negativity in the 613 patients who achieved CRi/CRh within 42 days of the start of Cycle 2 is underway. Conclusions: Analysis of pooled patient-level data confirms the survival advantage of achieving MRD-negative CR as previously reported in 3 independent, cooperative group trials using different induction regimens. The prognostic value of MRD negativity in NPM1m AML is consistent across a broad range of cutoff definitions in both BM and PB providing further evidence for the value of NPM1 MRD as a surrogate endpoint for clinical trials.

Comprehensive Age-Stratified Impact of NPM1 Mutation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with NPM1 mutation (MT) in the absence of FLT3-ITD is a favorable prognostic risk group in various stratification schemes, including the recently published machine learning-based, clinical-feature agnostic analysis of AML mutations (Awada et al., Blood 2021). However, prior large clinical trials based large registry data (Ostronoff et al., JCO 2015) observed that the positive prognostic impact of NPM1MT is not general as it does not seem to extend to elderly patients above 65 ys. in contrast to prior smaller studies. In this group, a formal, extensive analysis has not been performed on patients treated outside clinical trials. AML cohort from Karmanos Cancer Institute (n=52) was enhanced by a publicly available metanalytic cohort consisting of various sub-studies (Awada et al., Blood 2021) to a total of 2,165 AML patients to identify and validate the age-stratified analyses of the impact of NPM1MT in AML. Only patients whose NPM1 mutation status was available were included in the study. Baseline clinical and molecular characteristics were noted. Chi-square test was used to study various parameters as described, and Kaplan Meir curves were used for survival analyses. In total, 25% (533/2,165) of the patients carried NPM1MT. Male: Female ratio was 0.92. NPM1MT was enriched in primary (p) AML compared to NPM1WT [94 vs. 74%, p=0.0001], whereas there were less secondary (s) AML in NPM1MT compared to NPM1WT [6 vs. 26%, p=0.0001]. NPM1MT was less associated with abnormal karyotype versus NPM1WT [16 vs. 63%, p<0.0001]. Median variant allele frequency (VAF) in evaluable patients was 38% [range, 5-60%], and all mutations were canonical frameshift. Median overall survival (OS) was significantly better in NPM1MT compared to NPM1WT patients [24 vs. 17.5 mo., p=0.003]. When grouped by FLT3-ITD mutational status, NPM1MT patients had a better OS compared to NPM1WT in FLT3-ITDNEG group [29 vs. 17 mo., p=0.00045]. Whereas there was no difference in OS in the FLT3-ITDPOS group [15 vs. 21 mo., p=0.461]. NPM1MT patients with pAML had a better OS compared to NPM1MT patients with sAML [31 vs 9.6 mo., p=0.001]. When stratified by age groups, NPM1MT/FLT3-ITDNEG patients had a significantly higher OS than NPM1WT/FLT3-ITDNEG patients in the 55-65 year age group (75 vs. 20 mo., p=0.002). After splitting this group, the OS benefit was preserved in the 55-60 (58.5 vs. 16 mo., p=0.041) and 60-65 year group (mOS not estimable vs. 21 mo., p =0.011). There was no difference in OS for NPM1MT/FLT3-ITDNEG vs. NPM1WT/FLT3-ITDNEG patients in the age groups <55 years (not estimable vs. 155 mo. p=0.066), 65-75 years (13 vs. 13.1 mo., p=0.46), and >75 years (3.3 vs. 5.2 mo., p=0.32). NPM1MT patients with lesser co-occurring mutations had a greater OS [≤3 vs. >3 total mutations, 51 vs. 18 mo., p=0.046]. The median age was 60.5 years compared to 66.4 years in the two populations, respectively (p=0.001). The most common associated mutations in <65 vs. >65 year with NPM1MT were DNMT3A (38% vs. 43%, p=0.27), IDH2 (23% vs. 25%, p=0.61), TET2 (13% vs. 27%, p=0.0001), FLT3-ITD (21% vs. 16%, p=0.16), IDH1 (19% vs. 15%, p=0.25), NRAS (13% vs. 7%, p=0.03), SRSF2 (5% vs. 15%, p=0.0002), WT1 (10% vs. 4%, p=0.01), PTPN11 (5% vs. 8%, p=0.18), CEBPA (5% vs. 6%, p=0.63), and ASXL1 (1% vs 7%, p=0.0006). In comparing dominant vs. non-dominant NPM1MT, OS was not different in NPM1MT/FLT3-ITDNEG patients [46.5 vs. 21 mo. P=0.42); however, there was a trend towards better survival in the NPM1MT/FLT3-ITDPOS group [OS not estimable vs. 22 mo., p=0.06 resp.]. In sum, extensive analysis of the age-stratified impact of NPM1MT in AML revealed intricate details of prognostic groups. We validated the prior findings of the not favorable impact of NPM1MT in older (>65 ys.) AML patients. We also found no difference in the frequently associated negatively impacting (such as FLT3) mutations. Since this dataset broadly represents patients treated outside clinical trials, it underscores whether they were undertreated or truly ineligible for intensive induction therapy. If the former is true, older NPM1MT AML should be stringently selected for aggressive therapeutic or targeted interventions, which could enhance their survival in this otherwise positive prognostic group. Our ongoing analysis, to be presented at the ASH meeting, will determine whether the less intense therapy may be the culprit and identify any subgroup that would respond better. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Persistence of Mutations in Complete Remission Including DNMT3A, TET2 and ASXL1 Mutations Is Associated with Worse Prognosis in Patients with Acute Myeloid Leukemia Treated in ALFA 0702 Study

