Genetic Risk Classification Research Articles

Risk stratification in the clinical application of minimal residual disease assessment in acute myeloid leukemia.

In acute myeloid leukemia (AML), further investigation is warranted to integrate measurable residual disease (MRD) with genetic characteristics for formulating a dynamic prognostic system for predicting response and selecting appropriate postremission therapeutic strategies. The authors incorporated MRD with genetic risk classification and assessed its impact on transplantation decision making within different risk cohorts, comprising 769 patients with newly diagnosed AML across three clinical trials. Only patients who achieved complete remission (CR) within two courses of chemotherapy were selected. In the favorable-risk and intermediate-risk groups, patients who underwent transplantation according to the protocol experienced significant 3-year overall survival (OS) benefits compared with those who did not (favorable-risk group: hazard ratio [HR], 0.38; 95% confidence interval [CI], 0.20-0.73l p=.004; intermediate-risk group: HR, 0.53; 95% CI, 0.33-0.85; p=.008). In the intermediate-risk group, early detection of MRD positivity, even after the initial course of chemotherapy, was associated with a significantly elevated cumulative incidence of relapse (47.2% vs. 36.0%; p=.009) and a notable extension of OS with allogeneic hematopoietic stem cell transplantation (HR, 0.47; 95% CI, 0.28-0.79; p=.004). Conversely, patients who achieved MRD negativity at either of the two time points had comparable OS in the favorable-risk and intermediate-risk groups, regardless of whether they underwent transplant or not. In the adverse-risk group, allogeneic hematopoietic stem cell transplantation led to improvements in OS irrespective of MRD status (HR, 0.51; 95% CI, 0.38-0.69; p<.001). Early clearance of MRD demonstrated significant prognostic value, particularly for patients in the favorable-risk and intermediate-risk groups. Positive MRD status after two courses of intensive chemotherapy were associated with a higher relapse rate and inferior OS, necessitating allogeneic hematopoietic stem cell transplantation.

Validation and refinement of the 2022 European LeukaemiaNet genetic risk classification of acute myeloid leukaemia patients receiving allogeneic haematopoietic cell transplantation.

Validation and refinement of the 2022 European LeukaemiaNet genetic risk classification of acute myeloid leukaemia patients receiving allogeneic haematopoietic cell transplantation.

Genetic risk classification for adults with AML receiving less-intensive therapies: the 2024 ELN recommendations

AbstractThe European LeukemiaNet (ELN) genetic risk classifications were developed based on data from younger adults receiving intensive chemotherapy. Emerging analyses from patients receiving less-intensive therapies prompted a proposal for an ELN genetic risk classification specifically for this patient population.

Genetic risk stratification and outcomes among treatment-naive patients with AML treated with venetoclax and azacitidine

AbstractThe European LeukemiaNet (ELN) acute myeloid leukemia (AML) genetic risk classification systems are based on response to intensive chemotherapy; their ability to discriminate outcomes in older patients treated with venetoclax-azacitidine may be suboptimal. This pooled analysis of the phase 3 VIALE-A trial (NCT02993523) and phase 1b study (NCT02203773) examined prognostic stratification according to the 2017 and 2022 ELN risk classifications and derived new molecular signatures differentiating venetoclax-azacitidine–treated patients based on overall survival (OS). Overall, 279 patients treated with venetoclax-azacitidine and 113 patients treated with placebo-azacitidine were analyzed. The ELN 2017 or 2022 prognostic criteria classified most patients as adverse-risk AML (60.2% and 72.8% for venetoclax-azacitidine and 65.5% and 75.2% for placebo-azacitidine, respectively). Although outcomes with venetoclax-azacitidine improved across all ELN risk groups compared with placebo-azacitidine, ELN classification systems poorly discriminated venetoclax-azacitidine outcomes. By applying a bioinformatic algorithm, new molecular signatures were derived differentiating OS outcomes with venetoclax-azacitidine. The mutational status of TP53, FLT3 internal tandem duplication (FLT3-ITD), NRAS, and KRAS categorized patients into higher-, intermediate-, and lower-benefit groups (52%, 25%, and 23% of patients, respectively), each associated with a distinct median OS (26.5 months [95% confidence interval (CI), 20.2-32.7]; 12.1 months [95% CI, 7.3-15.2]; and 5.5 months [95% CI, 2.8-7.6], respectively). ELN prognostic classifiers did not provide clinically meaningful risk stratification of OS outcomes in patients treated with venetoclax-azacitidine. TP53, FLT3-ITD, NRAS, and KRAS mutation status allows the classification of these patients into 3 risk groups with distinct differences in median OS. These trials were registered at www.clinicaltrials.gov as #NCT02993523 and #NCT02203773.

Enhancing personalized medicine through molecular diagnostics: Implications for nursing care

The advent of molecular diagnostics, exemplified by technologies such as Polymerase Chain Reaction (PCR) and gene sequencing, represents a pivotal shift towards personalized medicine, fundamentally altering the landscape of healthcare delivery and nursing care. This paper delves into the transformative potential of genetic screening and risk assessment, disease typing and classification, and predictive analysis of treatment responses, highlighting the critical role of molecular diagnostics in facilitating a more nuanced understanding of individual health profiles and tailoring treatment accordingly. The integration of genetic data into treatment planning enables healthcare practitioners to optimize therapeutic strategies, significantly improving patient outcomes. However, this advancement is not without challenges. The ethical considerations surrounding data privacy, the complexity of genetic information, and issues of cost and accessibility are scrutinized, underscoring the need for robust ethical guidelines, continuous professional education, and policy interventions to ensure equitable access to these technologies. Through a comprehensive review, this paper illustrates the intricate interplay between molecular diagnostics and personalized nursing care, advocating for an interdisciplinary approach to harness the full potential of these technological advancements in improving patient care.

