Acute Myeloid Leukemia Subgroups Research Articles

Chromatin Profiles Are Prognostic of Clinical Response to Bortezomib-Containing Chemotherapy in Pediatric Acute Myeloid Leukemia: Results from the COG AAML1031 Trial

The addition of the proteasome inhibitor bortezomib to standard chemotherapy did not improve survival in pediatric acute myeloid leukemia (AML) when all patients were analyzed as a group in the Children’s Oncology Group phase 3 trial AAML1031 (NCT01371981). Proteasome inhibition influences the chromatin landscape and proteostasis, and we hypothesized that baseline proteomic analysis of histone- and chromatin-modifying enzymes (HMEs) would identify AML subgroups that benefitted from bortezomib addition. A proteomic profile of 483 patients treated with AAML1031 chemotherapy was generated using a reverse-phase protein array. A relatively high expression of 16 HME was associated with lower EFS and higher 3-year relapse risk after AML standard treatment compared to low expressions (52% vs. 29%, p = 0.005). The high-HME profile correlated with more transposase-accessible chromatin, as demonstrated via ATAC-sequencing, and the bortezomib addition improved the 3-year overall survival compared with standard therapy (62% vs. 75%, p = 0.033). These data suggest that there are pediatric AML populations that respond well to bortezomib-containing chemotherapy.

Abstract 6206: Molecular stratification of AML: Integrative genomic and immune profiling points to high risk subgroup and targetable molecular drivers

Abstract Background: Acute Myeloid Leukemia (AML) is a complex hematological malignancy characterized by clinical diversity. The ability to accurately subtype patients can inform prognosis and guide therapeutic approaches. With the integration of multi-omics data, it is possible to elucidate the molecular mechanisms underpinning unique AML subtypes, with intent to identify potential drug targeting pathways in a precision medicine strategy. Method: RNAseq datasets from Beat-AML (discovery, N=462), TCGA-AML (validation, N=173), and Leucegene (validation, N=452) were analyzed through non-negative matrix factorization to delineate distinct transcriptomic-driven molecular subtypes within AML. A curated set of the top 1,000 most variable genes was utilized to minimize analytical noise. GSVA was used for pathway analyses across Hallmark, KEGG, Reactome, and the immune microenvironment was characterized via xCell. Recurring DNA variants were examined in each subtype. Clinical outcomes of each subtype were analyzed using the Kaplan-Meier method and assessed by a log-rank test. These insights were subsequently validated in two independent cohorts - TCGA AML and Leucegene. The most influential components of immunological effector mechanisms, which characterize poor prognostic subpopulation, were assessed using bootstrap forest partitioning as a feature selection technique. Results: Our Beat-AML analysis identified 5 distinct clusters. Cluster 3 consisted of 20% of AML cases (n=86), and displayed a worse overall survival with a HR (95% CI) of 1.41 (1.03, 1.92, P=0.03). This cluster was characterized by three co-mutations: FLT3, NPM1, and DNMT3A. Pathway analysis highlighted biological processes key to antibody therapy intervention, specifically complement pathways, NK cell cytotoxicity, and FCGR-mediated phagocytosis as enriched within this subpopulation. Concurrently, oncogenic (PI3K/AKT/MTOR, TGFB, NOTCH) and metabolic pathways (fatty acid and oxidative phosphorylation) were activated. Immune profiling indicated increased levels of M2 macrophages. Using bootstrap forest partitioning, C2 and ITGAM were identified as key genes defining cluster 3. These observations were confirmed in TCGA AML and Leucegene cohorts. Conclusion: Our comprehensive analysis identified a distinct high-risk AML subgroup characterized by unique genomic, transcriptomic, and immunological patterns. The combined presence of NPM1/FLT3-ITD and DNMT3A mutations in cluster 3—each previously attributed with varied prognostic outcomes—warrants in-depth biological investigation. With these consistent AML subgroup results identified across three independent cohorts (total of 1,087 patients), we highlight the complexities within the molecular landscape of AML and emphasize the need for tailored therapeutic approaches - a transformative shift in AML treatment. Citation Format: Kubra Karagoz, Lauren Brady, Brandon Higgs, Han Si. Molecular stratification of AML: Integrative genomic and immune profiling points to high risk subgroup and targetable molecular drivers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6206.

Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia

AbstractEpigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

Integrative single-cell expression and functional studies unravels a sensitization to cytarabine-based chemotherapy through HIF pathway inhibition in AML leukemia stem cells.

Relapse remains a major challenge in the clinical management of acute myeloid leukemia (AML) and is driven by rare therapy-resistant leukemia stem cells (LSCs) that reside in specific bone marrow niches. Hypoxia signaling maintains cells in a quiescent and metabolically relaxed state, desensitizing them to chemotherapy. This suggests the hypothesis that hypoxia contributes to the chemoresistance of AML-LSCs and may represent a therapeutic target to sensitize AML-LSCs to chemotherapy. Here, we identify HIFhigh and HIFlow specific AML subgroups (inv(16)/t(8;21) and MLLr, respectively) and provide a comprehensive single-cell expression atlas of 119,000 AML cells and AML-LSCs in paired diagnostic-relapse samples from these molecular subgroups. The HIF/hypoxia pathway signature is attenuated in AML-LSCs compared with more differentiated AML cellsbut is more expressed than in healthy hematopoietic cells. Importantly, chemical inhibition of HIF cooperates with standard-of-care chemotherapy to impair AML growth and to substantially eliminate AML-LSCs in vitro and in vivo. These findings support the HIF pathway in the stem cell-driven drug resistance of AML and unravel avenues for combinatorial targeted and chemotherapy-based approaches to specifically eliminate AML-LSCs.

How Induction Cycles before Remission Influence Long-Term Survival in Acute Myeloid Leukemia