Background: Only few studies have validated the use of molecular minimal measurable residual disease evaluation using error-corrected NGS (NGS-MRD). The prognostic relevance of detection of isolated DNMT3A, TET2 or ASXL1 (DTA) mutations post-induction remains controversial. Benchmarking of NGS-MRD with respect to other molecular MRD tools (such as WT1 expression and NPM1 mutation) has yet to be conducted. Material and methods: All patients included in the ALFA-0702 study who reached cytological complete remission (CR) or CR with incomplete platelets recovery after one induction course and with available material were included (n=189). Standard NGS sequencing of 67 genes was performed at diagnosis. Error-corrected NGS was performed on post-induction bone marrow samples using a custom panel including all mutations identified at diagnosis (n=735 mutations in 51 genes). Median sequencing depth in CR was 24695x after UMI deduplication. A threshold of 0.1% VAF was set as cut-off value for NGS-MRD detection for all targets. WT1 expression data in CR were available in 100 patients with initial WT1 over-expression. NPM1-MRD was evaluated in 67 patients by error-corrected NGS. For this evaluation, one alternative read was considered sufficient to define positivity. Leukemia free survival (LFS) and overall survival (OS) probabilities were evaluated since CR. Results: Median age at diagnosis was 46 years. ELN2017 risk was favorable in 52 (27%) patients, intermediate in 75 (40%) and unfavorable in 60 (32%). With a median follow-up from CR of 47 months, median LFS and OS was 35 and 41 months respectively. At diagnosis, gene mutations were detected in 181 (95%) patients. The median number of mutations per patient was 4 (range 1-10). Ninety-one patients had no target detected (NGS-MRD neg) and ninety had at least one target detected (NGS-MRD pos) including 42 patients with detection of DTA mutations only (DTA-MRD). NGS-MRD pos patients were significantly older (p=0.0018) than NGS-MRD neg patients and harbored a higher median number of mutations at diagnosis (4 vs 3, p=0.0021). There were no significant differences in terms of demographics and clinical presentation (age, WBC, number of mutation) between patients with DTA-MRD and non-DTA-MRD patients. In univariable analyses, NGS-MRD pos was associated with a lower probability of LFS (4-years LFS: 44% vs 68%, p= 6e-04) and OS (4-years OS: 57% vs 81%, p= 9e-04). There was no impact of the initial number of mutations on LFS nor OS. There were no significant differences in LFS and OS between DTA-MRD and non-DTA-MRD patients (figure 1). When adjusting analysis on ELN2017 status, MRD-NGS pos remained associated with shorter LFS (HR 2.15[1.34-3.44] , p=0.001) and OS (HR 2.41[1.38-4.20] p=0.002). As a continuous variable, the number of mutations detected in CR was associated with shorter LFS and OS. Specifically, detection of ≥2 mutations predicted poorer LFS (HR, p=3e-05) and OS (HR, p=4 e-05), compared with detection of only 1 mutation (figure 2). Sixteen patients were positive for WT1 MRD evaluation, and 84 were negative (WT1 neg). Among the WT1 neg patients 37 were NGS-MRD pos and 47 were NGS-MRD neg. When adjusting for both ELN17 and WT1 expression in CR, there was no significant impact of MRD-NGS positivity on LFS (HR=1.62 [0.85 - 3.09]) nor OS (HR=1.28 [0.59 - 2.74]) In the 67 patients with NPM1 mutation, 41 patients had NPM1 undetectable MRD (including 21 NGS-MRD pos and 20 NGS-MRD neg) and 26 had detectable NPM1-MRD (including 16 NGS-MRD pos and 10 NGS-MRD neg). When adjusting for both ELN status and NPM1-MRD, NGS-MRD pos was associated with a non-significant trend towards lower LFS (HR=2.34[0.96-5.71] p=0.063). Discussion and conclusions: In the homogeneously treated ALFA-0702 population, MRD-NGS positivity after the first induction course predicts shorter LFS and OS independently of ELN17 risk stratification. Detection of a higher number of mutations in CR is associated with a worse prognosis. Contrary to previous studies accruing older patients treated less intensively persistence of DTA mutations has similar prognostic relevance than non-DTA mutations. Future studies will be necessary to determine the superiority or addivitity of NGS-MRD with respect to available molecular MRD tools. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Expression of mi-RNAs Involved in the Hematopoietic Niche Microenvironment in Newly Diagnosed AML Patients