Predicting cytogenetic risk in multiple myeloma using conventional whole-body MRI, spinal dynamic contrast-enhanced MRI, and spinal diffusion-weighted imaging

ObjectivesCytogenetic abnormalities are predictors of poor prognosis in multiple myeloma (MM). This paper aims to build and validate a multiparametric conventional and functional whole-body MRI-based prediction model for cytogenetic risk classification in newly diagnosed MM.MethodsPatients with newly diagnosed MM who underwent multiparametric conventional whole-body MRI, spinal dynamic contrast-enhanced (DCE-)MRI, spinal diffusion-weighted MRI (DWI) and had genetic analysis were retrospectively included (2011–2020/Ghent University Hospital/Belgium). Patients were stratified into standard versus intermediate/high cytogenetic risk groups. After segmentation, 303 MRI features were extracted. Univariate and model-based methods were evaluated for feature and model selection. Testing was performed using receiver operating characteristic (ROC) and precision-recall curves. Models comparing the performance for genetic risk classification of the entire MRI protocol and of all MRI sequences separately were evaluated, including all features. Four final models, including only the top three most predictive features, were evaluated.ResultsThirty-one patients were enrolled (mean age 66 ± 7 years, 15 men, 13 intermediate-/high-risk genetics). None of the univariate models and none of the models with all features included achieved good performance. The best performing model with only the three most predictive features and including all MRI sequences reached a ROC-area-under-the-curve of 0.80 and precision-recall-area-under-the-curve of 0.79. The highest statistical performance was reached when all three MRI sequences were combined (conventional whole-body MRI + DCE-MRI + DWI). Conventional MRI always outperformed the other sequences. DCE-MRI always outperformed DWI, except for specificity.ConclusionsA multiparametric MRI-based model has a better performance in the noninvasive prediction of high-risk cytogenetics in newly diagnosed MM than conventional MRI alone.Critical relevance statementAn elaborate multiparametric MRI-based model performs better than conventional MRI alone for the noninvasive prediction of high-risk cytogenetics in newly diagnosed multiple myeloma; this opens opportunities to assess genetic heterogeneity thus overcoming sampling bias.Key points• Standard genetic techniques in multiple myeloma patients suffer from sampling bias due to tumoral heterogeneity.• Multiparametric MRI noninvasively predicts genetic risk in multiple myeloma.• Combined conventional anatomical MRI, DCE-MRI, and DWI had the highest statistical performance to predict genetic risk.• Conventional MRI alone always outperformed DCE-MRI and DWI separately to predict genetic risk. DCE-MRI alone always outperformed DWI separately, except for the parameter specificity to predict genetic risk.• This multiparametric MRI-based genetic risk prediction model opens opportunities to noninvasively assess genetic heterogeneity thereby overcoming sampling bias in predicting genetic risk in multiple myeloma.Graphical

Interactions of genetic variations in FAS, GJB2 and PTPRN2 are associated with noise-induced hearing loss: a case-control study in China

BackgroundThis study aimed to screen and validate noise-induced hearing loss (NIHL) associated single nucleotide polymorphisms (SNPs), construct genetic risk prediction models, and evaluate higher-order gene-gene, gene-environment interactions for NIHL in Chinese population.MethodsFirst, 83 cases and 83 controls were recruited and 60 candidate SNPs were genotyped. Then SNPs with promising results were validated in another case-control study (153 cases and 252 controls). NIHL-associated SNPs were identified by logistic regression analysis, and a genetic risk model was constructed based on the genetic risk score (GRS), and classification and regression tree (CART) analysis was used to evaluate interactions among gene-gene and gene-environment.ResultsSix SNPs in five genes were significantly associated with NIHL risk (p < 0.05). A positive dose-response relationship was found between GRS values and NIHL risk. CART analysis indicated that strongest interaction was among subjects with age ≥ 45 years and cumulative noise exposure ≥ 95 [dB(A)·years], without personal protective equipment, and carried GJB2 rs3751385 (AA/AB) and FAS rs1468063 (AA/AB) (OR = 10.038, 95% CI = 2.770, 47.792), compared with the referent group. CDH23, FAS, GJB2, PTPRN2 and SIK3 may be NIHL susceptibility genes.ConclusionGRS values may be utilized in the evaluation of the cumulative effect of genetic risk for NIHL based on NIHL-associated SNPs. Gene-gene, gene-environment interaction patterns play an important role in the incidence of NIHL.

RADAR Trial: MRD Response Adapted Trial for Newly Diagnosed Transplant Eligible Myeloma Patients

Background: Achieving and sustaining minimal disease negativity improves clinical outcomes in newly diagnosed transplant eligible myeloma (NDMM). Outcomes in NDMM are influenced by levels of minimal residual disease (MRD) post therapy and genetic risk by FISH or molecular profiling prior to therapy. Trials exploring response adapted post transplant strategies using MRD as a prognostic tool are going. We provide an update of the current UK-MRA RADAR study. Study Design/Methods: The UK-MRA RADAR study is a national multicentre risk adapted response guided multiarm multistage (MAMS) phase II/III trial which will recruit 1400 patients with NDMM eligible for ASCT. Eligible patients receive induction with 4 cycles of RCyBorD (lenalidomide, cyclophosphamide, bortezomib, dexamethasone), followed by high-dose melphalan and stem cell rescue. Patients with high-risk disease receive Isatuximab in addition to RCyBorD during induction. Genetic risk classification is performed using standard of care FISH testing at local sites. Participants enter the standard or high risk pathway based on presence of atleast two markers: t(4;14), t(14;16), t(14;20), del(17p), del(1p) and gain (1q21). MRD analysis in RADAR uses a multiparametric flow cytometry panel with CD319 and Cd229 used to augment post-isatuximab sample analysis at a central laboratory, with negativity defined at threshold of 10 -5(Figure 1). For standard risk patients, the study addresses two pivotal MRD-related questions: 1) For those with sustained MRD negativity after 12 months of isatuximab maintenance, can treatment be stopped safely to allow patients a treatment-free interval thereby reducing treatment burden? 2) For patients with persistent detectable disease after ASCT, does intensification of consolidation and maintenance therapy augment conversion to MRD negativity compared to lenalidomide maintenance? High risk participants receive intensified consolidation and maintenance regimens post_ASCT aligned to OPTIMUM/MUKnine ( NCT03188172). A further amendment is planned to explore the activity of a BCMA bispecific antibody in high risk patients pre and post transplant. It is hypothesised this will improve both depth (MRD negativity) and ability to sustain response (PFS). Feasibility and benefits of multimodal MRD incorporating imaging and blood-based testing will be tested in the new high risk pathway. In RADAR, we will also gather information on how patients respond to receiving information about their genetic risk assignment and treatment response (MRD). This is a unique opportunity to understand the patients perspective on personalised medicine, which has implications for implementation in clinical care, and future trial design. Trial Progress. RADAR opened to recruitment in May 2021 and is currently recruiting across 87 hospital sites with a further 16 sites in set up. 504 patients have been recruited; 87 are classified as high risk on FISH analysis ( 375 standard risk, 15 unable to determine, 19TBC, 8 withdrawn before FISH analysis). 220 have reached D+100 MRD assessment. Trackable phenotypes were available in 98.2% of patients. Discussion: The RADAR study simultaneously tests escalation and de-escalation strategies, according to disease response, and using MRD to guide post transplant therapy in NDMM patients. Use of central flow cytometry laboratory for MRD has proven feasible in patients to date. Recruitment is on target and outcomes will provide RCT-level evidence for the adaptive use of MRD in treatment allocation and intensification post transplant. The platform design enables adaptation with ability to test new strategies for high risk patients and for standard risk patients who are MRD positive post-transplant. Funding This trial is funded by Cancer Research UK (C9203/A24078), Sanofi Genzyme and Celgene : A BMS company. Sanofi Genzyme and Celgene: A BMS company provide study drug and unrestricted educational grants to support study coordination. This work is also supported by Core Clinical trials unit infrastructure from Cancer Research UK ( C7852/A25447). The funder of the study had no role in study design, data collection, data analysis, data interpretation or writing of the abstract