Background ： Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by accumulation and expansion of immature myeloid cells in the bone marrow, peripheral blood and other tissues. Despite the progress made in mechanisms and approval of new therapeutics, the clinical outcome of AML remains suboptimal. The achievement of complete remission (CR) after induction chemotherapy is critical because the prognosis of refractory AML was especially unfavorable. However, little is known about the association between remission after first induction therapy and the overall survival (OS) in different subgroups of AML. Study Design and Methods: We retrospectively analyzed patients with newly diagnosed AML from January 2019 to August 2022. The diagnosis and treatment strategies were based on European Leukemia Net (ELN) criteria and National Comprehensive Cancer Network (NCCN) guideline. Composite complete remission (CRc) rate (CR+CR with incomplete recovery [CRi]) were assessed at the completion of induction or re-induction therapy. Minimal residual disease (MRD) was assessed using multiparameter flow cytometry with a minimum sensitivity of 0.1%. OS was measured from the day of diagnosis until death or the last follow-up date. Results : In total, 679 patients were included and the median age was 59.0 (range, 14.0-93.0) years old. There were 56.8% (386/679) of patients who achieved CRc after the induction therapy (group A), 18.1% (123/679) achieved CRc after the re-induction therapy (group B). The median follow-up period was 24.2 months. Even though there was no significant difference between group A and group B on OS, group A exhibited improved OS when censored at allogenic hematopoietic stem cell transplantation (HSCT) compared to that in group B (group A vs group B:3-year OS rates 65.7% vs 51.1%，p=0.004，Figure A). We further explored the effects of remission depth on survival. The results showed that, after induction therapy, patients achieved CR exhibited superior OS compared with that in CRi (CR vs. CRi, 3-year OS rates 69.4% vs 51.6%, p=0.006). Also, compared with these patients who failed to achieved MRD negativity within re-induction therapy, patients achieved MRD negativity within re-induction therapy showed superior OS when censored at HSCT (MRD- vs. MRD+, 3-year OS rates 64.7% vs 34.9%, p=0.040). After the first reduction therapy, patients achieved CRc but MRD positive benefit from HSCT (HSCT vs Non-HSCT, 3-year rates 83.0% vs 57.9%, p=0.017), but in patients with MRD negativity, HSCT did not improve the survival outcomes (HSCT vs Non-HSCT, 3-year rates 76.8% vs 65.7%, p=0.078). The next-generation sequencing (NGS) results were available in 520 patients. Based on the 2022 ELN risk stratification, the 3-year OS rates were 60.3%, 44.2% and 35.0% in favorable, intermediate and adverse group, respectively. Moreover, there were 43, 47, 60 patients in favorable, intermediate, and adverse subgroups underwent HSCT subsequently. In intermediate risk subgroup, patients who failed to achieve CRc after the first induction therapy, HSCT may serve as an effective intervention to prolong survival (3-year rates 100% vs 12.1%, P<0.001,). On the contrary, patients who achieved CRc in induction therapy, the overall survival were not influenced by HSCT status (3-year rates 81.5% vs 31.7%, p=0.153, Figure B). Conclusion: Our finding indicated that remission after the first cycle of induction therapy may be a prognostic factor for OS. Moreover, earlier and deeper remission may lead to improved survival. For those patients in intermediate risk group, who achieved CRc in induction therapy or achieved CRc with MRD negativity within re-induction therapy, HSCT may not influence the survival outcome.

CEBPA-Driven Expression of the Transcriptionally Inactive deltaTP73 Isoform Phenocopies TP53mutated Poor Risk and Drug-Resistant Acute Myeloid Leukemia

Mutations in TP53 ( TP53mut) are present in roughly 10% of acute myeloid leukemia (AML) patients and represent a unique subgroup of patients with very poor prognosis. While the exact mechanisms of TP53mut-driven leukemogenesis remain elusive, transcriptional changes associated with suppression of the TP53 signaling pathway are considered to confer a dismal prognosis on TP53mut patients. Using molecular profiling of a large cohort of AML patients (n=823) we identify a new poor prognostic subgroup in AML that is TP53 WT but shares strong similarities with TP53mut patients. Patients included in this new group present increased expression of the oncogenic TP73 gene isoform ΔNp73,which lacks the transactivation domain but still binds to chromatin. We find that the transcriptional and metabolic program of ΔNp73 high AMLs strongly resembles that of TP53mut AML, being enriched for stemness signatures like “17LSC” and “LSC UP”. In line with TP53mut AML, ΔNp73 high/ TP53 WT frequently co-occurs with U2AF1, SRSF2, TET2, and RUNX1 mutations . Lentiviral overexpression (OE) of ΔNp73 in healthy CD34 + cells increased cell proliferation and stemness. ΔNp73-OE in TP53 WT AML models, imposed drug resistance against several standard-of-care cytotoxic therapies in AML (e.g., AraC, venetoclax (VEN) ± azacytidine (AZA), and FLT3 inhibitors), while drug resistance was not further enhanced upon ΔNp73-OE in TP53mut AML models. These results were validated using ex vivo treated primary AML samples (n=80), whereby ΔNp73 high samples were more resistant to several cytotoxic therapies, including VEN+AZA . Lentiviral ΔNp73-OE in primary APL samples (n=10) allowed efficient engraftment in vivo using xenograft mouse models with colonization of secondary organs and splenomegaly, which was not observed in the empty vector controls. To unravel mechanisms via which ΔNp73controls oncogenic transformation we performed RNA-seq and ChIP-seq to identify direct transcriptional targets of TP53, ΔNp73, and the transcriptionally active isoform TAp73. These studies revealed that ΔNp73 and TP53 compete for the same target genes, whereby ΔNp73-OE resulted in complete loss of TP53 binding and strong downregulation of these loci, including downregulation of known TP53 target genes CDKN1A, BBC3, and GAS6, but also of TP53 itself. Gene set enrichment analyses of molecular programs in ΔNp73-OE cells revealed that these were associated with the terms “LSC UP”, “CEBPA targets”, and “SIGNAL TRANSDUCTION BY TP53”, indicating that ΔNp73 phenocopies TP53mut AML. Chromatin accessibility data on AML patients revealed a differentially accessible region in intron 2 (+24 Kb after the transcription start site, referred to as peak B) of the TP73 gene, with unknown regulatory functions on TP73 expression in AML. To access the potential enhancer functions of this region driving ΔNp73 expression we generated CRISPR/Cas9-KO MOLM13 cells lacking the peak B region (MOLM13-KO). MOLM13-KO cells exhibited reduced ΔNp73 expression, and the ability to up-regulate ΔNp73 expression upon high dose AraC-induced stress was abrogated. Since CEBPA was a transcription factor predicted to bind to Peak B, we investigated whether CEBPA could regulate the expression of the different TP73 isoforms. CEBPA-KD in AML cells resulted in decreased ΔNp73 levels and restored the TAp73 expression, suggesting that CEBPA controls the balance of the distinct TP73 isoforms. Pharmacological inhibition of CEBPA using the FDA-approved drug Guanfacine (GFC), diminished ΔNp73 levels and restored drug sensitivity against several cytotoxic agents in ΔNp73-OE AML cells. Moreover, the combination of GFC+VEN displayed strong synergistic effects in a cohort of ex vivo treated AML blasts (n=20), including patients with TP53mut, which are often resistant to VEN. Here, we describe a new AML patient subgroup that is TP53 WT but behaves similarly to TP53mut AML due to the expression of the ΔNp73 isoform that impairs TP53 signaling. This patient subgroup displays a very poor prognosis and is resistant to standard-of-care drugs used for AML patients. We identify a novel enhancer region that drives the expression of oncogenic TP73 isoforms, which is controlled by CEBPA, and inhibition of CEBPA by GFC enhances sensitivity to cytotoxic therapies in TP53mut-like AMLs. Our insightsfurther refine patient stratification and provide alternatives for treatment for the most dismal group of AML patients.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Acute Myeloid Leukemia (AML) Patients with Myelodysplasia (MDS) Related-Gene Mutations in First Complete Remission (CR1) Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplant (HCT)