Introduction Acute myeloid leukemia (AML) is a genetically heterogeneous disease and the most common type of acute leukemia in adults. Despite the progress made in the last years in AML therapy, induction treatment failure and disease relapse still remain the main challenges in clinical practice. miRNAs are involved in the pathogenesis of AML as well as influence the differentiation and proliferation of normal hematopoietic cells. The expressions of specific miRNAs have been identified as potential biomarkers for predicting prognosis both in solid tumors and hematological malignancies. Methods The expression of 12 miRNAs involved in hematopoiesis and the microenvironment of the hematopoietic niche: miR-15a-5p, miR-34a-5p, miR-125b-5p, miR-139-5p, miR-146-5p, miR-181a-5p, miR-199b-5p, miR-204-5p, miR-218-5p, miR-299-5p, miR-424-3p, miR-452-5p was determined in duplicates using qPCR in bone marrow samples of newly diagnosed AML patients. The miRCURY LNA SYBR® Green PCR Kit (Qiagen) was used to carry out qPCR. miRNAs expression was normalized using the mean expression of all miRNAs in a given sample. The normalization was performed using the formula ∆Ct = Ct(reference) − Ct(miRNA of interest). This approach results in higher values for higher miRNA expression. Differential expression analysis was carried out using a t-test. Cox proportional hazard regression analyses of clinical factors and miRNAs for predicting overall survival in AML patients were performed. Results To the study group we enrolled 45 (23 women and 22 men) newly diagnosed AML adults with the mean age 45.1 ± 12.8 years. Among them, 13.9% patients (pts) had a low-risk, 44.2% intermediate-risk and 41.9% high-risk AML according to EuropeanLeukemiaNet (ELN) 2017 risk stratification (Döhner et al. 2017). In the study group, 16.7% of pts had NPM1 mutation, whereas 30.2% of pts had FLT3-ITD or FLT3-TKD mutation. The majority of patients (88.9%) received intensive treatment: DA-60 (daunorubicin 60mg/m2 + cytarabine 200mg/m2) regimen (50%), followed by DAC (DA-60 + cladribine 5mg/m2) (40%) and DA-90 (daunorubicin 90mg/m2 + cytarabine 100mg/m2) (10%). Azacitidine in standard dose was used as the low-intensity regimen. There were no statistically significant differences in miRNAs expression according to complete remission (CR) achievement. Patients with high-risk AML had higher expression of miR-125b-5p (fold change- FC=2.3, p=0.017) and lower expression of miR-34a-5p (FC=0.3, p=0.029), miR-199b-5p (FC=0.5, p=0.039) and miR-15a-5p (FC=0.6, p=0.043) as compared to low- and intermediate-risk pts. In patients with NPM1 mutation, miR-34a-5p, miR-146a-5p and miR-15a-5p were significantly upregulated, whereas miR-139-5p, miR-424-3p and miR-452-5p were downregulated. FLT-3-mutated patients had significantly lower expression of miR-424-3p (FC=0.2, p=0.019) comparing to FLT-3-unmutated controls. In patients with unfavorable karyotype, upregulation of miR-125b-5p (FC=2.8, p=0.002) and miR-139-5p (FC=3.0, p=0.027) was observed meanwhile lower expression of miR-34a-5p (FC=0.3, p=0.035) in these patients was detected. The median overall survival (OS) in the study cohort was 12.3 months. In univariate Cox regression analysis, hematopoietic cell transplantation-comorbidity index (HCTCI) score >0 (HR 2.86, 95%CI: 1.15-7.12, p=0.024), white blood cell (WBC) count at diagnosis (HR 1.01, 95%CI: 1.00-1.01, p=0.033), and higher expression (>median) of miR-199b-5p (HR 0.31, 95%CI: 0.12-0.81, p=0.027) (Fig.1) were factors influencing the outcome. In multivariate model, higher expression of miR-199-5p (HR 0.27, 95%CI: 0.08-0.91, p=0.036) retained their significant protective effect in the context of established prognostic factors. Conclusions Our preliminary results demonstrate that the expression of miRNAs involved in the microenvironment niche is deregulated in AML patients. The upregulation of miR-199-5p at the diagnosis is associated with longer overall survival of AML patients. Significantly lower expression of miR-199-5p was reported in ELN high-risk group. These data indicate that miR-199-5p may be a potential prognostic factor in AML patients. Larger studies are needed to confirm our observation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Functional Implications of MYC Variants As a New Class of Pathogenic Variants in AML