Identification of Characteristics and Prognostic Impact of FUS-ERG and AML1-MTG16 Fusion Genes in Adult AML Patients

Background FUS-ERG and AML1-MTG16 are two rare fusion genes in acute myeloid leukemia (AML), characterized by similar karyotypic abnormalities, namely t(16;21)(p11;q22) and t(16;21)(q24;q22), respectively. A previous research has demonstrated that FUS-ERG represents a high-risk subtype associated with an extremely poor prognosis, while AML1-MTG16 has favorable outcomes in pediatric cohort. Despite this valuable insight, a comprehensive systematic review of these fusion genes in the adult population remains absent. Method In this study, we conducted an extensive collection of AML patients with FUS-ERG or AML1-MTG16 fusion genes, who were reported in Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, PubMed and China national knowledge infrastructure (CNKI) from 1988 to 2023. Patients lacking detailed survival data or those who did not receive treatment were excluded. Additionally, patients diagnosed and treated in our institution between 2018 and 2023 were included if they were identified as carrying FUS-ERG or AML1-MTG16 fusion genes. The reference cohort comprised of 669 adult AML patients (excluding the t(16;21) cases and acute promyelocytic leukemia) admitted to our institution from 2016 to 2021. All patients within the cohort were classified into 3 categories based on the 2022 ELN genetic risk classification, namely favorable (F), intermediate (I) and adverse (A) risk. These patients were then compared with the FUS-ERG and AML1-MTG16 groups to explore their respective clinical features and prognosis. Results A total of 800 patients were included for analysis (Table 1). Patients with FUS-ERG (n=111) exhibited significant distinctions, including a younger onset age (34 years vs. 59 years), lower platelet count (30×10 9/L vs. 51×10 9/L), higher bone marrow blasts proportion (80.0% vs. 60.0%), a higher relapse rate (78.6% vs. 44.7%) and a higher incidence of trisomy 8 (16.4% vs. 9.0%), trisomy 10 (9.1% vs. 0.3%) and complex karyotype (37.8% vs. 11.6%), when compared to the reference cohort (p&amp;lt;0.01). Meanwhile, patients with AML1-MTG16 (n=20) primarily presented as the secondary AML (70.0%), and displayed a higher prevalence of trisomy 8 (40.0%) and complex karyotype (35.0%) than reference cohort (p&amp;lt;0.01). The FUS-ERG patients had a less median overall survival (OS) when compared to the reference cohort (13 vs. 35 months, p&amp;lt;0.001). The median OS in subgroups with ELN 2022 F-risk vs. ELN 2022 I-risk vs. FUS-ERG was not reached (NR) vs. 33.6 months vs. 13.0 months (p&amp;lt;0.001) and the median OS of FUS-ERG group was even inferior to ELN 2022 A-risk group (13.0 vs. 16.8 months, p=0.015). Furthermore, the FUS-ERG patients also displayed a less median disease-free survival (DFS) than the reference cohort (7.5 vs. 20.5 months, p&amp;lt;0.001). The median DFS in subgroups with F-risk vs. I-risk vs. A-risk vs. FUS-ERG was NR vs. 19.8 vs. 13.3 vs. 7.5 months (p&amp;lt;0.001). In contrast, patients with AML1-MTG16 had a median OS of 18.7 months and a median DFS of 16 months, presenting a relatively unfavorable OS trend and indistinctive DFS, when compared to the reference cohort (OS: p=0.083, DFS: p=0.612), F-risk (OS: p&amp;lt;0.001, DFS: p=0.128), I-risk (OS: p=0.162, DFS: p=0.835) and A-risk (OS: p=0.877, DFS: p=0.731). Moreover, the AML1-MTG16 group had a better DFS than the FUS-ERG group (14.0 vs. 7.5 months, p=0.016), but their OS was comparable (16.0 vs. 7.5 months, p=0.219). For adult patients with FUS-ERG (Table 2) multivariate analysis revealed that age &amp;gt;60 years (HR=2.96, 95%CI: 1.14-7.73, p=0.026), WBC &amp;gt;100×10 9/L (HR=2.98, 95%CI: 1.24-7.13, p=0.014) and the presence of monosomy (HR=2.61, 95%CI: 1.21-5.66, p=0.015) were independent risk factors for OS, while monosomy was associated with poor DFS (HR=3.72, 95%CI: 1.69-8.20, p=0.001). However, receiving HSCT could significantly improve both OS (HR=0.22, 95%CI: 0.11-0.42, p&amp;lt;0.001) and DFS (HR=0.31, 95%CI: 0.16-0.57, p&amp;lt;0.001). Pediatric FUS-ERG patients presented similar clinical features, exhibiting a better median OS (19.5 vs.13 months, p=0.014) and a comparable median DFS (9.5 vs. 7.5 months, p=0.496), when compared to the adult population. Conclusion Both FUS-ERG and AML1-MTG16 were identified as high-risk subgroups in adult AML anddiffered from the results in pediatric cohort. Several factors including age, WBC count, HSCT and monosomy influenced the prognosis of adult AML patients with FUS-ERG.

Molecular Measurable Residual Disease in Patients with Newly Diagnosed m IDH1 Acute Myeloid Leukemia Treated with Ivosidenib + Azacitidine

Background: Ivosidenib (IVO) + azacitidine (AZA) improved complete remission (CR) rates and overall survival (OS) relative to placebo (PBO) + AZA in patients (pts) with newly diagnosed mutant isocitrate dehydrogenase 1 (m IDH1) acute myeloid leukemia (AML) in the pivotal AGILE study (Montesinos et al. NEJM 2022). Prior analyses showed clinical responses to IVO+AZA were associated with deep clearance of m IDH1 (limit of detection [LOD]: 0.02-0.04% variant allele frequency [VAF]), as well as clearance of baseline co-mutations below the threshold of conventional next-generation sequencing (NGS) (LOD: 2.0% VAF) (Döhner et al. Blood 2022; Daigle et al. Blood 2021). Measurable residual disease (MRD), a negative prognostic marker, following chemotherapy is often detected via multiparametric flow cytometry or quantitative polymerase chain reaction although both techniques have their limitations. (Heuser et al. Blood 2021). NGS can detect a wide range of AML mutations and studies show that NGS-assessed MRD is highly predictive of both relapse and survival (Thol et al. Blood 2018; Tsai et al. Blood Adv 2021). However, a limitation of NGS is difficulty in distinguishing between somatic leukemia-associated mutations and clonal hematopoiesis of indeterminate potential (CHIP) (Bacher et al. Blood Cancer J 2018). We aimed to perform an NGS analysis of MRD responses in the AGILE study. Methods: All AGILE pts who had a best overall response (BOR) of CR, CR with incomplete count recovery, or CR with incomplete platelet recovery, and had ≥1 on-treatment bone marrow mononuclear cell (BMMC) sample available were included. Suggested timepoints for MRD testing included day 1 of treatment cycles 3, 5, 7, 9, 11, 14, 20, 26, and 32. Baseline and on-treatment BMMC DNA samples were analyzed using a diagnostic 51-gene myeloid NGS panel (LOD: 3% VAF) and a 26-gene AML MRD panel, respectively. For variants detected at baseline, the LOD was 0.1% VAF; for variants not detected at baseline (either emerging mutations or if baseline samples were unavailable), the LOD was 0.5% VAF. All variants above the applicable LOD with known or potential clinical significance were considered evidence of positive MRD, aside from DNMT3A, TET2 and ASXL1 (“DTA”) mutations to reduce the risk of false-positive MRD due to CHIP. Results: The analysis set comprised 173 samples from 33 IVO+AZA-treated pts and 10 PBO+AZA-treated pts (Fig. 1). Clinical response data were based on a March 18, 2021 primary analysis data cut; OS data were based on an updated June 30, 2022 data cut. The median number of baseline mutations was 4 (range: 1-10) per patient, median number of MRD assessments was 3 (range: 1-9) and median follow-up period was 189 days (range: 49-875). Ten (30.3%) IVO+AZA-treated pts became MRD-negative (MRD neg), compared with 2 (20.0%) PBO+AZA-treated pts. All pts attaining an MRD neg response had a BOR of CR (10 [34.5%] of 29 IVO+AZA-treated pts in CR; 2 [22.2%] of 9 PBO+AZA-treated pts in CR). Seven (70.0%) of the 10 MRD neg IVO+AZA-treated responding pts converted to an MRD neg response by day 1 of cycle 7 (C7D1). Fewer pts with an MRD neg response had an Adverse risk at baseline according to European LeukemiaNet 2022 genetic risk classification versus those pts with an MRD-positive (MRD pos) response (26.3% vs 73.7%) (Table 1). In the IVO+AZA arm, rates of event-free survival at 12 months and OS at 24 months were numerically higher in MRD neg vs MRD pos responders (80.0% vs 60.6% and 88.9% vs 72.3%, respectively) but did not reach statistical significance. All 5 (15.2%) IVO+AZA-treated pts with confirmed relapses had detectable MRD at the last completed assessment prior to relapse. One pt converted from an MRD neg to an MRD pos response (“MRD relapse”) 169 days prior to overt clinical relapse. Among the 23 IVO+AZA-treated pts without an MRD neg response, 16 were alive as of the updated data cut; OS duration ranged from 20.3-48.9 months. Conclusions: IVO+AZA induced molecular MRD negativity in approximately one third of responding pts with newly diagnosed m IDH1 AML; MRD neg responses were most often observed on or before C7D1. Interestingly, several pts had durable clinical responses and OS despite ongoing MRD positivity, potentially due to persistent CHIP rather than true MRD, illustrating the complexity of interpreting NGS MRD data. Additional analyses, including associations between specific molecular alterations, MRD response, and OS are ongoing and will be presented.

Analysis of Post-Transplant Clinical Outcomes and Prognostic Factors in FLT3-ITD Mutated Acute Myeloid Leukemia Patients Under ELN 2022 Risk Stratification

Background In 2022 European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML), one of the most important changes made to the genetic risk classification of AML is that the FLT3-ITD allelic ratio (AR) is no longer considered in the risk classification; consequently, AML with FLT3-ITD (without adverse-risk genetic lesions) are now categorized in the intermediate-risk group, irrespective of the allelic ratio or concurrent presence of NPM1 mutation. Aims To investigate the survival outcomes of FLT3-ITD mutated AML patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), based on prognostic stratification defined by the 2017 and 2022 ELN guidelines, and to analyze the risk factors affecting transplant prognosis. Methods A total of 163 FLT3-ITD mutated AML patients underwent allo-HSCT were enrolled in this study. Bone marrow aspirates were obtained, cytogenetic and molecular characteristics were collected at the time of diagnosis. Genetic risk category was classified according to the 2017 and 2022 edition of the European LeukemiaNet (ELN) recommendations. HLA-haploidentical donor transplants were performed if no HLA matched sibling or unrelated donor was available. Patients received myeloablative conditioning with a modified Bu/Cy/Flu/Ara-c (busulfan/cyclophosphamide/fludarabine/cytarabine) or MCBC (melphalan/cladribine/busulfan/cyclophosphamide) preparative regimen. The therapeutic process and clinical outcomes were retrospectively analyzed. Results We assessed 163 adults with FLT3-mutated AML (median age 37 years) allografted between 2017 and 2022 in our clinical center. The median follow-up of alive patients was 24.9 months. The 2-year relapse-free survival (RFS) and overall survival (OS) were 67.2% and 71.9%, respectively. The 2-year incidences of relapse and non-relapse mortality were 27.9% and 12.5%, respectively. There was no significant difference in survival outcomes between the intermediate and high-risk groups after transplantation, both defined by the 2017 and 2022 ELN guidelines. High FLT3-ITD allelic ratio (≥0.5) at initial diagnosis was associated with elevated WBC counts ( P&amp;lt;0.001), but did not affect the response ratefollowing induction chemotherapy ( P=0.059) . Patients with high allelic ratio were more likely to benefit from maintenance therapy after transplantation ( P=0.048 for OS). In univariate analysis, presence of NPM1 mutation and the allelic ratio of FLT3-ITD did not affect any of the transplant outcomes. In multivariate analysis, MRD-negative at transplantation, occurrence of chronic GVHD and posttransplant maintenance therapy improved OS and RFS. Conclusion Allo-HSCT can overcome the poor prognosis in patients with FLT3-ITD mutated AML. Neither the presence of NPM1 mutation nor the FLT3-ITD allelic ratio affected survival. Prevention of disease relapse after transplantation remains a challenge. The main prognostic factors affecting transplant outcomes included pre-transplant MRD status and the application of maintenance therapy. Thus, introduction of FLT3 inhibitors as post-transplant maintenance therapy and the development of highly sensitive MRD assays will further reduce disease recurrence and improve survival.

Survival Outcomes Associated with Molecular and Cytogenetic Alterations in AML Patients Treated with Hypomethylating Agent and Venetoclax

Molecular profiling has improved prognostication and helped identify novel therapeutic targets in AML. Nonetheless, it has also created a complex web of molecular profiles and cytogenetic abnormalities and in many cases it remains unclear which specific alterations primarily drive leukemogenesis. We sought to review our patients (pts) treated with a hypomethylating agent (HMA) + Venetoclax (Ven), to evaluate molecular and cytogenetic profiles with their associated outcomes. We identified 142 pts with AML treated with HMA + Ven at Northwell Health between January 2019 and December 2023. Through retrospective chart review, we obtained patient and disease characteristics, treatment regimens, clinical outcomes as well as molecular and cytogenetic profiling (using commercial AML panels) prior to the initiation of HMA + Ven. Bone marrow biopsy pathology reports were reviewed to identify the subtype of AML and grouped into De novo AML, AML with MDS related changes (AML-MRC) and therapy-related AML (t-AML). Median relapse free survival (mRFS) and median overall survival (mOS) in months (m) as defined by ELN 2022 (Dohner et al, Blood 2022), were estimated with Kaplan-Meier curves and compared utilizing log-rank testing. T-test was used to evaluate significant differences between categorical data. Of the 142 pts identified, the median age was 77. The majority of patients had adverse (n = 99) or intermediate risk (n = 31) genetics as defined by the 2022 ELN genetic risk classification. The overall response rate (ORR) was 42% with mOS of 9.6m. AML-MRC patients (n=80) had mOS of 9.1m and mRFS of 11.6m with 61 pts receiving HMA + Ven as frontline treatment. 47 pts with De novo AML had mOS of 13.4m and mRFS of 16.1m with 36 pts receiving HMA + Ven as frontline therapy. Only 15 pts had t-AML with mRFS of 12.3m and mOS of 8.8m with 10 pts receiving HMA + Ven as frontline therapy. There was no significant difference in mRFS or mOS between these groups, although there was a trend favoring De novo AML. 47 different molecular alterations were identified among the 142 patients. Figure 1 illustrates outcomes associated with the 16 most prevalent molecular alterations found in our AML population. Mutational burden was similar between AML types (2.7 vs 3.0 vs 2.4 mutations per pt). De novo AML was associated with an increased mutation burden in signaling molecules (60%) and transcription factors (47%) compared to AML-MRC (29%, 31%). AML-MRC had a higher occurrence of mutations in epigenetic regulatory genes (79%) and splicing factors (31%) compared to De novo AML (55%, 19%). Mutations in splicing factors were associated with a significant increase in both mRFS and mOS(p = 0.01) compared to pts without mutations. Pts with DNMT3A, NPM1 or SRSF2 mutations had the highest ORR to HMA + Ven (63%, 64% and 63% respectively). RUNX1, TP53, complex cytogenetics and del 7q were associated with worse ORR (46%, 36%, 46% and 42% respectively) as well as decreased mOS. SRSF2, mutated in both AML-MRC and De novo AML, not only had an elevated ORR, but improved mRFS and mOS. Contrarily, mRFS and mOS were not significantly improved despite the high ORR in DMT3A mutated pts. DNMT3A was mutated most often in AML-MRC and was associated with a higher rate of other epigenetic regulatory gene co-mutations. The same is true for patients with KMT2A mutations (ORR 50%, mOS 2.8m, mRFS 6.1m) which suggests ineffectiveness of HMA+ Ven in maintaining remissions with this alteration with possible early selection for resistant clones. RUNX1 mutations were associated with an improved mRFS (16.1m) despite lower mOS (8.8m). NPM1 mutations had the highest mRFS of 34.4m. The survival advantage may be secondary to the absence of adverse cytogenetics in 88% of these pts. IDH2 mutations was associated with a significantly improved mOS compared to unmutated IDH2; however this may be confounded by the use of an IDH2 inhibitor at relapse. Our review of molecular alterations in AML pts identified several molecular alterations such as SRSF2, DNMT3A, RUNX1 which we found to be associated with favorable responses when treated with HMA + Ven. In addition, we demonstrated that NPM1 mutation remains favorable in patients treated with HMA + Ven. Our findings help contribute to the ever-growing, complex molecular and genomic profiling of AML patients.

The Impact of DNMT3A Mutation on Survival of AML Patients Receiving Allogeneic Hematopoietic Cell Transplantation in First Remission Depends on the Karyotype and Co-Occurring Mutations: On Behalf of the EBMT Acute Leukemia Working Party

DNMT3A gene mutations are more frequent in cytogenetically normal acute myeloid leukemia (AML) and frequently associated with NPM1 mutation and internal tandem duplication (ITD) of the FLT3 gene. However, DNMT3A mutation is not included as a distinct prognostic group in the recent European Leukemia Net (ELN) 2022 genetic risk classification of AML. In the context of allogeneic hematopoietic cell transplantation (allo-HCT), the prognostic value of DNMT3A is still not well studied. We evaluated, through the European Society for Blood and Marrow Transplantation (EBMT) registry, the impact of DNMT3A mutation, according to the karyotype, and NPM1 and FLT3-ITD mutation status, on post-transplant outcomes in AML patients receiving allo-HCT in first complete remission (CR1). We identified a total of 1374 adult AML patients (51% female, median age 57 years; range 18-78 years) with ELN-2022 intermediate- or poor-risk cytogenetics and available DNMT3A, FLT3ITD and NPM1 mutation status, who underwent their first allo-HCT in CR1 between 2013 and 2021. Of these, 631 (46%) patients had DNMT3A mutation. Patients with mutant DNMT3A were more likely to be female (57% versus 46%, p&amp;lt;0.001), to have normal cytogenetics (75% versus 54%, p&amp;lt;0.001), FLT3-ITD co-mutation (50% vs 23%, p&amp;lt;0.001), NPM1 co-mutation (60% vs 21%, p&amp;lt;0.001), and to receive a myeloablative conditioning and in vivo T cell depletion. After a median follow-up of 2.3 years, the 2-year leukemia free survival (LFS) and overall survival (OS) were 62% and 69%, respectively. The subsequent analysis was split according to baseline karyotype (normal or abnormal). Among 870 patients with normal cytogenetics, 472 (54%) patients had DNMT3A mutation. The 2-year relapse incidence (RI), LFS and OS were 24%, 63% and 70% respectively, in patients with DNMT3A mutation compared to 18%, 69% and 74% in patients with wild type DNMT3A. Among these patients with normal karyotype, multivariable analyses (MVA) were done within the 4 groups of FLT3-ITD and NPM1 mutations. Within the group of 324 patients with both FLT3-ITD and NPM1 positives, 244 were triple-positive (75%), LFS and OS were not significantly different with and without DNMT3A mutation (2y LFS: 62% vs 63%, HR: 0.98, p=0.95; 2y OS: 70% vs 73%, HR: 1.17, p=0.58). Conversely, among 146 patients with NPM1 mutation without FLT3-ITD, LFS was significantly lower for 91 patients with DNMT3A mutation (2y LFS: 68% vs 87%, HR: 2.70, p=0.02) but OS was not significantly different (2y OS: 79% vs 87%, HR: 2, p=0.13). On the other hand, among 318 with wild type NPM1 and without FLT3-ITD, LFS and OS were not significantly different for 108 patients with DNMT3A mutation compared to 210 patients with wild type DNMT3A (2y LFS: 62% vs 67%, HR: 1, p=0.98; 2y OS: 65% vs 71%, HR: 1.02, p=0.94). Conversely, among 82 patients with FLT3-ITD and wild type NPM1, 2-year LFS and OS were 53% and 58% respectively, for 29 patients with DNMT3A mutation compared to 71% and 76% respectively, for 53 patients with wild type DNMT3A. The number of patients didn't allow to perform MVA in this group. Finally, among 504 patients with abnormal cytogenetics, 159 (31%) patients had DNMT3A mutation. On MVA, DNMT3A mutation did not significantly affect LFS or OS. Poor-risk cytogenetics was associated with worse LFS, OS and increased RI. Older age negatively affected LFS and OS. In conclusion, in AML patients allografted in CR1, the impact of DNMT3A mutation depends on the karyotype and co-occurring mutations. Our data suggest that DNMT3A mutation negatively affects post-transplant survival selectively in patients with normal karyotype and either NPM1 mutation without FLT3-ITD, or patients with normal karyotype, FLT3-ITD and wild type NPM1. DNMT3A mutation shows no impact on post-transplant outcomes in other settings.

Efficacy and Safety of Decitabine Combined with Modified CAG Regimen in Patients Aged ≥ 70 Years with Newly Diagnosed Acute Myeloid Leukemia

To evaluate the clinical efficacy and safety of decitabine combined with modified CAG regimen (D-CAG regimen) in patients aged ≥70 years with newly diagnosed acute myeloid leukemia (AML). The clinical data of 59 AML patients (≥70 years old) who were newly diagnosed and treated in the Hematology Department of the First Affiliated Hospital of Nanjing Medical University from November 2010 to June 2021 were retrospectively analyzed. Among the 59 AML patients, 28 were males and 31 were females, with a median age of 74 (70-86) years. The complete remission (CR) rate was 69.4% (34/49), and the median duration of CR was 10.7 (0.6-125.4) months after 2 courses of D-CAG treatment. According to the British Medical Research Council (MRC) classification, there was only one patient in the favorable-risk group, and the CR rate was 71.8% (28/39) in the intermediate-risk group, and 55.6% (5/9) in the adverse-risk group, respectively. There was no statistical difference in the CR rate between the intermediate-risk and adverse-risk group. Referring to ELN 2017 genetic risk classification, CR rate was 88.2% (15/17) in the favorable-risk group, 45.5% (5/11) in the intermediate-risk group, and 66.7% (14/21) in the adverse-risk group. There was no significant difference in CR rate between the favorable-risk and adverse-risk categories, but both were significantly higher than that in the intermediate-risk group (P <0.05). Next-generation sequencing (NGS) analysis showed that 11 gene mutations with a frequency of more than 10%, including TET2 mutation (35.6%), ASXL1 mutation (30.5%), NPM1 mutation (28.8%), FLT3-ITD mutation (27.1%), DNMT3A mutation (22.0%), IDH1 mutation (15.3%), CEBPA single mutation (13.6%), TP53 mutation (13.6%), IDH2 mutation (11.9%), RUNX1 mutation (11.9%), and NRAS mutation (10.2%). There were no statistical differences in mutation frequency of these 11 genes between CR group and non-CR group. Compared with normal karyotypes, patients with complex karyotypes were more likely to develop TP53 mutations (P <0.001), while FLT3-ITD and DNMT3A mutations were more likely to occur in patients with normal karyotypes (P =0.04, P =0.047). The median follow-up, overall survival (OS), and event-free survival (EFS) of all the patients was 11.7 (1.5-128.2) months, 12.3 (1.5-128.2) months, and 8.5 (1.5-128.2) months, respectively. The median OS and EFS of CR patients were 19.8 and 13.3 months, respectively, which were significantly longer than 6.4 and 5.7 months in patients experiencing treatment failure (P < 0.001, P =0.009). In regard to genes with mutation frequency >10%, there were no statistical differences in CR rate, median OS, and median EFS between mutated and wild-type patients by Chi-square test and survival analysis. Univariate analysis showed that age, hemoglobin, lactate dehydrogenase, cytogenetics and CR were factors affecting prognosis, while multivariate analysis showed that only CR failure was an independent adverse prognostic factor for OS. The major adverse reactions to D-CAG regimen were grade 3-4 myelosuppression, pulmonary infection, and fever (infection focus was not identified). D-CAG regimen is safe and effective in the treatment of AML patients ≥70 years old, and can partially improve the prognosis of elderly and high-risk patients.

Comparing Genetic Risk and Clinical Risk Classification in Luminal-like Breast Cancer Patients Using a 23-Gene Classifier.

A 23-gene classifier has been developed based on gene expression profiles of Taiwanese luminal-like breast cancer. We aim to stratify risk of relapse and identify patients who may benefit from adjuvant chemotherapy based on genetic model among distinct clinical risk groups. There were 248 luminal (hormone receptor-positive and human epidermal growth factor receptor II-negative) breast cancer patients with 23-gene classifier results. Using the modified Adjuvant! Online definition, clinical high/low-risk groups were tabulated with the genetic model. The primary endpoint was a recurrence-free interval (RFI) at 5 years. There was a significant difference between the high/low-risk groups defined by the 23-gene classifier for the 5-year prognosis of recurrence (16 recurrences in high-risk and 3 recurrences in low-risk; log-rank test: p &lt; 0.0001). Among the clinically high-risk group, the 5-year RFI of high risk defined by the 23-gene classifier was significantly higher than that of the low-risk group (15 recurrences in high-risk and 2 recurrences in low-risk; log-rank test: p &lt; 0.0001). This study showed that 23-gene classifier can be used to stratify clinically high-risk patients into distinct survival patterns based on genomic risks and displays the potentiality to guide adjuvant chemotherapy. The 23-gene classifier can provide a better estimation of breast cancer prognosis which can help physicians make a better treatment decision.

The transcriptomic landscape of elderly acute myeloid leukemia identifies B7H3 and BANP as a favorable signature in high-risk patients.

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

Risk-Adapted Therapy Directed According to Response (RADAR, UK-MRA Myeloma XV) - Comparing MRD-Guided Treatment Escalation and De-Escalation Strategies in Patients with Newly Diagnosed Myeloma Suitable for Stem Cell Transplantation

Background. Outcomes in newly diagnosed multiple myeloma (MM) are influenced by genetic risk by FISH or molecular profiling determined prior to therapy (Shah. V et al, 2022, DOI: 10.1038/leu.2017.179), and depth of response after therapy as determined by IMWG and minimal residual disease (MRD, De Tute, RM et al, 2022, DOI: 10.1200/JCO.21.02228). Results of OPTIMUM/MUKnine (NCT03188172) provide compelling evidence for intensification of therapy in patients with genetically determined high-risk disease, but strategies to incorporate MRD as a predictive tool to reactively adapt therapy according to response are yet to be proven. Study Design / Methods. The UK-MRA RADAR study is a national, multi-centre, risk-adapted, response-guided multi-arm, multi-stage (MAMS) phase II/III trial which will recruit 1400 patients with newly diagnosed MM eligible for ASCT. Eligible patients receive induction with 4 cycles of RCyBorD (lenalidomide, cyclophosphamide, bortezomib, dexamethasone), followed by high-dose melphalan and stem cell rescue. In the current protocol, post-ASCT treatment is determined by genetic risk classification and MRD status (Figure 1). Genetic risk classification in RADAR is performed using standard-of-care NHS FISH testing at local sites. Laboratory reports are reviewed by the Trial Management Group and participants enter the standard or high-risk pathway based on the presence of at least two of the markers: t(4;14), t(14;16), t(14:20), del(17p) and gain(1q). Participants who cannot be categorised after two attempts are classed as "Unable to Determine" and treated on the standard-risk pathway. MRD analysis in RADAR uses a multi-parametric flow panel with CD319 and CD229 used to augment post-Isatuximab sample analysis at a central laboratory, with negativity defined at threshold of 10-5. For standard-risk patients, the study addresses two pivotal MRD-related questions: 1) For those with sustained MRD negativity after 12 months of isatuximab maintenance, can treatment be stopped safely to allow patients a treatment-free interval thereby reducing treatment burden? 2) For patients with persistent detectable disease after ASCT, does intensification of consolidation and maintenance therapy augment conversion to MRD negativity compared to lenalidomide maintenance? For high-risk participants the original study question was: do intensified consolidation and maintenance regimens post-ASCT show activity in prolonging disease control? With new data from OPTIMUM, however, RADAR was amended so that high risk participants receive isatuximab with cycles 2-4 of induction, 4 cycles of RBorIsaD consolidation and R-Isa maintenance until progression (Figure 1). At the same time del(1p) was added as a high-risk marker. The amended question for the high-risk pathway is: does induction, consolidation and maintenance incorporating isatuximab, show activity of prolonging disease control? Trial Progress. RADAR opened to recruitment in May 2021 and is currently recruiting at 37 NHS sites with a further 40 sites in setup. 156 patients have been recruited; 20 are classified as high risk on FISH analysis (116 standard risk, 5 unable to determine, 15 TBC). Proportions of patients with each high-risk lesion are shown in Table 1. 27 patients have completed ASCT, and 10 have reached D+100 MRD assessment. Of the standard risk patients reaching the D+100 timepoint, 4 are MRD positive and 4 MRD negative and have been randomised to consolidation and maintenance options accordingly. Assessments of COVID-19 vaccination, immunity and infection are being recorded within the RADAR trial and the effect of isatuximab will be examined. Discussion. The RADAR study simultaneously tests escalation and de-escalation strategies, according to disease response, and using MRD to guide therapy in transplant-eligible MM. Use of local results for genetic risk stratification has proven feasible in 96.5% of patients to date. Recruitment is on target and outcomes will provide RCT-level evidence for the adaptive use of MRD in treatment allocation. Finally, the platform design enabled adaptation, in this case, to include CD38 antibody for high-risk patients, ensuring up-to-date treatment based on latest evidence, keeping pace with evolving standard of care in the UK, and without altering the scientific MRD-related questions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of WT1 Mutation on AML of Different Risk Groups Based on 2022 European Leukemianet (ELN) Risk Classification

Background The Wilms tumor 1 (WT1) gene, located on chromosome 11p13 exists in multiple isoforms. It has been implicated in the regulation of cell survival, proliferation and differentiation, and may function both as a tumor suppressor and an oncogene. Approximately 15% of acute myeloid leukemia (AML) patients carry WT1 mutations, and WT1 mutation have been shown to be independent predictor of worse clinical outcome in some but not all adult AML studies. Some studies suggested that NPM1-AML & AML with CEBPA double-mutation accompanied with WT1 mutation can alter their original favorable prognosis. Recently, a revised European LeukemiaNet (ELN) genetic risk classification has been published and the prognostic significance of WT1 mutation in three risk groups according to this 2022 ELIN risk classification is still unclear. Objective To investigate the prognostic significance of WT1 gene mutations in acute myeloid leukemia (AML) patients with different risk of the 2022-ELN risk classification. Methods 502 patients de novo AML diagnosed between March 2015 and July 2021 who underwent next-generation sequencing (NGS) tests and had MICM precise typing at the Peking University Institute of Hematology were enrolled in this study. The patients received chemotherapy ± hematopoietic stem cell transplantation (HSCT), and they were stratified into three groups according to the 2022 version of ELN-AML risk stratification. Results Among 502 patients, a total of 71 (14.1%) patients carried WT1 mutations, of which 57 patients had mutations at a single locus, 8 patients had mutations at double sites, 5 patients had co-mutations at 3 sites in the WT1 gene, and 1 patient had mutations at 4 sites. Comparing the incidence of AML-related mutations between the WT1 mutation group and the wild-type group, the results showed that the concomitant rate of NPM1 mutations was lower in the WT1 mutation group than in the WT1 wild-type group (14.1% vs 27.1%, p=0.028), and the concomitant rate of CEBPA-bZIP mutations was higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.9%, p<0.001). The concomitant rate of CEBPA double mutation was also higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.4%, p<0.001), and the concomitant rate of GATA2 mutation was higher in the WT1 mutation group than in the WT1 wild-type group (14.1% vs. 4.4%, p=0.004). In addition, the WT1 gene expression level (WT1/ABL1) was higher in the WT1 mutation group than in the WT1 wild group at diagnosis (22.0% vs 13.5%, p=0.004). All patients were further divided into 6 groups based on 2022 ELIN risk classification and whether they received HSCT. There were 158 (31.5%) patients in the low-risk chemotherapy group, 73 (14.5%) in the low-risk transplantation group, 72 (14.3%) in the intermediate-risk chemotherapy group, 58 (11.6%) in the intermediate-risk transplantation group, 78 (15.5%) in the high-risk chemotherapy group, and 63 (12.5%) in the high-risk transplantation group. Univariate and multivariate COX regression analyses were performed including WT1 mutation, FAB classification, age, sex, and karyotype stratification indicators. In 58 patients who were intermediate-risk and received HSCT, patients with WT1 mutation resulted in higher cumulative incidence of relapse(CIR) in compared with those without WT1 mutation (52.3% vs 33.5%, HR=9.28, p<0.001). In the remaining 5 groups of patients, WT1 mutation seemed have no significant effect on overall survival(OS), event-free survival (EFS), or CIR in patients (p-values were all less than 0.05). Conclusion Among intermediate-risk AML patients in the 2022 version of ELN stratification who received transplantation, the CIR after transplantation was higher in AML patients with WT1 mutation. It seemed that WT1 mutation was an independent risk factor for post-transplantation recurrence, and allogeneic HSCT cannot efficiently overcome the poor prognosis of WT1 mutation in the intermediate-risk group. Intermediate-risk AML with WT1 mutations still requires more active strategies to prevent relapse. Of course, the impact of WT1 mutation on prognosis needs to be further confirmed by expanding the sample size.

Disruption of Intracellular Metal Homeostasis As a Vulnerability in SF3B1 Mutated AML

Acute Myeloid Leukemia (AML) is a heterogeneous disease in which outcome is highly dependent on cytogenetics and mutational profile. Adverse risk AML, recently defined and updated in the 2022 European LeukemiaNet (ELN) genetic risk classification, is associated with a poor overall survival. To identify novel therapeutic approaches for AML, we screened a library of 10,000 compounds across 56 genetically diverse primary AML specimens. We then looked for active compounds with increased activity towards adverse risk AML samples and with low cytotoxicity to normal hematopoietic cells. Among the hits identified, we were able to validate that S767 molecule is preferentially active on adverse risk AML samples. Additionally, we found a strong association between SF3B1 mutations and sensitivity to S767. To gain insights into S767 mechanism of action, we performed a CRISPR/Cas9 genome-wide functional screen, and identified multiples genes involved in mitochondrial processes both in synthetic rescue and synthetic lethal genes. In particular, knock-down of enzymes involved in heme biogenesis and iron-sulfur cluster biosynthesis appear to interfere with S767 cytotoxic activity. Given the central role of iron in these two synthetic pathways, and the structure of S767, we hypothesized that our molecule binds and disrupts intracellular iron homeostasis. Using UV-spectrometry analysis, we determined that S767 has metal affinity and binds to Fe2+, but also Cu2+ and Zn2+. We then quantified intracellular metal concentrations by ICP-MS (inductively coupled plasma mass spectrometry) and found that S767 exposure significantly decreases intracellular iron concentrations while increasing that of copper. Since metals are critical cellular cofactors involved in multiple enzymatic reactions, disruption of intracellular metal homeostasis can affect many processes. Mitochondrial respiratory chain is particularly sensitive to these perturbations as copper and iron-sulfur clusters are present in complexes I-IV of the electron transport chain and are essential for mitochondrial complexes function. By measuring oxygen consumption rate in leukemic cells exposed to S767, we found that the molecule indeed disrupts mitochondrial respiration. SF3B1 is a splicing factor recurrently mutated in myeloid malignancies including myelodysplastic syndromes (MDS) and AML. Heterozygous mutations typically affect SF3B1 splicing functions and induces consequently mis-splicing of target mRNAs, including the ABCB7 iron-sulfur cluster transporter. Analysis of the Leucegene transcriptomic data of primary AML specimens revealed mis-splicing and downregulation of ABCB7 in SF3B1 mutated patients. Strikingly, ABCB7 was one of the top synthetic lethal gene identified by chemogenomic CRISPR/Cas9 screen following exposure to S767 molecule. Given that ABCB7 exports iron-sulfur clusters out of the mitochondria to be assembled by cytosolic proteins and plays a role in iron homeostasis, we hypothesized that its aberrant expression in SF3B1 mutated AML participates to the high sensitivity of these leukemias to S767. We thus identified a novel molecule with selective activity in AML. Our data suggest that S767 is not a protein-targeting molecule but rather acts through the disruption of intracellular metal homeostasis, and ultimately affects mitochondrial respiration. Its mechanism of action highlighted a vulnerability in SF3B1 mutated AML which present a defect in ABCB7 expression. Importantly, SF3B1 mutations are now recognized in the 2022 ELN risk classification as an adverse prognostic marker if they are associated with intermediate or adverse risk AML, giving a unique opportunity to develop novel therapeutic approaches for this subgroup of patients with poor clinical outcome. In addition, SF3B1 mutations are found in 25% of MDS cases, which are associated with ring sideroblasts and accumulation of mitochondrial iron, and that could also benefit from such therapeutic approach.

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.