Introduction: Acute myeloid leukemia (AML) with myelodysplasia-related gene mutations is a new disease entity introduced in the International Consensus Classification of Myeloid Neoplasms 2022 (ICC 2022). Myelodysplasia-related gene mutations include ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 . Allogeneic hematopoietic stem cell transplant (HCT) represents a curative option for subgroups of AML patients, but not for all. In a prior study (Song et al.) reported the survival benefit of allogeneic HCT in 59 patients with AML with secondary type mutations (STM) including SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2. Allogeneic HCT could abrogate adverse prognosis in AML with STMs, and reduce its mortality risk by 58% (Song et al. BMT 2022). In the present study, we aim to evaluate the frequency of this new disease entity of AML with MDS-related mutations and evaluate the prognostic impact of allogeneic HCT in patients with MDS-related mutations achieving CR1. For comparison of the benefit from HCT, the mantel-Byar test was applied to take into account time-dependent covariate of HCT in CR1 to avoid immortal bias. Patients and Methods: We conducted a retrospective study that included 1,228 adult patients who were diagnosed with AML from January 1997 to January 2020and achieved CR1 in 5 centers (Toronto, Vancouver, and Montreal, Canada; Hwasun and Seoul, South Korea). Next-generation sequencing was performed as their standard clinical practice using targeted sequencing panel in each institution. As defined in ICC 2022, AML with myelodysplasia-related gene mutations was defined as the case of AML with ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 mutation(s). We considered allogeneic HCT in CR1 as a time-dependent covariate and used a Mantel-Byar test to avoid immortal bias. We identified 278 pts (22.6%) who had at least one MDS-related gene mutation at diagnosis. Relapse-free survival (RFS) at 2 years was selected for the primary endpoint, defined as the time from CR1 to relapse or death from any cause. EZR software version 1.41 was used throughout the study for statistical analysis. Results: The median follow-up duration among survivors after achieving CR1 was 16.5 months (mo) (Range: 1.3-217.9 mo). Between the HCT vs non-HCT group, there were no differences in sex, cytogenetic risk or other somatic mutation except NPM1 mutation which was more frequently detected in non-HCT group (p <0.001). As expected, younger pts were more frequently noted in HCT group, p=0.001). First, considering HCT as non-time dependent covariate, we have compared RFS and OS between HCT vs non-HCT groups. The 1 year RFS rate was superior in the HCT group (77.7%) to that in non-HCT group (49.9%, p<0.0001; Fig.1A). A similar trend was noted for the 1-year OS rate presenting higher OS rate in the HCT group (83.8%) than in the non-HCT group (72.7%, p=0.0003). Next, when HCT in CR1 was taken into account as a time-dependent (td) covariate, Mantel-Byar analysis showed a consistent finding of superior RFS in HCT group to non-HCT group: time dependent covariate of HCT in CR1 was found to be favorable (Hazard ratio [(HR) of 0.45, (95% C.I. [0.31-0.65]; p=0.00003)] as shown in Fig.1B which is a Simon-Makuch plot. In multivariate analysis (MVA) for RFS, time-dependent covariate of HCT in CR1 retained its statistical significance, thus concluding that HCT in CR1 is an independent prognostic factor for RFS (HR: 0.44, 95% CI [0.29-0.68], p =0.0002) together with other variables found to be adverse on RFS including monosomal karyotype (p=0.03), IDH1 mutation (p=0.01), and age > 60 years (p=0.002); while CEBPA (p=0.02) and NPM1 (p=0.049) mutations were found to be favorable on RFS in MVA. Conclusion: In our cohort of 1228 AML patients, we have identified 278 AML patients with myelodysplasia-related gene mutations (22.6%). We have confirmed that allogeneic HCT in CR1 is strongly recommended in a subgroup of AML with myelodysplasia-related gene mutations based on the favorable impact of HCT in CR1 on RFS from the present study. This result supports the urgent referral of this patient group for allogeneic HCT in CR1.

Heterogeneous Gene Fusion Transcripts Found in t(7;12)(q36;p13) Acute Myeloid Leukemia but with Similar Gene Expression Profile

Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.

The Presence of Somatic Mutations on Specific Pathways Such As Chromatin Modifier, Spliceosome, and Myeloid Transcription Factor at Diagnosis Was Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplantation (HCT) in 1,228 Patients with Acute Myeloid Leukemia in First Complete Remission (CR1)