MYC, a well-known oncogene, is implicated in a majority of cancers, but there is little data on its role in myeloid malignancies. We recently described a novel internal tandem duplication of MYC gene (MYC-ITD) in a subset of patients with AML. As the prevalence of MYC aberrations have not been well documented in AML, we interrogated the genome, transcriptome and associated clinical and laboratory data from the largest cohort of pediatric AML patients to define the prevalence, frequency of co-occurring cytomolecular alterations and outcomes associated with MYC variants. Mutation Profiling: Diagnostic specimens from 1,806 patients with AML (age 0-29 years) enrolled on COG Phase III trials were retrospectively interrogated using contemporary NGS techniques or a PCR based "Tandem Repeat" assay for MYC-ITD. Conventional karyotyping and mutational analysis, including NGS, were used to determine additional cytogenetic and molecular abnormalities. Of the 1,806 patients studied, MYC aberrations were identified in 58 patients (3.2%) with 50% (N=29) harboring MYC-ITD, 46.5% (N=27) with a SNV or indel (MYCSNV/indel) and 2 patients (3.5%) with dual MYC-ITD and SNV. For MYC-ITD, tandem duplications occurred in exon 3 (Max binding domain) with ITD lengths ranging from 252-588 bp. The majority of MYCSNV/indel (77%) were identified at the P72-P78 hotspot with 33.3% (N=9) harboring the P74>L mutation and 18.5% (N=5) having the T73>N mutation. Functional Studies: Given the novelty of the ITD variant, we initially characterized the functional properties of MYC-ITD in vitro. Similar to wild-type (MYCWT), MYC-ITD is localized to the nucleus and requires Max to bind to specific DNA sequences (E-boxes). However, in contrast to MYCWT, MYC-ITD fails to recognize sequences containing a single E-box in vitro, but exhibits higher affinity toward sequences with two or more E-boxes. MYC-ITD is consistently more efficient in activating promoters with multiple E-boxes than MYCWT. Thus, MYC-ITD may possess different DNA-binding and protein-interacting properties, potentially leading to altered transcriptional regulation and biological output. Given the alterations in DNA binding properties, we evaluated the functional consequences of MYC-ITD in our cord blood stem cell/endothelial cell (EC) co-culture system. Normal cord blood stem cells were transduced with MYC-ITD vs. MYCWT and grown in an EC co-culture system. MYC-ITD transduced cord blood stem cells showed significantly higher proliferation compared to MYCWT or control GFP-transduced cells with proliferation advantage of 37-and 45-fold after 14 and 21 days in co-culture, respectively (p<0.001). Clinical Impact: Analysis of cooperating cytomolecular lesions revealed a striking difference between MYC-ITD and MYCSNV/indel. MYC-ITD was highly enriched in core-binding factor (CBF) AML found in 86% (N=25)[(N=18, 62%) with inv(16) and (N=7, 24%) with t(8;21)] (Figure 1A), vs. 26% in the MYCWT cohort and had a paucity of somatic mutations. Conversely, MYCSNV/indel was enriched in NUP98-NSD1 fusions (N=7, 27%), FLT3-ITD (N=10, 38%) and NPM1 mutations (N=9, 33%) (Figure 1A), which was significantly higher than the WT cohort. Patients with MYC-ITD had a morphologic CR rate after course 1 of 92.3% vs. 80% (p=0.249) in patients with MYCSNV/indel with a corresponding relapse rate of 24% and 52% (p=0.046). Lower CR and higher relapse reflects the higher proportion of patients with high-risk lesions (FLT3-ITD, NUP98-NSD1 and WT1) in MYCSNV/indel and favorable CBF in MYCITD. Those with MYC-ITD and MYCSNV/indel had a 5-year event free survival (EFS) of 70% vs. 38.5%, respectively (p=0.018) with a corresponding overall survival (OS) of 76% and 47% (p=0.023) similar to patients with favorable or high risk disease (Figure 1B). Conclusion: Here we define MYC aberrations as a new class of pathogenic variants in childhood AML. We demonstrate functional and clinical characterization of a novel MYC-ITD and contrast this novel variant to that of established pathogenic MYCSNV/indel. We show the high prevalence of co-occurring core-binding factor fusions with MYC-ITD, and high frequency of co-occurring NUP98-NSD1, FLT3-ITD and NPM1 mutations in MYCSNV/indel. Patients with MYC-ITD have superior clinical outcomes while patients with MYCSNV/indel have clinical outcomes similar to high-risk patients, likely due to the associations with the disease associated fusions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes with Allogeneic Hematopoietic Stem Cell Transplantation in NPM1-Mutated Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis

Introduction Acute myeloid leukemia (AML) has poor outcomes despite long term advancement in its diagnosis and treatment. The introduction of molecular genetic markers in AML has produced a major effect on therapeutic approach used in these patients. The genetic aberration in the Nucleophosmin (NPM1) gene is the most common mutation found in AML and is associated with "favorable" prognosis. Allogenic hematopoietic stem cell transplant (Allo-HSCT) is potentially a curative treatment option. Herein, we present a systematic review and meta-analysis with the aim of exploring outcomes with allo-HSCT in NPM1-mutated AML. Methods A detailed literature search was performed for the systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. A population, intervention, comparison, and outcome table were developed, and three electronic databases (PubMed, Cochrane Register of Controlled Trials, and Clinical trials.gov) were searched using MeSH terms and keywords for "Transplantation Hematopoietic Stem Cell" AND "Nucleophosmine Protein, Human" AND "Acute Myeloid Leukemia". A total of 137 articles were screened and original studies reporting patients with AML having NPM1 mutation who underwent Allo-HSCT were included while reviews, duplicate, and non-relevant articles were excluded. A total of twelve studies were included. Quality evaluation was done using the NIH quality assessment tool. The inter-study variance was calculated using Der Simonian-Laird Estimator. The pooled analysis was conducted using the 'meta' package by Schwarzer et al. in the R programming language (version 4.16-2). A random-effects model was used to estimate the proportions with 95% confidence intervals (CI). Results A total of 769 patients from twelve studies were included for this systematic review and meta-analysis. The median age of patients was 53 (21-79) years and 46.5% (303/651) were males as reported by ten studies. The donor type was matched related donor (MRD) 38%, matched unrelated donor (MUD) 61%, haploidentical 0.6%, and cord blood donor (CBD) 0.2%. The median follow-up was 46 (2-96) months. At a median follow-up of 2 (2-5) years, the pooled overall survival (OS) was 71% (95 % CI 0.66-0.73, p= 0.15, I2=31%, n= 710) while the pooled event free survival (EFS) and pooled relapse rate (RR) was 64% (95 % CI 0.59-0.68, p= 0.06, I2=45%, n= 741) and 25% (95 % CI 0.19-0.31, p <0.01, I2=70%, n= 695), respectively. The pooled incidence of acute graft versus host disease (GvHD) was 39% (95 % CI 0.16-0.65, p<0.01, I2=96%, n= 380) and the pooled incidence of chronic GvHD was 46% (95 % CI 0.35-0.58, p= 0.02, I2=71%, n= 301). The pooled incidence of non-relapse mortality (NRM) was 15% (95 % CI 0.08-0.23, p= 0.02, I2=63%, n= 298) at a median follow-up of 2 (2-5) years. Conclusion NPMI-mutated AML has favorable outcomes with allogeneic hematopoietic stem cell transplant and has good overall and event free survival with lower incidence of relapse and non-relapse mortality. Further randomized clinical trials are needed to consolidate these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Machine Learning Allows the Identification of New Co-Mutational Patterns with Prognostic Implications in NPM1 Mutated AML - Results of the European Harmony Alliance

Background: Mutations of NPM1 are generally considered as a favorable prognostic marker in acute myeloid leukemia (AML), although patients present with distinct co-mutations that might influence outcomes. The recently updated European LeukemiaNet classification (ELN2022) does now group NPM1mut AML patients with FLT3-ITD (regardless of the allelic ratio) as intermediate prognosis, while NPM1mut without FLT3-ITD is part of the favorable group. However, the mutational landscape of NPM1mut AML is more complex and analysis of a large cohort of patients might allow further dissection to identify less frequent co-mutational patterns of prognostic and predictive importance. Aims: To identify clinically significant co-mutational patterns in NPM1mut AML to further refine risk stratification models for this subset of patients. Methods: From the HARMONY Alliance database, a total of 1093 intensively treated NPM1mut patients were selected. Patients treated with targeted therapies (e.g. FLT3 or IDH1/2 inhibitors) were not included and NPM1 MRD information was not available at this stage of the analysis. A machine learning algorithm was developed in order to identify combinations of up to 4 co-mutated genes with potential impact on overall survival (OS). A heuristic search algorithm was implemented and bootstrap sampling was applied to estimate the impact of all possible gene combinations on OS. In addition, a global dashboard was developed where it was possible to compare mutational combinations with Kaplan Meier and Cox regression models. Finally, clinically significant co-mutational patterns were summarized in a novel risk stratification model for NPM1mut AML, which was validated using a publicly available external patient cohort (Awada et al, Blood, 2021). Results: The study population of 1093 NPM1mut AML patients included 57% females and median age was 53 years. Regarding ELN2022 classification, 57% of patients were classified into the favorable, 42% into intermediate and only 1% into adverse risk groups. The most frequent co-mutations were DNMT3A (54%) and FLT3-ITD (42%), followed by NRAS, FLT3-TKD and TET2 (20% each). Mutations on IDH1 (13%) and IDH2 (15%) showed a similar behavior for all the analyses performed, so for simplification purposes we will refer to IDHmut when any of them was mutated and to IDHwt when both were wildtype. The triple combination of NPM1mut + FLT3-ITD + DNMT3Amut identified a subgroup of patients with adverse prognosis (2-year OS of 33%), similar to patients with TP53mut. Of note, not all FLT3-ITD patients carried an intermediate