Introduction Allogeneic stem cell transplant (HCT) brings survival benefit to certain subgroups of acute myeloid leukemia (AML) patients, although it may not be suitable for all cases. Decision for HCT referral is based on the ELN risk grouping, which classifies pts into favorable, intermediate, and adverse risk categories. While HCT is recommended for pts in the intermediate and adverse risk, those in the favorable risk are deferred to receive HCT. However, currently, there is a lack of data directly demonstrating benefits of HCT in specific mutation groups. Patients and Method In this retrospective study, out of 1477 newly diagnosed AML pts 1,228 pts achieved CR1 and had undergone baseline next-generation sequencing (NGS) at diagnosis from January 1997 to January 2020. The data were collected from five different centers located in Toronto, Vancouver, and Montreal, Canada, as well as Hwasun and Seoul, South Korea. NGS was performed in each institution with each institution's standard targeted sequencing panel. To assess the effectiveness of HCT, particular attention will be given to the time from achieving complete remission 1 (CR1) to HCT. To handle immortal time bias, the Mantel-Byar method was used to depict outcomes during the follow-up period. Kaplan-Meier curves were avoided as they can introduce immortal bias, considering the period of time before treatment as part of the treatment arm. Instead, the MBT and landmark methods were employed to address this issue. Allogeneic HCT was treated as a time-dependent variable to prevent immortal bias. Gene mutations were grouped into 6 biologic pathways, including DNA methylation, chromatin modifiers, cohesion complex, activated signaling, tumor suppressor, spliceosome, and myeloid transcription factor (TF) mutations. In the multivariate (MVA) analysis, any mutations with a frequency of >5% were included as well as TP53 which has very grave prognosis. The primary objective of this study is to identify specific groups of AML pts who can derive significant benefits from receiving HCT in CR1, treating HCT as a time-dependent (td) covariate in the analysis. Statistical analyses were conducted using EZR version 1.41. Results The median duration of follow-up among survivors after achieving CR1 was 22.6 months (range: 1-217.9 months). Among the 593 pts, who underwent allogeneic HCT in CR1, the HCT group was composed of younger pts, with a median age of 50 years compared to 56 years in the non-HCT group (p < 0.001). Also, there were fewer pts with NPM1 mutation in the HCT group, accounting for 20.7% compared to 32.4% in the non-HCT group (p < 0.001). Apart from these characteristics, the two groups were similar in other parameters. The 2-year overall survival (OS) was higher in the HCT group, at 66.4%, compared to 54.6% in the non-HCT group (p < 0.0001). Similarly, the 2-year RFS was higher in the HCT group, with a rate of 64.8%, compared to 52.1% in the non-HCT group (p =1.82e-15). Furthermore, HCT as a time-dependent covariate was found to be favorable (Hazard ratio [HR] 0.76, 95% C.I. [0.63-0.91]; p=0.003), as demonstrated in the Simon-Makuch plot (p = 0.003). In the MVA, HCT as a time-dependent covariate retained its favorable impact on RFS (HR 0.67, [0.56-0.82]; p<0.0001). The MVA showed several prognostic factors besides HCT such as NPM1 and CEBPA mutations as favorable factors for RFS, being classified under the European Leukemia Network (ELN) favorable risk group. Conversely, FLT3-ITD, DNMT3A, IDH1, KIT, and TP53 mutations, monosomal karyotype, age > 60 years, and white blood count (WBC) at diagnosis > 55,600/mm^3 were associated with worse RFS. In terms of RFS, the greatest benefit of HCT was observed in the pts having mutations in ASXL1 (HR 0.34 [0.15-0.78]), BCOR (HR 0.29 [0.14-0.61]), IDH1/2 (HR 0.62 [0.42-0.92]), and RUNX1 (HR 0.17 [0.08-0.40]) but no significant benefit in those with KRAS (HR 1.59), NRAS (HR 1.21), PTPN11 (HR 1.23), and WT1 (HR 1.30). According to the biological pathway of somatic mutations, the greatest benefit of HCT in terms of RFS was observed in pts with mutations in the chromatin modifier (HR 0.37 [0.22-0.62]), spliceosome (HR 0.32 [0.16-0.65]), and myeloid TF pathway (HR 0.52 [0.34-0.79]). Conclusion Allogeneic HCT exerts a greater benefit on RFS in AML pts with mutations in the chromatin modifier, spliceosome, and myeloid TF pathways. Further study is warranted to replicate this finding.

Efficacy and Safety Profile of CPX-351 in Secondary Acute Myeloid Leukemia (sAML): A Systematic Review

Secondary Acute Myeloid Leukemia (sAML) is a subgroup of Acute Myeloid Leukemia (AML) associated with poor outcomes despite recent advancements in chemotherapy. CPX-351 (United States: Vyxeos®), a 1:5 liposomal encapsulation of daunorubicin and cytarabine was approved in 2017 by the US Food and Drug Administration (FDA) for the treatment of newly diagnosed Secondary AML. Previously, the conventional 7+3 Regimen (7 days of cytarabine and 3 days of daunorubicin) was considered first line for the treatment of sAML. However, the pivotal study that led to approval of CPX-351 demonstrated increased Overall Survival (OS) with CPX-351. We report a systematic review of published literature on the efficacy and safety of CPX-351 in patients with sAML, including data from post-market real-world experiences of patients. Materials and Methods Following PRISMA guidelines, we performed a comprehensive literature search for articles published after 2017 using PubMed, Embase, Clinical Trials.gov, Cochrane Library, and Web of Science. Initially 63 articles were identified, and after a thorough screening, we finalized 9 studies including 767 patients, illustrated in Table 1. We analyzed data from patients who received CPX-351 chemotherapy as first-line therapy for sAML. MeSH terms and keywords were 'CPX-351' and 'Leukemia, Myeloid, and Acute'. Results The median age of patients was 65.9 years. Patients could receive up to two cycles of induction, and those who achieved complete remission (CR) or complete remission with incomplete recovery (CRi) could receive up to 2 cycles of consolidation with CPX-351. CPX-351 treatment showed median overall survival (mOS) of 12.20 months, CR rate of 43.66%, and an overall remission rate (ORR) of 51.1%. Hematopoietic Stem Cell Transplantation (HSCT) was performed in 39.1% patients. The mean time to absolute neutrophil count >500/mL (mANC) and platelet count >50,000/mL (mPC) recovery in patients who achieved OR was 34.4 days and 32.9 days, respectively. The most frequently reported greater than grade 2 adverse events (AEs) were febrile neutropenia (NF), opportunistic infections including pneumonia, and hypoxia. Conclusion In sAML patients, the CPX-351 regimen is more effective and well tolerated as compared to the conventional 7+3 Regimen. The real-world data also points to CPX-351 as an efficient therapeutic agent for sAML patients facilitating HSCT in many patients with promising outcomes after transplantation. CPX-351 is a treatment of choice for patients aged >60 years. Future randomized prospective trials with longer follow-up periods are needed to consolidate these findings, and help us better understand the efficacy and survival outcomes of CPX351 regimens for sAML treatment.

Comparable Survival for Fludarabine and Treosulfan Vs. Fludarabine and TBI As Conditioning Regimens in Patients with AML and MDS Undergoing Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) is a standard treatment option for patients (pts) with acute myeloid leukemia (AML) and myelodysplastic neoplasia (MDS) with relapse remaining the main cause of treatment failure. For AML pts transplanted in complete remission (CR) or MDS pts, the combination of fludarabin with fractionated total body irradiation (8 GyTBI, “FluTBI”) is an established conditioning regimen. Based on the favorable outcome data reported for fludarabine/treosulfan (“FluTreo”), especially for older and/or comorbid pts, this regimen has become a reliable dose reduced conditioning therapy prior to alloSCT and a valuable alternative to other TBI or chemotherapy-based regimens. Methods We conducted a retrospective analysis of 215 pts with AML in CR and 96 pts with MDS who underwent first alloSCT between 2010 and 2022 with a median follow-up (FU) of survivors of 34 months (range: 1 - 124 months). After conditioning therapy with FluTreo (150mg/m 2 fludarabin, 30g/m 2 treosulfan) or FluTBI (120mg/m 2 fludarabin, 4x2Gy TBI) pts were allografted from matched related (MRD; n=65), 10/10 human leukocyte antigen (HLA)-matched unrelated (MUD; n=200), or 9/10 HLA-matched unrelated donors (MMUD; n=46). For AML pts, any detectable molecular/cytogenetic alteration prior to alloSCT was classified as potentially measurable residual disease. Propensity score matching allowed us to identify 55 pair-matched FluTBI and FluTreo pts (nearest matching for age at alloSCT, sex, disease, HCT-CI score, ECOG, with a caliper distance of 0.5). Results Before matching, pts characteristics differed significantly between FluTreo and FluTBI in terms of age at alloSCT (median 64 vs. 47 yr), underlying disease (AML: 59% vs. 88%), ECOG (≥2 15% vs. 6%) and HCT-CI-Score (≥3 50% vs. 25%). In the AML subgroup, pts with measurable residual disease prior to alloSCT were significantly more frequent in the FluTreo group (68% vs. 48%, p<.001). GvHD prophylaxis consisted of calcineurin inhibitor and methotrexate/MMF in all pts. In addition, pts transplanted from unrelated donors received ATLG (Neovii). In the FluTreo group, more patients were transplanted from MRDs (24% vs. 14%, p.013). After matching, patient characteristics were well balanced with respect to age (median 57 vs. 55 yr, p.11), disease (AML/MDS) ( p .8), HCT-CI score ( p .24), sex ( p .85), donor types (MRD: 25.5% vs. 14.5%, MUD: 58.2% vs. 67.3%, MMUD: 16.4% vs. 18.2%, p .36) and measurable residual disease prior to alloSCT ( p .58). Risk groups for AML and MDS pts at diagnosis were comparable across both groups (ELN for AML and IPSS-R for MDS). Relapse incidences (RI) at 3 yrs were similar for FluTreo and FluTBI pts in the unmatched (25% vs. 25%, p.8) and matched cohorts (28% vs. 28% p .7). The non-relapse mortality (NRM) rate was <10% trending towards a lower NRM rate for the FluTBI group in the unmatched cohorts (at 1 yr: 8% vs. 5%, p .07) and a significant difference after matching (at 1 year 2% vs. 9% p .03). Overall survival (OS) for the whole cohort showed a trend towards an inferior outcome for FluTreo (3 yr: 71% vs. 79% p .06), after matching, no difference could be observed (3 yr: 80% vs. 71% p .54). Leukemia-free survival (LFS) was comparable across conditionings in the unmatched (59% vs. 66% p .42) and the matched cohorts (70% vs. 59% p .42). Irrespective of conditioning therapy, pts with measurable residual disease prior to alloSCT had comparable OS and LFS. However, for the matched cohort, pts with measurable disease treated with FluTreo showed a trend towards an inferior OS and LFS after 3 yr in contrast to FluTBI pts (OS: 66% vs. 80%, p .13; PFS: 58% vs. 67%, p .3). No significant differences could be observed in the incidence of acute onset graft versus host disease (GvHD) grade 2-4 at day 100 after alloSCT. Similarly, occurrence of chronic GvHD at 3 yr did not differ in the matched cohorts (32% vs. 43%, p .4). Conclusion With a meaningful median follow up of 34 months, our data for dose-reduced conditioning with either FluTreo or FluTBI highlight favorable and comparable survival rates of >70%. RI and NRM were low for both regimens. For pts with AML, who received FluTBI, measurable residual disease prior to alloSCT seemed to have no effect on outcome, while for the FluTreo cohort measurable residual disease prior to alloSCT showed a trend towards lower survival rates. This finding should be evaluated in a prospective trial comparing TBI and treosulfan based conditioning.

Identification of Acute Myeloid Leukemia Cell Surface Therapeutic Targets Using Single Cell RNA Sequencing Supported By Surface Proteomics

Background: Acute myeloid leukemia (AML) comprises diverse genomic subgroups and remains hard to treat in most patients. Despite breakthroughs in the therapeutic arsenal in recent years, clinical usage of therapeutic antibodies or chimeric antigen receptor T (CAR-T) cells has been lagging in contrast to other hematological malignancies. In fact, CD33 represents the only antibody-based strategy approved for this disease to date, highlighting the need to identify new promising targets. AML cells span a wide range of aberrant myeloid differentiation programs, complexifying the identification, by bulk genomics, of targets expressed in the most immature leukemic cells. Aims and Methods: To identify the expression landscape of surface proteins in immature leukemic cells, we performed single-cell RNA sequencing (scRNA-seq, 10x 3' Reagent Kits) of primary human AML cells from 20 specimens of the Leucegene cohort enriched in intermediate and adverse genetic backgrounds ( KMT2A-rearranged n=5, chromosome 5 and/or 7 deletions (abn5/7, n=5) complex karyotype (n=4), NPM1/DNMT3A/FLT3-ITD triple-mutant (n=3) and others (n=3)). A Random Forest classifier was developed to unbiasedly classify AML cells into distinct differentiation stages using normal bone marrow-derived scRNA-seq data from the Human Cell Atlas (HCA) consortium. Genes were scored based on their probability of coding for proteins expressed at the cell surface using the SPAT algorithm developed by our group (https://doi.org/10.1101/2023.07.07.547075), retaining high score ones. To validate surface expression, we concomitantly analyzed the surface proteome (hereafter named surfaceome) of 100 primary human AML samples from the Leucegene cohort, including all 20 samples profiled by scRNA-seq. Results: After quality control, we profiled and characterized 103 690 high quality cells (mean of 5185 cells/sample). We trained a Random Forest classifier to annotate cells in a two step process, first identifying plasma cells based on a restricted list of genes abundantly expressed in these cells and subsequently assigning the remaining cells to one of 33 cell types. We performed a five-fold cross validation of the model and subsequently determined the accuracy of our classifier to be 92% on the test subset of the HCA data. Applied to our AML cell collection, a total of 35 053 cells (34%) were unbiasedly classified as Hematopoietic Stem Cell (HSC)-like, corresponding to the most phenotypically immature leukemic cells in each patient sample (ranging from 4 to 74 %). Accordingly, HSC-like AML cells preferentially express genes associated with normal HSCs, such as CD34, FAM30A, and SPINK2, and globally lack expression of mature lineages defining genes, further validating our classifier. The proportion of HSC-like cells varied among AML subgroups, and was lowest in KMT2A-r AML (median 19%) and highest in abn5/7 samples (46%). Integration of our AML atlas using Harmony algorithm preserved differentiation hierarchies across samples, with most cell types, including HSC-like cells, occupying a defined area in the low dimensional embedding. To identify new surface antigens specifically expressed in immature leukemic cells, we compared the high (≥8) SPAT score gene expression profile of AML HSC-like cells with that of normal HSC cells (HCA), and identified 60 genes significantly overexpressed in AML immature cells. Of those, 39 genes were also detected at the protein level by the surfaceome analysis, supporting their predicted expression at the cell surface in AML samples. 59% of these 39 genes (n=23) were detected in over 80% of the specimens analyzed by the surfaceome, and thus are nearly universally expressed in our AML cohort. To identify targets of therapies that could be repurposed, we next evaluated the relevance of our findings by querying the Thera-SAbDab database. Most interestingly, 8 of the 39 AML specific HSC markers are targeted by therapeutic antibodies FDA-approved or in clinical trials for the treatment of AML (n=4, IL3RA, FLT3, CD37 and TNFRSF10B) or other indications (n = 4). Conclusion Our genetically diverse AML single-cell atlas, supported by mass spectrometry, enables the identification of both subset-specific and pan-AML surface protein genes. These represent potential targets for antibody based strategy development or therapy repurposing in AML.

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<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<0.01). The FUS-ERG patients had a less median overall survival (OS) when compared to the reference cohort (13 vs. 35 months, p<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<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<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<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<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 >60 years (HR=2.96, 95%CI: 1.14-7.73, p=0.026), WBC >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<0.001) and DFS (HR=0.31, 95%CI: 0.16-0.57, p<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.

Preferential Efficacy of Remission Maintenance with HDC/IL-2 in Age Groups of Patients with Chemoresponsive or Normal Karyotype AML

Background Relapse in complete remission (CR) remains a leading cause of morbidity and mortality in acute myeloid leukemia (AML). Previous phase 3 trial data show that combinatorial immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) reduces relapse incidence in the post-consolidation phase, in particular among younger adult AML patients (<60 years old) in CR1 (Brune et al., Blood 108:88). Recent post-hoc analyses identified subgroups of responding patients including (1) those achieving CR1 after the first induction course of chemotherapy (referred to as chemoresponsive AML) and (2) those carrying leukemic cells of normal karyotype (NK-AML), i.e. devoid of structural or numerical chromosomal aberrations. Here we report analyses of treatment efficacy in a phase 3 trial within age groups of patients with chemoresponsive AML or NK-AML. Methods Three hundred and twenty AML patients (18-84 years old, median 57) who were ineligible for allogeneic stem cell transplantation were randomly assigned to receive HDC/IL-2 or no treatment (control) for remission maintenance in a phase 3 trial. Patients were recruited in Europe, Israel, Oceania, and USA. Treatment was initiated in CR after the completion of consolidation chemotherapy and comprised 10 consecutive 3-week cycles of HDC/IL-2 for 18 months (mo) with 3- or 6-week rest periods. In each cycle, patients received HDC at 0.5 mg and human recombinant low-dose IL-2 (aldesleukin; 16,400 IU/kg) s.c. twice daily (n=160) or no treatment (control; n=160). After 18 months of treatment, all patients were monitored for >18 additional mo (median follow-up 48 mo). Results Among study subjects in CR1 203/261 (78%) of evaluable patients attained CR after the first induction cycle (chemoresponsive AML) and 128/220 (58%) had NK-AML. Within both subgroups, analysis of efficacy by age at diagnosis implied preferred benefit of HDC/IL-2 vs. control in patients 40-59 years old (Figure 1) with independent efficacy in patients 40-49 and 50-59 years old (Table 1). The treatment arm was not significantly superior over the control arm for LFS or OS in the youngest patients (18-39 years old) or in patients >60 years old (P>0.2, logrank test, for all comparisons (LFS and OS)) within subgroups of chemosensitive AML or NK-AML. Conclusions We speculate that the observed benefit of HDC/IL-2 in patients responding swiftly to initial chemotherapy may be explained by more efficient immune-mediated elimination of leukemic cells at a lower burden of leukemia. The preferential efficacy of HDC/IL-2 in NK-AML points towards efficacy in subgroups of leukemic mutations that are less common in the youngest group of patients. The reason for the lack of significant efficacy in patients >60 years old, despite achievement of CR1 after the initial chemotherapy and despite having NK-AML, remains unknown but may be explained by fading anti-leukemic immunity in old patients. Overall, these findings may be considered for the selection of patients suitable for immunotherapy with HDC/IL-2 in an emerging landscape of strategies for relapse prevention in AML.

Clonal Dynamics and Deterministic Clinical Fate Mapping of Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukemia with TP53 Disruption

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) with multi-hit TP53 comprise the highest risk subgroup of all myeloid malignancies. TP53 aberration is recognized as a diagnostic category in the 2022 International Consensus Classification (ICC) and the 5 th edition of the WHO. However, these systems do not discern among the subtypes of TP53 aberrations regarding response to therapy. In this study, we explore the clonal dynamics that underlie such prognostic heterogeneity within distinct subgroups of TP53-aberrant MDS and AML, with a specific focus on the effect of hypomethylating agent (HMA) vs. araC-based chemotherapy on TP53 variant allele frequency (VAF). Methods: The UMass Leukemia Registry from the UMass Data Lake identified 76 patients harboring TP53 aberration(s) plus ICD-10 code of either D46.9 (MDS and its subentities) or C92.00 (AML and its subentities) between 2011-2023. Data was integrated with hematopathology records. TP53 exome sequencing was performed with coverage depth 500X to 6500X, and VAFs were adjusted for copy number variation (CNV), as previously described (Patel SA et al., Leuk Lymphoma 2021; 62: 3348-60). Patients with serial bone marrow biopsy samples were included. We defined multi-hit TP53 status as per the ICC: two or more distinct TP53 mutations with VAF ≥ 10%, or a single TP53 mutation plus one of the following abnormalities: (1) del(17p13.1), (2) TP53 VAF >50%, (3) copy-neutral loss-of-heterozygosity, or (4) any complex karyotype. Clonal dynamics were assessed for MDS vs. AML and for varying TP53 allelic states as a function of therapeutic intervention. Clinical fate mapping was performed based on management decisions at various points of clinical care. Results: For patients with TP53-aberrant MDS, 17 patients underwent serial bone marrow biopsies during treatment. Of these, 11 (64.7%) had multi-hit status. Eight patients (47.1%) underwent hematopoietic cell transplant (HCT), and 7 of these 8 patients had received HMA-based front-line therapy. For patients with MDS, we assessed the effect of HMA-based therapy on TP53 VAF. The mean TP53 VAF (sum of VAFs) was significantly lower after HMA-based treatment (35% vs. 12.6%) ( p = 0.0075) (Panel A). For patients with TP53-aberrant AML, 19 patients underwent serial bone marrow biopsies. Of these, 14 (73.7%) had multi-hit status. Only 2 patients (10.5%) underwent HCT. Five patients (26.3%) received araC-based induction, while 11 patients (57.9%) initially received HMA-based induction. For patients with AML with multi-hit TP53, we assessed the effect of HMA-based vs. araC-based front-line therapy on TP53 VAF. HMA therapy led to mean fold reduction in TP53 VAF (pre-treatment to post-treatment) of 11.7 ± 5.8 ( p = 0.019), while araC-based therapy actually led to mean fold increase (but not statistically significant) in TP53 VAF (pre-treatment to post-treatment) of 2.6 ± 1.05 ( p = 0.319) (Panel B). HMA-based treatment resulted in significantly greater reduction in the VAF in aberrant clones and subclones compared to araC-based treatment. Compared to AML, most patients with MDS (62.9%) proceeded with palliative intent therapy at the time of diagnosis. Although patients with AML (53.7%) attempted to proceed with curative intent at the time of diagnosis, only 14.6% eventually proceeded with HCT, largely due to clinical decompensation or disease progression during front-line therapy. Among HCT recipients for MDS ( n =10), 7 (70%) remained in remission. Among HCT recipients for AML (n = 6), 4 (66.7%) remained in morphologic leukemia-free state. Conclusion: There is limited literature on clonal and subclonal diversity in patients with MDS or AML with TP53 aberrations. Such considerations constitute an important therapeutic issue, as certain interventions may only eliminate a fraction of a patient's mutant cells. In our study, HMA- and araC-based regimens had differential effects on various subclones: HMA was more effective in reducing the TP53 mutational burden and increasing patient ability to proceed with HCT. AraC-based therapy appeared less effective, perhaps due to selection pressure in favor of TP53-mutant cells. Prospective trials are required to identify optimal regimens that lead to the best long-term outcomes, and single-cell resolution might assist with more definitively assessing dynamics of clonal response to treatment.

Olutasidenib for the Treatment of mIDH1 Acute Myeloid Leukemia in Patients Relapsed or Refractory to Hematopoietic Stem Cell Transplant, Prior mIDH1 Inhibitor, or Venetoclax

Introduction. Olutasidenib is a small molecule, oral, mutated-IDH1 (mIDH1) inhibitor approved for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML). In the pivotal cohort of the registrational phase 1/2 trial, 51/147 (35%) patients achieved a complete remission (CR) or CR with partial hematologic recovery (CRh) to olutasidenib, with a duration of response of 25.9 months and an overall response rate (ORR) of 48%. The phase 1/2 study included 10 cohorts for a total of 335 patients, and there were patient subsets that were R/R to previous hematopoietic stem cell transplant (HSCT), ivosidenib (IVO) or venetoclax (VEN). We conducted post hoc analyses to better understand the response to olutasidenib in these poor-prognosis subgroups of mIDH1 AML. Methods. This Phase 1/2, open-label, multi-center study enrolled patients with confirmed AML or intermediate, high-, or very high-risk myelodysplastic syndromes/neoplasms (MDS). Patients received olutasidenib 100mg QD, 150mg QD or 150mg BID monotherapy or the combination of olutasidenib 150mg BID plus azacitidine. Patients were grouped by prior therapy. Baseline demographics and disease characteristics are shown in Table 1. The study was not powered to statistically evaluate subpopulations; descriptive statistics are provided. Data cutoff date is June 2021. Results. The post-HSCT group (n=31) had a relatively high median baseline bone marrow blast count (58%) and a median of 4 prior treatment regimens ( Table 1). Six (19%) of these patients had a CR (including the 1 patient with MDS), and 3 (10%) patients had a CR with incomplete count recovery (CRi) resulting in a 29% composite complete remission (CRc) rate. One patient had a morphologic leukemia-free status, resulting in a 32% ORR. Half the responders received monotherapy, and half received a combination of olutasidenib and azacitidine. Two patients were enrolled in the monotherapy maintenance cohort while in CR to prior HSCT; one of these patients maintained a CR for 13 months before progressing, and the other patient progressed and discontinued due to a new central nervous system disease ( Table 2). For the 10 patients with responses (ORR), the median duration of response was 7.1 months (range 1-23.4+). At data cutoff, 2 patients were ongoing responders, 3 patients proceeded to a second HSCT, and 3 to donor lymphocyte infusion (DLI). The post-IVO group (n=9) had a high median baseline bone marrow blast count (83%) and a median of 4 prior treatment regimens ( Table 1). Two patients (22%) achieved a CR; both CRc and ORR were 22% ( Table 2). Both responders had received olutasidenib in combination with azacitidine. One responder proceeded to HSCT after 3.1 months, and one responded for 5.6 months and then had disease progression. Response rates in the post-VEN group (n=20) included CR in 6 patients (30%), CRh in 1 (5%), and CRi in 2 (10%) resulting in a CRc of 9 (45%) and an ORR of 45% ( Tables 1 and 2). One responder received combination olutasidenib and azacitidine. Two patients were enrolled in the monotherapy maintenance cohort while in CRi to prior treatment; both improved their response to a CR. Median duration of response was not reached and ongoing at 28.5 months. At data cutoff, 5 patients continued treatment, and 15 had discontinued. Conclusions. This descriptive analysis suggests that olutasidenib alone or in combination with azacitidine may induce complete remissions in patients with mIDH1 AML or MDS that is R/R to VEN, IVO or even HSCT. Further investigation of these difficult-to-treat subpopulations is needed. Although only a small number of patients receiving maintenance therapy were included in this analysis, the data show that maintenance of a CR and even improvement of response from CRi to CR is possible with olutasidenib.

Independent Prognostic Value of DNA-Based Minimal Residual Disease Assessment in Unselected Population-Based Cohort of Pediatric Acute Myeloid Leukemia

Monitoring of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) can assess response to treatment and predict relapse. Current MRD monitoring methods in childhood AML - flow cytometry and quantitation of fusion transcripts - have significant shortcomings and do not cover the entire spectrum of patients. We demonstrated (Lukes et al, Hemasphere 2020) the feasibility of MRD monitoring using DNA-based primary targets in selected subgroups of AML. Now we extended this approach to the unselected consecutive population of pediatric AML. We included 133 children (0-18) out of 135 consecutively diagnosed with AML in Czechia (2012-2022), all treated on non-MRD-based protocols. For genetic characterization, we used algorithm consisting of screening for recurrent gene fusions and mutations by PCR and targeted-NGS followed by transcriptome sequencing (WTS). Targeted-NGS (or PCR) was performed to identify genomic fusion sequences. 130 patients were eligible for MRD monitoring (3 patients died early). MRD was preferentially monitored by DNA-based qPCR (alternatively by amplicon NGS or qRT-PCR). MRD levels were expressed relative to diagnosis. Primary genetic aberration was found by targeted screening in 102 children; remaining 31 were further investigated using WTS. In 27 children, WTS identified rare, novel or atypical primary genetic aberrations not covered by the targeted screening. Primary genetic aberration was found in 97% of cases (129/133). Majority of AMLs (81%) were classified into common subtypes: AML with KMT2Ar, PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, mutation (m) of GATA1 , CEBPA or NPM1. Recurrent but rare aberrations in pediatric AML were found in 16 patients: UBTFm, RUNX1m, HOXA10 translocation, KAT6A::CREBBP, KAT6A::LEUTX, DEK::NUP214, BCR::ABL1, NUP98::NSD1 and CBFA2T3::GLIS2. In 5 patients, fusion genes were found, previously described sporadically (SPFQ::ZFP36L216, XPO1::TNRC1817) or not at all in AML (ETV6::CTNNB1, FUS::FEV, ZEB2::RUNX1). Only secondary aberrations were identified in remaining 4 patients. In 82 patients (of 84 examined), genomic fusion sequence was found and qPCR designed and used. Of 41 children carrying genetic aberrations other than fusions, in 29 quantification system was successfully implemented. Altogether, quantification system for MRD monitoring was established in 122 patients, with primary aberrations as DNA targets in 116 patients, reaching sensitivity of 10 -4 in 112 and 5x10 -4 in 4 patients. Four and two children were monitored with sensitivity of 10 -4 using secondary aberrations as DNA targets or by qRT-PCR-based quantification of fusion transcripts, respectively. MRD clearance significantly differed among genetic subtypes: while GATA1m AML had the fastest response, in contrast to patients with prognostically favorable genetic subtypes - CBFB::MYH11, RUNX1::RUNX1T1, CEBPAm and NPM1m - who were treated predominantly on the standard risk (SR) arm of the AML-BFM 2012 Registry Protocol and none achieved molecular remission (mREM) at D28 (70% vs 0%, p < 0.0001). Significant proportion of patients (25-89% within individual subtypes) did not achieve mREM even after the last block of chemotherapy. The response of patients with KMT2Ar AML and AML classified into remaining subtypes (predominantly intermediate- and high-risk) was overall faster compared to prognostically favorable subtypes on SR arm (KMT2Ar D28 mREM 33% vs 0% at D28, p < 0.0001). KMT2A::MLLT10/3 had faster clearance then other KMT2Ar (mREM D28 44% vs. 0% in other KMT2Ar, p = 0.02). In a multivariate analysis including (cyto)genetic risk and treatment, D28 MRD was the only significant predictor of outcome using both the 10 -3 (p = 0.006 for EFS and 0.012 for OS) and 10 -2 levels (p = 0.004 for EFS and 0.01 for OS) for stratification. Similarly, in an alternative multivariate analysis model, D56 MRD (positive at any level versus negative) was also the only significant predictor of outcome independent of risk and treatment (p = 0.005 for EFS and 0.01 for OS) In summary, we present a strategy for MRD monitoring in pediatric AML that is technologically feasible, real-life applicable to vast majority of all patients, and has clear prognostic significance. Supported by National Institute of Cancer Research No. LX22NPO5102 funded by the European Union-Next Generation EU, and AZV grant NU20-07-00322.

Real-World Treatment Patterns Among Patients with Newly Diagnosed (ND) Acute Myeloid Leukemia (AML) with TP53 Mutation in the United States (US)

Introduction: A unique subgroup of AML is defined by the presence of the TP53 mutation, with worse overall survival (OS) and limited therapeutic options (Döhner et al, Blood 2022; Daver et al, J Hematol Oncol 2023). Clinical trials are essential for establishing the efficacy and safety of new treatment options in AML; yet, they may not be generalizable to patients with TP53 mutation, who are often older adults with comorbidities and prior treatment exposure. Thus, the optimal treatment for TP53 mutated AML is unclear. Real-world data (RWD) can provide insights into demographic and clinical characteristics, as well as the utilization, effectiveness, and safety of treatment regimens used in routine clinical practice. This RWD study aimed to describe the characteristics, first line treatment patterns, and outcomes in patients with ND-AML with TP53 mutation in a real-world cohort of adult patients in the US. Methods: A retrospective observational study was conducted using the COTA real-world database, a de-identified source of longitudinal electronic health record (EHR) data pertaining to the diagnosis, clinical management, and outcomes of patients with cancer. The study population included adult patients (≥ 18 years) with ND-AML and confirmed TP53 mutation status (positive or negative) per Next-Generation Sequencing (NGS), who were diagnosed between January 1, 2013, and March 20, 2023. Patients were excluded if key study data were missing (i.e., if TP53 mutation status was unknown, or if the testing methodology was unspecified), or if an AML-defining recurrent genetic abnormality superseded TP53 in the hierarchical classification of the International Consensus Classification. First-line treatment regimens were mapped to relevant therapeutic categories (e.g., intensive chemo [IC] with cytarabine, hypomethylating agent with venetoclax [HMA+ven]) and summarized across clinical and demographic patient characteristics using descriptive analyses. Results: A total of 884 patients ND-AML with analysis of TP53 status were identified, including 173 TP53 positive (TP53+) and 711 negative (TP53-). Patients who were TP53+ were older, and otherwise demographically similar to those who were TP53- (see table). Testing for TP53 increased over time, with about 75% of tested patients being diagnosed in 2018-2022 versus 25% in 2013-17. All patients who were TP53+ were by definition ELN adverse risk group, whereas 36.8% of patients with TP53- AML were adverse risk. The most commonly used treatment regimens included hypomethylating agent (HMA) alone (17.9% of TP53+ and 11.8% of TP53-), HMA with venetoclax (HMA+ven) (28.9% TP53+, 20.3% TP53-), and IC w/cytarabine (26% TP53+, 46.8% TP53-). Patients with TP53+ AML were more likely to not receive any anti-leukemic treatment. Patients with TP53+ AML were less likely to achieve complete remission (CR) compared to those with TP53- AML (31.2% versus 46.1%, excluding patients in each subgroup who did not receive anti-leukemic treatment). In this cohort, OS was shorter for patients with TP53+ AML compared with those with TP53- AML. This difference was seen consistently in patients treated with the two most common regimens (IC w/cytarabine and HMA+ven) (Figure). OS appeared longer for patients treated with IC w/cytarabine in patients with or without TP53 mutation, but it should be noted that these patients were younger and generally more fit than patients receiving HMA+ven (61 years versus 75 years among patients with TP53+ AML). Conclusions: In patients with TP53+ AML, CR and OS are lower overall and when specific treatment options are compared to patients with TP53- AML. This difference was seen in patients receiving either of the two most commonly used treatment regimens. This study demonstrates the capability of using RWD to characterize the outcomes of a specific genetic subtype of ND-AML despite widespread testing for this marker only occurring in the latter half of the study timeframe. These findings are subject to the inherent limitations associated with observational data, including the challenges in collecting and analyzing real-world data (e.g., missing treatment data or other characteristics) which may result in bias or limited generalizability of study findings. Finally, this study further demonstrates that research into novel treatment of TP53+ AML is needed to improve patient outcomes.

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.