or adverse prognosis, as we were able to identify a subgroup (FLT3-ITD + IDHmut + DNMT3Awt) with excellent prognosis (2-year OS of 80%), which represented 4% of NPM1mut AML. However, in the absence of FLT3-ITD and the presence of DNMT3Amut, the addition of IDHmut decreased OS towards the intermediate risk (2-year OS 59%). Notably, not all DNMT3Amut patients carried an intermediate or adverse prognosis. In the absence of FLT3-ITD and with IDHwt, mutations on either NRAS, KRAS, PTPN11 or RAD21 revealed a subgroup with favorable prognosis even when DNMT3A was mutated (2-year OS 80%) and this group represented 11% of NPM1mut AML. This information is summarized in a 4-category risk stratification model (figure 1). The revised NPM1mut favorable group presented with a 3-year OS of 78%, which was 63%, 48% and 29% for intermediate-1, intermediate-2 and adverse risk groups respectively (p<0.001). Regarding 3-year relapse free survival (RFS), it was 71%, 59%, 39% and 26% accordingly (p<0.001). In the validation cohort, 3-year OS of was 73% for NPM1mut favorable group, being 59%, 40% and 22% for intermediate-1, intermediate-2 and adverse groups respectively (p<0.001). Multivariate OS analysis in NPM1mut AML identified the following independent prognostic factors: revised NPM1mut model (taking favorable group as reference, HR 1.77 for intermediate-1, HR 2.92 for intermediate-2 and HR 5.13 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.93, p<0.001), age >60 years (HR 1.84, p<0.001), WBC at diagnosis >100x103/uL (HR 1.59, p<0.001), and male gender (HR 1.21, p=0.04). Conclusion/summary: Analysis of large NPM1mut AML cohorts allows the identification of clinically significant co-mutational patterns. We propose a new genetic stratification model for NPM1mut AML that identifies 4 groups with different OS and RFS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes of Patients with NPM1+ Acute Myeloid Leukemia (AML) Enrolled in the CONNECT® Myeloid Disease Registry

Background Mutations in NPM1 (NPM1+) occur in ∼30% of patients with AML. NPM1+ has been associated with favorable outcomes following intensive chemotherapy (IC) and demonstrated positive prognostic value in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) mutations. Recently, several studies have reported differences in outcomes in patients with NPM1 mutations based on age and treatment. In this analysis we evaluated treatment patterns and outcomes in AML based on NPM1 mutational status and primary induction therapy in patients who were enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011). Methods Patients aged 55 years or older with AML were grouped by known NPM1 status (NPM1+ vs NPM1‒) and induction therapy (non-IC vs IC). Overall survival (OS) and event-free survival (EFS) were evaluated using the Cox model adjusted for age. For the EFS analyses, an event was defined as death, disease progression, or refractory to therapy. Results A total of 483 patients with known NPM1 status were assessed (NPM1+, n = 123; NPM1‒, n = 360). Most patients were male (NPM1+, 61.5%; NPM1‒, 60.7%) and white (NPM1+, 78.0%; NPM1‒, 85.6%; Table). Median age, Eastern Cooperative Oncology Group performance status (ECOG PS), and frailty group were similar in the NPM1+ and NPM1‒ groups. Favorable and adverse European LeukemiaNet (ELN) risk scores at baseline were different in the NPM1+ and NPM1‒ groups. The majority of patients received non-IC regimens as induction therapy (NPM1+, 69.1%; NPM1-, 75.0%). Overall, patients with NPM1+ AML had significantly longer median OS (26 mo) compared with patients with NPM1- AML (15 mo) (HR [95% CI]: NPM1+ vs NPM1- 0.58 [0.44-0.76]; P < 0.01). When analyzed by type of induction regimen (non-IC vs IC), although OS remained numerically longer for patients in the NPM1+ groups, the differences were not statistically significant. Among patients who received non-IC regimens, median OS was 19 and 14 mo in the NPM1+ and NPM1- groups, respectively (Figure). Among patients who received IC regimens, median OS was 24 and 16 mo in the NPM1+ and NPM1- groups, respectively. There was a significant improvement in EFS for patients in the NPM1+ group vs the NPM1- group (20 mo vs 11 mo; hazard ratio [HR] [95% CI], NPM1+ vs NPM1- 0.61 [0.47-0.80]; P < 0.01). This was also true for patients treated with non-IC regimens (15 vs 11 mo in the NPM1+ and NPM1- groups, respectively; HR [95% CI], NPM1+ vs NPM1- 0.67 [0.46-1.00]; P = 0.048). In contrast with the non-IC treated regimens, in patients treated with IC regimens, NPM1+ status was not associated with significantly longer EFS compared to NPM1- status. Patients with known NPM1 status were further evaluated for additional mutations and the following differences were noted: the frequency of mutations in tumor protein p53 (TP53) (1.6% vs 18.6%), RUNX1 (0.8% vs 14.8%), ASXL1 (2.4% vs 17.6%) IDH2 (8.9% vs 15.7%), and NRAS/KRAS (8.9% vs 14.3%) was lower in patients with NPM1+ vs NPM1- AML, respectively, whereas the frequency of mutations in FLT3-ITD (35.8% vs 17.1%), FLT3- tyrosine kinase domain (TKD) (14.6% vs 6.2%), DNMT3A (23.6% vs 15.7%), and IDH1 (14.6% vs 8.6%) was higher in patients with NPM1+ vs NPM1- AML, respectively. Conclusions Overall, patients with NPM1+ AML had longer OS and EFS than patients with NPM1‒ AML. This was observed among patients with NPM1+ AML for both non-IC and IC induction regimens. Patterns of co-mutations differed between the NPM1+ and NPM1- groups and is a likely contributing factor to differences between outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Use of an Allogeneic Leukemia-Derived Dendritic Cell Vaccine in MRD+ AML-Patients Results in MRD Conversion, Improved Relapse-Free Survival and Vaccine Induced Immune Responses to Tumor Antigens

Background. Maintenance therapy in AML patients with measurable residual disease (MRD) remains challenging. Immunotherapy could be an effective way to induce durable disease control. This phase II study aims to show efficacy of priming the immune system, using an allogenic leukemia-derived dendritic cell vaccine (DCP-001), to induce an effective anti-leukemic response to control or kill remaining malignant cells (ADVANCE-II, Clintrials.gov: NCT03697707). Methods. A total of 20 evaluable AML patients in first complete remission (CR1) with MRD positivity and ineligible for HSCT received 4 biweekly doses of 25e6 or 50e6 cells per intradermal vaccination (cells/vc) and 2 doses as boosts at week 14 and 18 of 10e6 cells/vc. MRD, as primary endpoint, was assessed at baseline, week 14, 20 and 32 by flow cytometry and/or molecular analyses. Evaluation of secondary endpoints relapse-free (RFS) and overall survival (OS) was done at data cut-off (18th July 2022). Peripheral blood samples were taken before and during treatment for analysis of vaccine induced responses as measured by IFNγ ELISPOT and general immune profiles by flow cytometry using a 40-marker panel. Results. Patients aged 34-79 (median 60y) had a cytogenetic risk status assessed of 4 better, 15 intermediate and 1 poor-risk, with mutations in NPM1 (n=10), CBFB-MYH11 (n=4), IDH2 (n=2), RUNX1-RUNX1T1 (n=2), TP53 (n=1) and CEBPA (n=1). All 20 patients have received 4 initial vaccinations, 17 patients received all booster vaccinations and 1 patient received one booster. No serious adverse events (AE) or severe AE (grade 3 or higher) related to the treatment have been reported. Related AEs are mainly injection site reactions, such as redness, warmth and swelling, occurring within 48 hours after intradermal administration. To date, median RFS and OS have not yet been reached (Figure 1), with a median follow up period of 15.4 (range 6.6- 16.8) months for the entire study population. Estimated RFS and OS at 6 months is 83.9% (range 64-94%) and 97% (range 79-99%), respectively. Estimated RFS and OS at 12 months is 70% (range 48-85%) and 87% (67-95%), respectively. MRD response (conversion or >10-fold decrease of MRD) was observed in 7 patients: 5 patients converted to negative and 2 showed a MRD decrease. Responses were observed within 14-20 weeks and durable in 4 out of 5 patients, remaining MRD negative up to week 32. Seven patients remained in CR with stable MRD levels and 6 patients relapsed before week 32. Three patients with stable MRD became eligible for HSCT during study and were transplanted successfully. Immune responses were observed to tumor associated antigens expressed by DCP-001; WT1, RHAMM and PRAME, with specific vaccine induced responses (VIR) in the majority of patients (17/20) and highest number of VIR per antigen observed in patients with MRD responses (Figure 2). Differences in immune profiles at baseline were observed; relapsed patients had a high number of CD8RO+ cells, in particular CD8 CM. On the contrary, MRD responders had the highest level of CD8RA+ cells at baseline, with CD8 EMRA cells at the highest level. Patients with stable MRD had comparable, but slightly lower CD8RO+ levels than MRD responders. Conclusion/discussion. Vaccination with DCP-001 resulted in durable MRD responses with five patients converting to MRD negativity, remaining relapse-free and alive during follow up. This translates in promising survival data with the median RFS for the full patient population not reached to date, at a median FU time of 15.4 months. Highest level of vaccine-induced T-cell responses to leukemia specific antigens were detected in MRD responders. Baseline levels of CD8 memory profiles were markedly different in MRD responders, which might be indicative that lower number of memory cells, and thus especially higher number of CD8 EMRA could reflect the ability to induce a tumor specific cytotoxic T-cell response after vaccination. Relapse on study was limited, with an estimated RFS at 6 months of 83.7%, which is higher than expected for this MRD+ AML patient population, which would be around 45% as shown for placebo-treated patients from a recent phase III study in a similar patient population (Quazar AML001). Immunotherapy with DCP-001 as AML maintenance therapy is safe and effective in triggering immune responses and converting patients from MRD positive to MRD negative, leading to promising relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dexaml-02 : A Phase II Study of Dexamethasone Added to Induction and Postremission Therapy in Older Patients with Newly Diagnosed AML. a French Innovative Leukemia Organization (FILO) Study

Background In a recent retrospective study, we have showed that the addition of dexamethasone to intensive chemotherapy was significantly associated with a better outcome in AML patients with hyperleukocytosis at diagnosis (Bertoli S, Haematologica 2018). Moreover, preclinical studies demonstrated that specific AML oncogenic alterations (RUNX1-RUNX1T1, RUNX1, NPM1, or SRSF2 mutations as well as NF-kB activation in leukemic stem cells) or therapeutic stress (cytarabine, FLT3 inhibitors) are associated with up-regulation of the inflammatory gene response and expression of the glucocorticoid receptor which induce sensitization to glucocorticoids (Récher C, Front Oncol 2021). Therefore, dexamethasone, used as a chemo sensitizer in combination with intensive chemotherapy, may improve treatment response and outcome regardless of the white blood cell count. Methods DEXAML-02 (LAM-SA 2018) is a non-randomized phase II multicenter trial to assess the impact on outcome of dexamethasone added to induction chemotherapy in older patients with AML (NCT03609060). Key inclusion criteria were: > 60 years of age, newly diagnosed AML (either de novo or therapy-related AML), favorable or intermediate cytogenetic risk according to MRC 2010 classification, ECOG performance status < 3; HCT-CI score < 3, adequate baseline organ function. Key exclusion criteria were: AML with adverse cytogenetic risk, AML arising from MDS, MPN or CMML, AML with BCR-ABL1, known active central nervous system leukemia. Induction chemotherapy regimen consisted in one cycle of idarubicin 8 mg/m²/day, D1 to D5, cytarabine 100 mg/m²/d, CIV D1 to D7, lomustine 200 mg/m²/d, orally at D1 and dexamethasone 10 mg/12h, IV, D1 to D3. The use of G-CSF, anti-fungal, anti-viral and anti-pneumocystis jiroveci prophylaxis were recommended. Patients who failed to reach complete remission (CR/CRi) after this single cycle of induction were off-study whereas CR/CRi patients were planned to receive up to 6 courses of mini-consolidation administrated every 30 to 45 days with idarubicin 8 mg/m², D1, cytarabine 50 mg/m²/12h, subcutaneous, D1 to D5, dexamethasone 20 mg/d, D1. The addition of midostaurin in patients with FLT3-ITD or FLT3-TKD mutations was allowed during induction, consolidation and maintenance. Allogeneic stem-cell transplantation was allowed after 2 to 4 cycles in patients with intermediate or adverse risk according to ELN 2017 classification. Patients with CBF-AML could receive 2-3 cycles of intermediate dose cytarabine (with dexamethasone) instead of 6 cycles of mini-consolidations according to investigator choice. Results From August 2018 to February 2020, 120 patients were included. 75 patients were male (62.5%). Median age was 70y (min-max, 61-80). Median WBC was 4.9 G/L (0.64-137). Most patients had an intermediate cytogenetic risk (91.7%). NPM1 and FLT3 mutations (per local analysis) were observed in 45 (37.5%) and 33 patients (27.5%), respectively. NGS of a 91 myeloid panel genes was centrally performed in 111 patients. 24 patients received midostaurin. Following the induction cycle, 100 patients (83.3%) achieved CR/CRi, 14 failed (11.7%) and 6 died early (5 %). In NPM1mut patients, 43 achieved CR/CRi (95.6%). During induction, grade III-IV adverse events were: infections (50.8%), mucositis (16.7%), gastro-intestinal (14.2%), hepatic (11.7%), hyperglycemia (10.8%), pulmonary (7.5%), cardiac (5.8%), cutaneous (5%) or bleedings (5%). Of the 100 CR/CRi patients, 86 entered the consolidation phase of whom 59 and 38 received 3 and 6 cycles, respectively. 23 patients received an allogeneic-SCT in first CR. The median follow up time was 31.4 months. Median RFS and OS were 20.2 and 30.7 months, respectively. In NPM1mut patients, median RFS was 20.1 months while median OS was not reached. Conclusion In older AML patients who are eligible for intensive treatment, adding dexamethasone to induction and consolidation chemotherapy is feasible and associated with a high response rate after a single induction cycle and encouraging overall survival. A historical comparison with the patient population of the LAM-SA 2007 trial (Pigneux A, JCO 2018) will be presented during the meeting to highlight a potential signal of activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal