High-risk Acute Myeloid Leukemia Patients Research Articles

Long-term follow-up of patients with acute myeloid leukemia undergoing allogeneic hematopoietic stem cell transplantation after primary induction failure

Primary induction failure (PIF) in acute myeloid leukemia (AML) patients is associated with poor outcome, with allogeneic hematopoietic stem cell transplantation (HCT) being the sole curative therapeutic option. Here, we retrospectively evaluated long-term outcomes of 220 AML patients undergoing allogeneic HCT after PIF who never achieved remission, and identified clinical and molecular risk factors associated with treatment response and ultimate prognosis. In this high-risk population, disease-free survival was 25.2% after 5 years and 18.7% after 10 years, while overall survival rates were 29.8% and 21.6% after 5 and 10 years of HCT, respectively. 10-year non-relapse mortality was 32.5%, and 48.8% of patients showed disease relapse within 10 years after allogeneic HCT. Adverse molecular risk features determined at initial diagnosis, poor performance status at the time of allogeneic HCT, and long diagnosis-to-HCT intervals were associated with unfavorable prognosis. Collectively, our data suggests that immediate allogeneic HCT after PIF offers long-term survival and cure in a substantial subset of cases and that high-risk AML patients who never achieved complete response during induction might benefit from early donor search.

Maintenance Therapy with Chidamide Plus Azacitidine after Allogeneic Hematopoietic Stem Cell Transplantation for High-Risk Acute Myeloid Leukemia Patients

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is theoretically the only curative option for high-risk acute myeloid leukemia (AML) patients. However, many patients relapse after allo-HSCT, and treatment options are very limited. Azacitidine, a DNA methyltransferase inhibitor exhibits anti-leukemia activity in myeloid diseases. Chidamide, a selective histone deacetylase inhibitor, effectively induces apoptosis of AML cells. They have synergistic inhibitory effect on AML cells when combined together. Therefore, we conducted a multicentre, prospective study (ChiCTR2300067593) to examine the tolerability and efficacy of a combination of both chidamide and azacitidine as a maintenance therapy for high-risk AML patients after allo-HSCT. Patients and methods： Patients ≤ 60 years of age with high-risk AML post- allo-HSCT were enrolled. The high-risk characteristics of AML were defined as one or more of the following criteria: a) Patients with poor prognosis according to the genetic risk stratification assessment of European Leukmia Net (ELN) in 2022; b) Primary refractory or recurrent AML; c) Secondary AML (secondary to MDS or treatment-related AML). All patients received Chidamide 5 mg/d combined with azacitidine 75 mg/m2/d for 5 days. The cycle interval was 6-8 weeks. A total of 6 cycles was recommended. Treatment started as early as 3 months after transplantation. The primary endpoint of this study was cumulative incidence of relapse (CIR). The secondary endpoints included rates of overall survival (OS) and event-free survival (EFS). Survival outcomes were estimated using Kaplan-Meier analysis. Results： Thirty-six patients have been recruited. The median age was 42 years (range, 16-59). Baseline characteristics are shown in Table 1. With a median follow-up time of 412（185-762）days, the 1-year CIR was 12.3 (6.5-18.1) %, the 1-year OS and EFS rates were 100.0% and 87.7 (81.9-93.5) %, respectively. The median OS and EFS time were not reached. Four patients relapsed, two died, and two are still alive and ready for the second transplantation. The most common adverse events (AEs) were thrombocytopenia reaction and gastrointestinal tract reaction. Grade 2 or 3 AEs were observed in 16.7% (6/36) and 25.0% (9/36) of the patients, respectively. No grade >3 AEs were noted. 9 patients (6/36,16.7%) developed grade 3-4 thrombocytopenia, without active hemorrhage. All of them received blood transfusion and thrombopoietin receptor agonist. The median persistence of thrombocytopenia was 3 (2-5) d. Chronic graft-versus-host-disease (cGVHD) occurred in 25.0% (9/36) of patients. Four cases (4/36, 11.1%) were complicated with infection. They were one case of intestinal infection, one case of pulmonary and EBV infections, one case of hepatitis B virus activation and one case of herpes zoster. And all infected patients recovered safely after anti-infection and symptom-oriented treatment. CMV activation was not observed. Non relapse mortality (NRM) was zero. Conclusions： We conclude that chidamide plus azacitidine can be administered safely after allo- HSCT with no evidence of an increased incidence of GVHD, and this combination may decrease the relapse rate in high-risk AML patients. This novel maintenance therapy may be a promising way to prevent relapse in high-risk AML patients.

Identification and Validation of Ferroptosis-Related Genes As Novel Prognosis Prediction Panel for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a main type of adult acute leukemia, especially in elderly population. The development of AML ascribed to various factors including chromosomal abnormalities, isolated gene mutations, etc. and the 5-year survival rate is around 30%. Currently, the most popular and precise assessment of AML is based on cytogenetic differences, and the patients with higher risk molecular mutations had inferior prognosis. However, with the development and application of new drugs, including bcl-2 inhibitors, many AML with high risks (such as FLT3, P53 mutated) also achieve better remission and longer survival. Up to date, many novel prognosis models are in process, with the better understanding of the initiation and development of leukemia. Ferroptosis, a widespread and ancient form of cell death driven by iron-dependent phospholipid peroxidation. Its involvement in AML has been reported, as leukemic cells frequently exhibit enhanced iron uptake and reduced iron efflux, resulting in elevated intracellular iron levels. Targeting iron homeostasis and inducing ferroptosis may provide novel insights into AML therapy. Herein, we integrated multi-gene information to identify a novel prediction model for ferroptosis-related genes (FRGs), and validated the accuracy of the model using clinical samples from our center (Figure A). In this study, we downloaded RNA-seq data and clinicopathological features of 151 AML patients from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) and 70 corresponding health samples from the Genotype-Tissue Expression (GTEx) database (https://gtexportal.org/). Retrieve the ferroptosis-related genes (FRGs) set from the FerrDb database (http://www.zhounan.org/ferrdb/current/). The difference analysis, LASSO regression and Cox regression were used to determine the FRGs signature to construct prognostic models (Figure B). We finally identified 10 FRGs signature (ACSF2, SOCS1, CDO1, MYB, LPIN1, DNAJB6, PSAT1, GPX4, MT1G, FH) to establish a risk scoring model, and AML patients were divided into high-risk and low-risk group according to the median risk score. The risk score for each sample was determined using the following formula: . Kaplan-meier analysis showed that the overall survival of patients in the high-risk group was significantly worse( P<0.001) (Figure C). Cox regression analysis, ROC and DCA results found that these features have good predictive ability for prognosis of AML (Figure D-F). We also grouped the TCGA AML patients into poor/adverse and favorable/intermediate group based on NCCN guideline (risk stratification based on genomic mutation background), and found that the survival differences based on classical risk model were not that obvious ( P=0.062) (Figure G) when compared to our model ( P<0.001 in Figure C). Which suggested that our model may have its superior in distinguishing real high risk AML patients. In order to validate the accuracy of our model, we also conducted a retrospective study on 30 AML patients and 13 healthy controls (bone marrow stem cell donor) from September 2019 to September 2022. Bone marrow mononuclear cells (BMNC) from our biobank were obtained, and the ten FRGs panel was applied. Results indicated that there was a significantly difference between normal control and AMLs in FRGs expression (Figure H), and the survival data of high-risk AML patients with higher FRGs level was significantly inferior than that in low-risk group as expected ( P<0.05) (Figure I). To search for possible therapeutic drugs for high-risk patients, we downloaded AMLs data from the GDSC database (https://www.cancerrxgene.org/) and calculated the IC50 of commonly used chemotherapeutic drugs. We found that bcl-2 inhibitor (Obatoclax Mesylate) and TKI (Gefitinib) may have less sensitivity in our high-risk group ( P<0.05), while some unconventional drugs (paclitaxe, dactinomycin, camptothecin, irinotecan and vinorelbine) may benefit in patients with higher FRGs expression (Figure J). In summary, we identified and validated 10 FRGs signature to well predict the prognosis and drug sensitivity of AML. Based on our findings, appropriate combination of new drugs at the time of treatment may achieve unexpected therapeutic outcomes, but still need to go through further verification.

Epitope Edited Hematopoietic Stem Cells to Enable Synergistic Immunotherapy Combinations for Acute Myeloid Leukemia

Despite treatment advances, acute myeloid leukemia (AML) is still associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CAR-T cells, bispecific and toxin conjugated antibodies (mAb), demonstrated clinical efficacy when targeting dispensable lineage antigens (Ag), such as CD19 in B-ALL, the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Suitable targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. Several AML immunotherapies are currently under development, but their use is restricted to a limited time window, likely insufficient for disease eradication. To address these issues, we reasoned that precise modification of the targeted epitopes in donor HSPC used in HSCT results in loss of mAb recognition, without affecting normal protein expression, regulation, and function ( Nature, accepted). Epitope editing allows targeting genes essential for leukemia survival regardless of shared expression or functional role in normal HSPC, thus minimizing the risk of tumor immune escape by Ag loss or downregulation. Cytokine receptors such as FLT3, KIT and CD123 are found in >85% of AML cases and their mutation (e.g., FLT3-ITD) or overexpression is associated with poor prognosis. We identified amino acid substitutions in the FLT3, KIT and CD123 extracellular domains that preserve physiologic surface expression, ligand-binding, kinase phosphorylation, colony-forming capacity, proliferative response, transcriptional and phospho-proteomic profile of CD34+ HSPCs but avoid detection by a therapeutic monoclonal Ab. Cells expressing these variants were resistant to CAR-T killing and did not induce CAR activation and proliferation during in vitro co-culture. We were able to introduce these mutations into CD34+ HSPCs by adenine base editing (ABE) with high efficiencies (90%, 85% and 75% for FLT3, KIT and CD123, respectively) without the need for dsDNA breaks. After xenotransplant into NBSGW mice, FLT3, KIT or CD123 epitope-editedHSPC sustained long-term multilineage hematopoiesis indicating editing and preserved functionality of HSCs. Upon treatment with FLT3 CAR-T in vivo, we observed sparing of human CD34+38- HSPCs, granulo-mono progenitors (GMP), B-cells and B-progenitors in the bone marrow of mice engrafted with FLT3-edited HSPCs compared to AAVS1 controls. Treatment with CD123 CAR-T cells showed protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. We next generated advanced in vivo models with co-engraftment of human HSPCs and patient-derived AML xenografts (PDX), to demonstrate the selective resistance of epitope edited HSPCs and their progeny, while PDX were eradicated by FLT3 or CD123 CAR-T. To further enhance therapeutic efficacy, we are now exploring the combination of epitope-edited HSPCs and pharmacological agents with potential synergistic effects with CAR-T cells. FLT3 tyrosine kinase inhibitors (e.g., Crenolanib) have the potential to enforce surface expression of FLT3 by impairing its intracellular recycling, thus enhancing CAR-T mediated killing, but their use may be limited by overlapping toxicities on healthy hematopoiesis, in particular in the post-HSCT setting. To assess if epitope-editing could prevent not only CAR- but also TKI-mediated toxicities, we treated mice co-engrafted with FLT3- or AAVS1- edited HSPCs and a FLT3-ITD+ AML PDX with combinations of FLT3-CAR and a 2-week course of Crenolanib. Leukemia burden was reduced by Crenolanib alone and eradicated by FLT3-CAR-T. Critically, we observed preservation of healthy hematopoietic lineages only in mice engrafted with epitope-edited HSPCs, including the CAR+Crenolanib group, while AAVS1 controls showed overlapping off-tumor toxicity when treated with the CAR+Crenolanib combination. In conclusion, we believe that epitope edited HSPCs may not only enable safe and effective CAR-T immunotherapies for AML, but also allow their combination with pharmacological blockade of leukemia survival/proliferative pathways to achieve synthetic lethality mechanisms, while still avoiding dose-limiting toxicities. Further exploration of immunotherapy-synergistic combinations will be fundamental to improve the outcomes of difficult-to-treat high-risk AML patients.

Uncovering the Role of CD59 As a Novel Therapeutic Target Against High-Risk Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy driven by acquired genetic mutations in hematopoietic stem and progenitor cells. Continued disease progression leads to the unregulated growth of immature cells of the myeloid lineage, resulting in impaired hematopoiesis, organ failure, and death. High-risk AML is a disease subtype, classified by cytogenetic abnormalities, TP53 mutations, chemotherapy resistance, and dismal overall survival. To uncover the driving factors of poor outcomes in high-risk AML, we performed a multivariable analysis of the BEAT-AML-2.0 patient cohort. We identified that overexpression of the membrane regulatory protein, CD59, serves as a prognostic marker for an increased risk of death. Furthermore, we uncovered that CD59 is overexpressed in high-risk AML patients, including samples with TP53 mutations and refractory disease. CD59 prevents the formation of the membrane attack complex of the complement system and its overexpression is linked with resistance to cancer therapies that activate the complement cascade. However, the intracellular role of CD59 is poorly understood and it is unexplored in AML. Given that the standard of care for AML treatment, which consists of a combination therapy of cytarabine and an anthracycline, does not activate the complement system, we hypothesize that CD59 confers a survival advantage to AML cells in a complement-independent manner. To study the role of CD59 in AML, we constructed doxycycline-inducible short-hairpin RNA (shRNA) expression vectors against CD59 (shCD59) and a non-targeting control (shNT) and transduced them into SKM-1, HL-60, EOL-1, and OCI-M1 AML cell lines. First, we examined the impact of CD59 knockdown on cell growth through a cell competition assay between shCD59 or shNT cells and untransduced cells by culturing them at a 1:1 ratio. We observed that upon the addition of doxycycline, shCD59 cells are outcompeted by untransduced cells whereas the percentage of shNT cells remains unchanged. Cell viability analysis revealed an increased level of cell death in shCD59 cells in comparison to shNT cells. Moreover, we quantified the level of cellular proliferation in shCD59 and shNT cells through 5-ethyl-2'-deoxyuridine (EdU) staining. We observed that shCD59 cells have a signification reduction in cellular proliferation in comparison to shNT cells, highlighting changes in both cellular division and survival pathways upon CD59 knockdown. Importantly, to rule out the impact of alternative complement activation on the observed phenotype, we repeated the previously described experiments in heat-inactivated fetal bovine serum supplement cell culture medium and observed an identical response, confirming a complement-independent role of CD59. Based on our findings, we investigated the mechanism through which CD59 exerts pro-survival signals by performing RNA-sequencing on shCD59 and shNT cells. Through differential expression and pathway analyses, we discovered shCD59 cells exhibit increased inflammatory signalling and apoptotic pathway activation. Conversely, mTOR and MYC signaling pathways are significantly downregulated upon CD59 knockdown, including a reduction in mitotic spindle activity, DNA repair machinery, and DNA replication checkpoints. These results indicate that upon CD59 knockdown, AML cells undergo mitotic catastrophe, driven by deficient cell-cycle checkpoints, DNA damage, and ultimately, cell death. We further examined changes in the signaling landscape of shCD59 and shNT cells by analyzing the cellular phospho-proteome through mass spectrometry. In agreement with our RNA-sequencing analysis, shCD59 cells have increased activation of DNA damage and inflammation markers, such as BCLAF1 and IKBKG, respectively. In contrast, shNT cells display elevated activation of mTOR pathway markers, including EIF4B, and cell cycle drivers, such as CDC20. In summary, we present CD59 overexpression as a driver of AML progression and poor clinical outcomes. Through functional and computational studies, we have shown CD59 expression is critical for cell growth and cell survival, and as such, identifying it as a novel therapeutic target. The survival outlook for high-risk AML patients has remained unchanged for the past 25 years. Our findings offer valuable insights that can significantly alter the current treatment landscape of AML.

Hidac Consolidation Cycles May Impede Stem Cell Transplant Planning for High-Risk Acute Myeloid Leukemia Patients

Background Acute myeloid leukemia (AML) is a clonal aggressive hematologic neoplasm characterized by arrested myeloid maturation, resulting in the accumulation of myeloid blasts in bone marrow, blood, and other tissues. Cytogenetic and molecular characteristics further stratify AML into favorable, intermediate, or high-risk classifications; with high-risk including DEK-NUP214, KMT2A-rearrangement, BCR-ABL1, GATA2, MECOM, deletion 5q, abnormal 17p, complex or monosomal karyotype, wild type NPM1 and FLT3-ITD, or mutated: RUNX1, ASXL1, or TP53. In fit and eligible patients, therapy for high-risk AML patients involves induction therapy, and response evaluation at count recovery. If remission is confirmed, it is ideal that the patient proceed straight to hematopoietic stem cell transplantation (HSCT). HSCT remains the only curative therapy currently available. The logistics of proceeding to transplant might necessitate consolidation chemotherapy usually involving high dose cytarabine (HiDAC). Methods We performed a retrospective cohort analysis and evaluated outcomes for high-risk AML patients treated at our transplant institution, the Georgia Cancer Center, who were diagnosed and received HiDAC consolidation therapy between November 2003 and December 2020. High-risk AML was defined as those patients, whose AML was positive for one (or more) of the following FLT3, monosomy 5,7 and/or complex cytogenetics (CG) defined as having greater than or equal to 3 CG changes). For those not transplanted, we reviewed the complications, if any, that would preclude a HSCT. Results A total of 93 patients diagnosed with high-risk AML were evaluated at our center from 2003 to 2020. Males comprised 46.2% (n=43) and females comprised 53.8% (n=50) of the cohort. Of these patients, the median age of the patient cohort was 61.5 years. 40.8% (n=38) received HiDAC consolidation. Among patients who received HiDAC consolidation and proceeded to undergo HSCT, the median age was 47.6 years. In contrast, patients who received HiDAC consolidation but were unable to undergo HSCT had a median age of 61.3 years. Among the patients who were unable to undergo HSCT due to death, the median age was 41.8 years. For patients who received HiDAC consolidation but did not undergo HSCT due to reasons other than death, the median age was 53.4 years. 55.3% (n=21) went on to HSCT, and others did not proceed to HSCT. Of the patients who did not receive HSCT, 76.4% (n=13), underwent HLA-typing, indicating consideration for transplantation. Of these 17 patients (44.7%, n=9) did not proceed to transplant due to infectious complications, the remaining 8 had noninfectious reasons for non-transplant. Of the 9 patients without HSCT due to infection, four had an infection that prevented them from receiving a transplant and five patients passed away prior to transplant due to septic shock or multi-organ failure. Infections included bacteremia from Klebsiella, E. Coli, Proteus, group B strep, vancomycin resistant E. Faecalis, and coagulase negative staph, pneumonia from Klebsiella, urinary tract infections from extended spectrum beta-lactamase resistant E. Coli, Klebsiella, and proteus mirabilis, fungal sinusitis, and Clostridium difficile colitis. Other reasons that prevented HSCT included neutropenic fever with an unknown cause, substance abuse, disease progression, and lack of follow-up. Conclusion The data from our study and the literature on this topic over the past 30 years suggest starting the process of HSCT during the induction admission. HSCT is the sole curative option for high-risk AML patients, and the concept of bridging the period between induction and HSCT with HiDAC consolidation appears to offer no additional benefit compared to IDAC. Moreover, this approach prevents a subset of this population from being fit enough to receive HSCT.

Antithymocyte Globulin in Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia: Evaluating Outcomes According to Cytogenetic Risk

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an essential therapeutic approach for the management of acute myeloid leukemia (AML). Nevertheless, graft-versus-host disease (GVHD) remains a significant complication, significantly affecting the recipients' quality of life and overall survival. Antithymocyte globulin (ATG) has demonstrated promise in preventing GVHD during allo-HSCT. However, concerns persist regarding its potential impact on disease relapse. This retrospective single-center study aims to evaluate the prognostic implications of ATG based on cytogenetic risk stratification in AML patients undergoing transplantation from an HLA-matched sibling. A total of 124 AML patients who underwent allo-HSCT from an HLA-matched sibling in their first complete remission state between 2003 and 2022 were eligible for the study. Among them, fifty patients received rabbit ATG at a dose of 2.5mg/kg/day for days -2 to -1, while 74 patients did not receive ATG. All patients received cyclosporine and methotrexate in addition to their respective conditioning regimens (ATG or no ATG). The ATG group consisted of 38 patients classified as having a non-high cytogenetic risk, and 12 patients classified as having a high cytogenetic risk. Conversely, the non-ATG group included 68 patients with a non-high cytogenetic risk and 6 patients with a high cytogenetic risk. After a median follow-up of 48 months, there were no significant differences in median overall survival between the ATG and Non-ATG groups, regardless of cytogenetic risk status (non-high cytogenetic risk; 12.4 months vs. NA, p = 0.578; high cytogenetic risk; 13.2 months vs. 14.2 months, p = 0.729, Figure A-1,B-1). The ATG group in the non-high cytogenetic risk group demonstrated a significant decrease in the 1-year cumulative incidence (CI) of chronic GVHD compared to the non-ATG group (21.7% vs. 54.4%, p = 0.001, Figure A-2; and in those with moderate to severe cGVHD, it was 21.6% vs. 51.5%, p = 0.003, respectively). Additionally, the 1-year Chronic GVHD-free and relapse-free survival (cGRFS) was higher in the ATG group of the non-high cytogenetic risk group (45.8vs. 26.5%; p = 0.048, Figure A-3). Moreover, there were no significant differences in 1-year CI of relapse and 1-year non-relapse mortality (NRM) within the non-high cytogenetic risk group (ATG vs. non-ATG; 29.7% vs. 20.6%, p = 0.225; 8.4% vs. 4.4%, p = 0.635, Figure A-4,5). In the subgroup analysis of patients with high cytogenetic risk, although there was no statistical significance, the ATG group showed a trend towards decreased 1-year CI of chronic GVHD and higher cGRFS compared to the non-ATG group (10.4% vs. 40.0%, p = 0.342; 31.2% vs. 16.7%, p = 0.929, Figure B-2,3). However, the ATG group exhibited a significantly higher 1-year CI of relapse compared to the non-ATG group (60.0% vs. 16.7%, p = 0.019, Figure B-4). In conclusion, our study findings indicate that ATG was effective in preventing chronic GVHD and demonstrated higher cGRFS in AML patients undergoing matched sibling donor HSCT. However, AML patients with high cytogenetic risk should carefully consider the use of ATG due to the associated higher risk of relapse. Further investigations are warranted to validate these findings and determine the appropriate dosage of ATG for high cytogenetic risk AML patients.

Reconsidering the Significance of Blast Cut-Offs in Differentiating between MDS and AML Biology

Background The diagnosis of and distinction between acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS) have long been based on blast counts, with the presence of ≥20% myeloblasts in marrow or peripheral blood required to define AML in World Health Organization classification. Until 2001, blast counts greater than 30% had been used to diagnose AML by the original French-American-British classification system. However, the 2022 International Consensus Classification proposed a new combined category of MDS/AML with 10-19% blasts. Here we report the real-world treatment pattern and outcomes of AML and high-risk MDS patients at an academic institution in the United States, with specific attention to the blast burden at initial disease diagnosis to validate accuracy and limitation of this new proposed change. Methods This is a retrospective study conducted at UCLA Health encompassing all diagnoses of AML and MDS made at academic campus and community practices. We evaluated adult patients with a diagnosis with either AML or MDS from 1/1/2016-7/31/2022. For patients with more than one bone marrow biopsy on record, we recognized the date of diagnosis of MDS or AML with a blast burden ≥ 10% by immunohistochemistry or flow cytometry as the first bone marrow biopsy. In light of the changing diagnostic definitions, we analyzed patients by cohorts of MDS/AML with 10-19% blasts, AML low-blast with 20-29% blasts, and AML high-blast ≥ 30% blasts. Survival analysis by blast count at diagnosis was calculated by Kaplan-Meier estimates. A Multivariable Cox proportional hazard model was used to model survival outcomes by blast cohort, ELN risk 2022, response to first treatment, and receipt of allogeneic hematopoietic cell transplantation (HCT). Results There were 422 patients at UCLA Health with a diagnosis of AML or MDS included in our analysis with blast burden ≥ 10%. Patient demographics are shown in Table 1. Median age at diagnosis for MDS/AML, AML low-blast and AML high-blast cohorts is 67, 66 and 64 respectively (p=0.02). Thirty two (66.7%) patients in MDS/AML cohort received non-intensive first line treatment, whereas 202 (67.8%) AML high-blast patients received intensive first treatment. For AML low-blast patients, 37 (48.7%) received intensive treatment, and 29 (38.2%) received non-intensive treatment. More patients with AML (either low- or high-blast) underwent allogeneic HCT compared to MDS/AML patients. By Kaplain Meier analysis (KM) using blast counts as the sole predictor (Figure 1), no significant difference in OS when following patients for up to five years from diagnosis (p=0.0985) was seen. The p-value for two-way KM comparisons for MDS/AML vs. AML low-blast is 0.9682, and for AML low-blast vs. AML high-blast is 0.112. By multivariable analysis, ELN Risk 2022 (p<.0001), allogeneic HCT (p<.0001), and Complete Response to initial treatment (p<.0001) were predictive of OS, but blast percentages were not (p=0.56). Conclusion In our study, we found similar survival outcomes in high-risk MDS and AML patients regardless of initial blast counts at diagnosis. Though not statistically significant, MDS/AML and AML with low-blast burden appear to share more similar outcomes. This could be due to increased secondary AML seen in the low-blast cohort, and implies potentially different disease biology between high blast burden AML and AML with dysplastic changes. With this observation, we emphasize caution on simply using ICC 2022 to apply traditional AML therapy to the new MDS/AML category. Both WHO and ICC classification in 2022 reflects the increasing potential of molecular profiling to subtype these diseases, which will be the future direction. Rather than moving the blast count cutoff between AML and MDS, it is more critical to emphasize inclusion of high-risk MDS patients on all AML clinical trials and perhaps the converse for low blast burden AML with dysplastic changes, which might allow better understanding of response to therapy and disease biology.

Venetoclax Plus Azacitidine Versus Azacitidine Alone As Post-Transplant Maintenance Treatment in High-Risk AML and MDS

Background: Disease recurrence is the main cause of transplantation failure for high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Exploring innovative approaches to prevent relapse in AML and MDS after Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is in urgent need. Both bcl-2 inhibitor venetoclax (VEN) and azacitidine (AZA) possess significant antitumor activity effects against myeloid neoplasms. The efficacy and safety of AZA alone as maintenance therapy in the posttransplant setting have been reported but the results are controversial. The combination of VEN and AZA has resulted in high response rates in treating AML patients. We conducted a prospective clinical trial to compare the utility and safety of the combination of VEN and AZA (VEN/AZA) and AZA alone as maintenance treatment in high-risk AML and MDS in the post-transplant setting. Methods: This is a prospective, multicenter, randomized clinical trial. Patients with AML or MDS who were evaluated as high risk of relapse and underwent allo-HSCT were randomly assigned into VEN/AZA group and AZA group by gender, age, and disease type based on the central randomization system. AZA was administered by subcutaneous injection for 5 days at a dose of 50 mg/m2. VEN was administrated at the dosage of 200 mg daily from day 1 to day 7. VEN/AZA maintenance therapy regimen comprised AZA and VEN used alternately every month. AZA alone group was administrated AZA every 2 months. Maintenance treatment lasted at least 1 year after transplantation unless relapse occurred. The primary endpoint of the study included overall survival (OS), disease-free survival (DFS), and cumulative incidence of relapse (CIR). The second endpoints included adverse events (AEs), nonrelapse mortality (NRM), incidence of acute graft versus host disease (aGVHD), and chronic GVHD (cGVHD). Results: From June 2022 to June 2023, 24 patients were given the study protocol. 9 patients (6 AML, 3 MDS) received VEN/AZA maintenance and 15 (7 AML, 8 MDS) patients received only AZA maintenance. The median initial time to receive maintenance treatment was 3.67 months after transplantation. The median follow-up time was 11.30 (95%CI, 8.18-14.42) months. Only 1 patient relapsed and died 3 months after relapse on the study. The overall survival (OS) and the cumulative incidence of relapse (CIR) at 500 days were 100% vs 75.0% (95% CI, 42.59%-100%, p=0.39) and 0% vs 12.5% (95% CI, 0.48%-44.48%, p=0.39) for VEN/AZA and AZA group, respectively. The most common adverse effects (AEs) were hematological toxicities. Grade 3 or 4 hematological toxicities were observed in 5/9 (55.5%) in the VEN/AZA and 7/15 (46.7%) in the AZA group. Conclusions: Our results suggested that both VEN/AZA and AZA maintenance therapy were tolerated and the combination of VEN and AZA as maintenance therapy may be feasible and safe in the posttransplant setting to prevent relapse for high-risk AML and MDS patients.

Sequential Conditioning Regimen with Thiotepa, Clofarabine and Busulfan (TEC/CloB2A2) and Post-Transplant Cyclophosphamide in Adults with Refractory AML: A Retrospective Monocentric Study

Background : Since almost 20 years, sequential allogeneic stem cell transplantation is proposed to fit and relatively young patients with refractory hematologic malignancies (mainly acute myeloid leukemia (AML)) and who will otherwise die from the disease. This procedure is characterized by the use of a renforced conditioning regimen combining a debulking chemotherapy followed after 3 days of rest by the conditioning regimen itself. However, the results overall remain dismal as only a small proportion of patients are finally cured. Recently, a combination of thiotepa, etoposide, cyclophosphamide (TEC) followed by fludarabine/busulfan/ATG (FB2A2) using any type of donor for refractory hematologic malignancies have shown some promising results (Dulery, 2018). As clofarabine may have better antileukemic effect than fludarabine, we have investigated in our patients with refractory AML a new sequential approach combining TEC+clofarabine/busulfan/ATG (TEC/CloB2A2). Methods : We have investigated in our Hematology Department a new sequential conditioning regimen replacing fludarabine by clofarabine as part of a TEC/FB2A2 regimen with the hope to obtain less relapse and better survival in a cohort of patients with refractory AML. The TEC/CloB2A2 regimen consisted of Thiotepa 5 mg/kg at day(d)-13, Cyclophosphamide 400 mg/m²/d from d-12 to d-9 and Etoposide phosphate 100 mg/m²/d from d-12 to d-9, followed after 3 days of rest, by Clofarabine 30 mg/m²/d from d-5 to d-1, Busulfan 3,2 mg/kg/d d-5 and d-4 and Thymoglobuline 2,5 mg/kg/d d-3 and d-2. GVHD prophylaxis consisted of high-dose of post-transplant Cyclophosphamide (PTCY) 50 mg/kg/d at d+3 and d+5 in combination with mycophenolate mofetyl and cyclosporine starting at d+6. Any type of donor have been considered. Results : Between February 2020 and August 2022, 12 patients have been treated. Patient characterisitcs are given in the Table. Median age was 58 years (y) old. The majority of patients had primary refractory AML (n=9). This was the first transplant for all patients. One patient needed desensibilization before conditioning due to high level of anti-HLA antibodies directed against the donor. All but 2 donors were haploidentical. One patient with a matched donor did not receive PTCY after transplant. Two patients died during the aplasia phase, one at d+6 of sepsis and multiple organ failure (MOF) and one at d+30 of disseminated fungal infection. The median time of neutrophils (> 1 Giga/L) and platelets (> 50 Giga/L) recoveries were 22 days (range : 12-137) and 34 days (range : 16-241), respectively. There were no primary or secondary graft failures. Responses were evaluated at a median of 57 days (range : 26-70) after transplant. Five complete remission (CR), 3 CR with incomplete platelet recovery (CRp) and two failures were documented for an overall CR/CRp rate of 67%. Only one cutaneous grade 2 acute GVHD occured after transplant but no chronic GVHD. Six out of 8 CR/CRp patients received maintenance treatment after transplant to prevent relapse using 5'azacytidine alone in 1, 5 azacytidine+ donor lymphocyte infusion (DLI) in 2, DLI alone in 1. The last two patients received maintenance as part of a trial. The three patients receiving DLI developped acute GVHD thereafter including 2 grade 2 and one grade 3. Six of the patients achieving CR/CRp relapsed (75%) at a median of 160 days (range : 48-344) post-transplant. At last follow-up (July 2023), only one patient is alive in CR (8%) at 795 days from transplant (SET/CLoB2A2 +PTCY and sibling donor, grade 3 acute GVHD after DLI). Causes of deaths were relapse in 6, and infections in five (disseminated fungal infection n=1, COVID-19 n=2, bacterial infection + MOF n=1 and + distress respiratory syndrome n=1). Non-relapse mortality (NRM) was 25% and 41% at 1y and 2y, respectively. 1-y and 2y overall survival and leukemia-free survival were both at 25% (9.3-66.6) and 8.3% (1.2-54.4), respectively. Conclusion : The sequential TEC/CloB2A2 conditioning regimen is not associated with better outcomes compared to the TEC/FB2A2 sequential conditioning regimen. Other sequential approaches and/or GVHD/maintenance strategies have to be considered in the future for these very high-risk refractory AML patients in order to obtain better graft-versus-leukemia effect and lesser toxicity.

Chidamide-Based Pre-Emptive Treatment for High-Risk AML Patients after Hematopoietic Stem Cell Transplantation: A Real-World Experience from Chinese Single-Center

Background: The treatment of relapsed or refractory acute myeloid leukemia ( AML) remains challenging. Measurable residual disease (MRD) persistence at transplantation or post-transplantation has been identified as the strongest risk factor for relapse and poor outcomes for patients with hematological malignancies (HMs). Chidamide, a novel oral histone deacetylase inhibitor, has shown potential therapeutic effects in various types of HMs by inducing cell apoptosis and regulating immunity. This study aimed to evaluate the efficacy and safety of Chidamide-based prophylactic or pre-emptive treatment for patients who are MRD positive before or who have sustained MRD positive after transplantation. Aim: The rate of 6 months of conversion to MRD- and relapse-free survival of high-risk AML after Chidamide-based treatment. Methods: From December 2020 to July 2023, 50 patients with high-risk HMs received chidamide-based therapy after allo-HSCT. 39 patients were diagnosed with AML ( 4 secondary AML), 2 with myelodysplastic syndromes, 6 with acute lymphoid leukemia, and 3 with mixed-phenotype acute leukemia. 46 patients (92%) with relapsed/refractory status or detectable MRD (MRD+) at transplantation or after allo-HSCT received chidamide as pre-emptive, and the remaining 4 high-risk patients with MRD− conducted as prophylactic intervention. Patients with detectable MRD before transplantation and those who remained positive for MRD after transplantation were defined as very high-risk AML (VHR-AML). Chidamide was administered at a dose of 5 mg per day orally, 6 times per week, lasting at least 1 year after engraftment. Additional agents included hypomethylating agents (HMAs), donor lymphocyte infusion (DLI) or tyrosine kinase inhibitor (TKI) in patients with Philadelphia chromosome-positive. In this study, 28 patients received chidamide monotherapy, 13 received chidamide combined with HMAs, and 9 cases with other therapy. Results: The median age of all patients was 38 years old (range 17-67), comprising 24 males and 26 females. Up to 1st July 2023, with a median follow-up period of 376 days (25-984d), the overall rate of MRD negative showed 64%. The rate of MRD- survival, Relapse-free survival (RFS) and overall survival (OS) were 56%, 66% and 76%, respectively. The 6 months MRD negative rate, RFS and OS rate were 86%, 82%, 86% and 88%, which maintained at 82%, 82% and 86% with 12 months follow-up, respectively. In 46 cases of the pre-emptive intervention arm, the overall MRD- rate was 61.7%. Among them, 83.3% of patients (20/24) who received chidamide monotherapy exhibited a much higher conversion to MRD− than those combined with HMAs (7/13, 53.8%) or other drugs (2/9, 22.2%) (P=0.004). In 4 cases who received chidamide monotherapy as prophylactic intervention, 3 patients sustained MRD negativity up to the last follow-up, except for one patient diagnosed with MPAL(B+M) relapsed after 1 year of HSCT with taking chidamide for 6 months. A total of 30 patients (including 25 AML) with VHR diseases remained in a status of MRD+ or non-remission after transplantation. Eighteen patients (60%) achieved MRD− and sustained for a median of 251 days (range 31-531 d). VHR-AML patients account for 94.4% (17/18) of MRD- responders(Figure 1A). Similarly, the results showed a higher rate of conversion from MRD+ to MRD− in chidamide monotherapy than the combination groups (Chidamide monotherapy, 81.8%; Chidamide + HMA, 63.6% and Chidamide + others, 25%, P=0.042,Figure 1B). It highlighted that treatment with chidamide resulted in increased MRD− response, especially for AML. On the contrary, of 7 patients with B-cell malignancy, including 5 B-ALL and 2 MPAL (B + M) patients, only 2 (28.6%) patients converted to MRD−. Reversible grade 3/4 neutropenia occurred in 38% of patients, and thrombocytopenia was observed in 24% during the first four treatment cycles. Five patients experienced grade 1/2 non-hematological adverse events, such as fatigue, gastrointestinal reaction, pneumonia and infection. No new-onset GvHD was reported in patients without a prior history of GvHD during chidamide treatment. Conclusion: The chidamide-based regimen is safe and effective in pre-emptive treatment for patients with high-risk relapsed AML after transplantation. Whether as a monotherapy or combined with HMA, chidamide shows significant efficacy in eliminating MRD, warranting further clinical research.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) >2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM <1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

Efficacy and Safety Analysis of Venetoclax Combined with Azacitidine in Real-World Treatment of Patients with AML

Objective: To evaluate overall survival (OS), response rate, time to first response, duration of response, depth of response, and safety in real-world treatment with Venetoclax (Ven) combined with Azacitidine (AZA) in patients with Acute Myeloid Leukemia (AML). Methods: From January 2019 to December 2021, 31 patients with AML who completed at least 2 courses of Ven (100-400mg, 28 days), the Ven dose was adjusted based on drug interactions particularly with azole antifungal prophylaxis, and AZA (75mg/m 2,7 days). Baseline characteristics: Median age 65 years (30-82 years), including 5 patients ≥75 years. ECOG score: 19 patients (61.3%) scored 0-1, 12 patients (38.7%) scored 2-3. Type of AML: 20 De novo cases (64.5%), 8 MDS or MPN conversion cases (25.8%), 3 primary AML-MRC cases (9.7%). Previous treatment: 22 patients (71%) were initially treated, 8 patients (25.8%) were relapsed/refractory, and 1 patient (3.2%) was maintained after remission with standard therapy. At baseline, grade 3 or greater cytopenia was associated: 3 cases were anemia (9.7%), 13 cases were neutropenia (41.9%), and 14 cases were thrombocytopenia (45.2%). Bone marrow original cell count: < 30% (10 cases), 30-50%(9 cases), ≥50% (12 cases). Cytogenetic risk: 1 case was low risk (3.2%), 19 cases were medium risk (61.3%), 10 cases were high risk (32.3%), and 1 case was no dividing phase (3.2%). Molecular mutations included: NRAS/KRAS mutation in 12 cases, DNMT3A mutation in 11 cases, NPM1 mutation in 9 cases, IDH1/2 mutation in 6 cases, FLT3-ITD mutation in 5 cases each, HOX11 positive, ASXL1, RUNX1 and NPM1+FLT3-ITD mutation in 3 cases each. Results: After a median of 3 courses of treatment (C2-C15), 3 of the patients underwent bridge hematopoietic stem cell transplantation after remission. The best efficacy was evaluated in CR+CRi: 22 patients (71%), PR: 5 patients (3 of whom were refractory to recurrence), SD: 3 patients (1 of whom was refractory to recurrence), and NR: 1 patient (refractory to recurrence). First-line efficacy was better than second-line or multi-line. Median time to start treatment to reach CR/CRi: 45 days, DOR: 314 days. The negative response rate of flow MRD in CR/CRi patients was 81.8%, including 11 cases of C1 conversion, 3 cases of C2 conversion, 3 cases of C3 conversion and 1 case of C4 conversion. Flow minimal residual disease (MRD) persisted in AML1-ETO subgroup. The early combined response rate of patients with important gene mutations: ASXL1 and RUNX1 mutation subgroup 100%, NPM1 mutation subgroup 77.78%, IDH1/2 mutation subgroup 83.33%, FLT3-ITD mutation subgroup 80%, HOX11 positive subgroup 80%, 66.67% in the NPM1+FLT3-ITD and NRAS/KRAS mutation subgroups, 53.55% in the DNMT3A mutation subgroup, and 1 patient with TP53 mutation was not in remission. The median OS of the first-line/ maintenance treatment group was 19 months, which was better than the median OS of the second-line or multi-line treatment group was 7 months. Safety: The incidence of tumolysis syndrome and early treatment-related mortality was 0; The incidence of early reduction or treatment interruption was higher (25.8%). Severe adverse reactions included sepsis in 2 cases, severe pneumonia in 2 cases and septic shock in 1 case. Conclusions: In the real world first-line treatment of high-risk AML patients with Ven combined with AZA, the disease control and survival were initially improved, and the early combined response rate (CR+CRi) of molecular subgroups IDH1/2, NPM1, RUNX1, ASXL1, FLT3-ITD and HOX11 were higher. The higher incidence of adverse events in early treatment and delayed treatment may be related to 41.9% of patients with grade 3 neutropenia at baseline, partly AML-MRC. So, based on Ven-28d/AZA, We still need to explore new therapeutic modalities which should balance the efficacy and safety.

Expression signature of ten small nuclear RNAs serves as novel biomarker for prognosis prediction of acute myeloid leukemia

This study aimed to screen for small nuclear RNAs (snRNAs) associated with the prognosis of acute myeloid leukemia (AML) by using The Cancer Genome Atlas (TCGA) whole-transcriptome sequencing dataset. A total of 130 AML patients from TCGA cohort with complete prognostic information and transcriptome data were enrolled in the current study. Comprehensive survival and functional enrichment analyses were performed to explore the prognostic value and potential biological functions of prognostic snRNAs in AML patients. In the current study, we screened 72 snRNAs that were notably associated with the clinical outcome of AML and developed an expression signature consist of ten snRNAs, that can be accurately applied to assess the overall survival of AML patients. Functional mechanism analysis revealed that this expression signature may be strongly linked to some classical tumor-associated pathways, such as Notch and Wnt pathways, as well as being closely related to B and T cell receptor pathways. Furthermore, we screened six compounds (chicago sky blue 6 B, 5230742, clorsulon, nefopam, nicardipine, and streptomycin) that may serve as targeted therapeutic drugs for AML using connectivity maps. Tumor immunoassays indicated significant differences in the immune microenvironment of the bone marrow tissue between high-risk and low-risk AML patients. Immune infiltration analysis also revealed significant differences in the abundance of multiple immune cells in the bone marrow of the two groups of AML patients groups. In conclusion, our results revealed a novel prognostic expression signature of AML consisting of ten snRNAs, and we conducted a preliminary exploration of its potential biological functions and tumor immunity.

Characteristics and outcomes of patients with acute myeloid leukemia admitted to intensive care unit with acute respiratory failure: a post-hoc analysis of a prospective multicenter study

BackgroundAcute respiratory failure (ARF) is the leading cause of intensive care unit (ICU) admission in patients with Acute Myeloid Leukemia (AML) and data on prognostic factors affecting short-term outcome are needed.MethodsThis is a post-hoc analysis of a multicenter, international prospective cohort study on immunocompromised patients with ARF admitted to ICU. We evaluated hospital mortality and associated risk factors in patients with AML and ARF; secondly, we aimed to define specific subgroups within our study population through a cluster analysis.ResultsOverall, 201 of 1611 immunocompromised patients with ARF had AML and were included in the analysis. Hospital mortality was 46.8%. Variables independently associated with mortality were ECOG performance status ≥ 2 (OR = 2.79, p = 0.04), cough (OR = 2.94, p = 0.034), use of vasopressors (OR = 2.79, p = 0.044), leukemia-specific pulmonary involvement [namely leukostasis, pulmonary infiltration by blasts or acute lysis pneumopathy (OR = 4.76, p = 0.011)] and liver SOFA score (OR = 1.85, p = 0.014). Focal alveolar chest X-ray pattern was associated with survival (OR = 0.13, p = 0.001). We identified 3 clusters, that we named on the basis of the most frequently clinical, biological and radiological features found in each cluster: a “leukemic cluster”, with high-risk AML patients with isolated, milder ARF; a “pulmonary cluster”, consisting of symptomatic, highly oxygen-requiring, severe ARF with diffuse radiological findings in heavily immunocompromised patients; a clinical “inflammatory cluster”, including patients with multi-organ failures in addition to ARF. When included in the multivariate analysis, cluster 2 and 3 were independently associated with hospital mortality.ConclusionsAmong AML patients with ARF, factors associated with a worse outcome are related to patient’s background (performance status, leukemic pulmonary involvement), symptoms, radiological findings, the need for vasopressors and the liver SOFA score.We identified three specific ARF syndromes in AML patients, which showed a prognostic significance and could guide clinicians to optimize management strategies.

Immune-related lncRNAs pairs prognostic score model for prediction of survival in acute myeloid leukemia patients

Acute myeloid leukemia (AML) is one of the most common malignant and aggressive hematologic tumors, and risk stratification is indispensable to ensure proper treatment. But immune-related long noncoding RNAs (ir-lncRNAs) pairs prognostic risk models used to stratify AML have yet to be reported. In this study, we established a prognostic risk model based on eight ir-lncRNAs pairs using LASSO-penalized Cox regression analysis and successfully validated the model in an independent cohort. According to risk scores, patients were divided into a high-risk group and a low-risk group. High-risk patients presented more tumor mutation frequency and higher expression of human leukocyte antigen (HLA)-related genes and immune checkpoint molecules. Gene Set Enrichment Analysis (GSEA) indicated that the transforming growth factors β (TGFβ) pathway was activated in the high-risk group; meanwhile, we found that TGFβ1 mRNA levels were significantly elevated in AML patients and correlated with poor prognosis, which is closely related to drug resistance. Consistently, in vitro studies found that exogenous TGFβ1 can protect AML cells from chemotherapy-induced apoptosis. Collectively, we developed an ir-lncRNA prognostic model that helps predict the prognosis of AML patients and provides valuable information about their response to immune checkpoint inhibitors, and we found that increased TGFβ1 levels resulting in chemoresistance may be one of the leading causes of treatment failure in high-risk AML patients.

Identification and validation of a novel CD8+ T cell-associated prognostic model based on ferroptosis in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly aggressive cancer with great heterogeneity and variability in prognosis. Though European Leukemia Net (ELN) 2017 risk classification has been widely used, nearly half of patients were stratified to "intermediate" risk and requires more accurate classification via excavating biological features. As new evidence showed that CD8+ T cell can kill cancer cells through ferroptosis pathway. We firstly use CIBERSORT algorithm to divide AMLs into CD8+ high and CD8+ low T cell groups, then 2789 differentially expressed genes (DEGs) between groups were identified, of which 46 ferroptosis-related genes associated with CD8+ T cell were sorted out. GO, KEGG analysis and PPI network were conducted based on these 46 DEGs. By jointly using LASSO algorithm and Cox univariate regression, we generated a 6-gene prognostic signature comprising VEGFA, KLHL24, ATG3, EIF2AK4, IDH1 and HSPB1. Low-risk group shows a longer overall survival. We then validated the prognostic value of this 6-gene signature using two independent external datasets and patient sample collection dataset. We also proved that incorporation of the 6-gene signature obviously enhanced the accuracy of ELN risk classification. Finally, gene mutation analysis, drug sensitive prediction, GSEA and GSVA analysis were conducted between high-risk and low-risk AML patients. Collectively, our findings suggested that the prognostic signature based on CD8+ T cell-related ferroptosis genes can optimize the risk stratification and prognostic prediction of AML patients.

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.

Polyploidy Is Necessary for Apoptotic Cell Death in TP53-Mut AML in Response to Polo-like-Kinase 4 (PLK4) Inhibition and Results in Caspase 3 Cleavage

Background Approximately twenty percent of patients with newly diagnosed acute myeloid leukemia (AML) have TP53 mutations, associated with complex karyotype and the highest risk of relapse(Döhner et al., 2017; Giacomelli et al., 2018). Although combination therapies, such as Ara-C + anthracyclines and Venetoclax (VEN) + hypomethylating agents (HMA) result in higher remission rates in patients with TP53-mutant AML, the mutant clone is never eliminated and invariably leads to relapse(Short et al., 2021; Takahashi et al., 2016). Novel targets and mechanisms associated with TP53-mut AML need to be identified. Results: We performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations of TP53-mutant or TP53-wild-type (wt) high-risk AML patients before and after treatment. Amongst overexpressed genes, we identified Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis and therefore of polyploidy, as a potential target highly expressed in TP53-mutant AML using differential gene expression analysis of TP53-wt vs TP53-mutant samples collected (TP53-mutant=36, TP53-wt=31, PLK4 LOG2FC=~2, p<0.001). We validated this finding using an independent RNA-seq dataset from the Munich Leukemia Lab of 726 AML samples (TP53-mutant=72, TP53-wt=654, PLK4 LOG2FC=0.48, p<0.001) and investigated the impact of PLK4 expression levels on AML patient outcomes in the context of TP53-status. High levels of PLK4 expression in AML are associated with poor outcomes (Figure 1). Targeting PLK4 by CFI-400945 (25nM, Treadwell Therapeutics, Toronto, Canada) in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 for 72 hours resulted in increased polyploid cells in TP53-mut cells compared to TP53-wt AML (> 2 fold, p< 0.0001). PLK4 inhibition triggered higher levels of cleaved caspase 3 in polyploid TP53-mut polyploid MOLM-13 cells compared to TP53-wt MOLM-13 cells (Figure 2). Polyploidization in TP53-mut MOLM-13 cells 48 hours post treatment with 15nM of PLK4 inhibitor was not reversible and retained higher apoptogenic activity in TP53-mut MOLM-13 cells even after wash out for additional 48 hours. To translate our findings to in vivo models, we transplanted CRISPR-generated TP53-wt, TP53-ko, and TP53-248 mutant MOLM-13 cells (Boettcher et al., 2019) into NSG mice (n=58: untreated n= 28, treated n=30), confirmed successful engraftment, and treated the mice with CFI-400945 (oral gavage 7.5 mg/kg, 5 times/week) for 21 days. Our preliminary data indicates that all treated mice showed improved survival compared to untreated mice. The median survival for treated TP53-wt vs untreated TP53-wt was 17.5 vs 14.5 days (20.7% increase, n=20 (untreated n=10, treated n=10), p=0.00086). The median for treated TP53-R248Q vs untreated TP53-R248Q was 15.0 vs 12.0 days respectively (25% increase, n=19 (untreated n=9, treated n=10), p=0.00039). The median OS for treated TP53-ko vs untreated TP53-ko was 11 vs 8 days respectively (37.5% increase, n=19 (untreated n=9, treated n=10), p<0.0001). Next, we performed a Cox proportional hazard model to investigate the contribution of TP53 genotype and PLK4 inhibition on overall survival. The resulting model (Likelihood ratio test=83.09 on 3 degrees of freedom and p<2.2e-16) showed a higher hazard ratio in recipients with TP53-R248Q and TP53-ko (~4.3, and ~1.2 hazard coefficients respectively) in comparison to TP53-wt (p=2.67e-14, and 0.000708 respectively). Additionally, treated mice had statistically significant lower hazard ratios compared to untreated (hazard coefficients=-2.47277 and p=8.38e-10). The data suggest that PLK4 inhibition is a promising treatment approach for AML with TP53 mutation. We are presently testing different dosing schedules in other aggressive TP53-mut AML models to further ascertain the efficacy of PLK4 inhibition. Conclusion: We established an association between PLK4 overexpression and poor survival outcomes in patients with newly diagnosed AML. TP53-mutant AML express higher levels of PLK4 in comparison to TP53-wt AML and targeting PLK4 induces polyploidy and apoptotic cell death predominantly in TP53-mutant AML. We demonstrate that targeting of PLK4 improves overall survival in TP53-mut and TP53-ko AML cells in vivo. Data suggest that targeting PLK4 represents a potential treatment approach for TP53-mut AML. An early phase, multi-center clinical trial is ongoing with CFI-400945 in AML (NCT04730258, TWT-202). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel cuproptosis-related LncRNA signature: Prognostic and therapeutic value for acute myeloid leukemia.

BackgroundCuproptosis is a type of programmed cell death that is involved in multiple physiological and pathological processes, including cancer. We constructed a prognostic cuproptosis-related long non-coding RNA (lncRNA) signature for acute myeloid leukemia (AML).MethodsRNA-seq and clinical data for AML patients were acquired from The Cancer Genome Atlas (TCGA) database. The cuproptosis-related prognostic lncRNAs were identified by co-expression and univariate Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) was performed to construct a cuproptosis-related lncRNA signature, after which the AML patients were classified into two risk groups based on the risk model. Kaplan-Meier, ROC, univariate and multivariate Cox regression, nomogram, and calibration curves analyses were used to evaluate the prognostic value of the model. Then, expression levels of the lncRNAs in the signature were investigated in AML samples by quantitative polymerase chain reaction (qPCR). KEGG functional analysis, single-sample GSEA (ssGSEA), and the ESTIMATE algorithm were used to analyze the mechanisms and immune status between the different risk groups. The sensitivities for potential therapeutic drugs for AML were also investigated.ResultsFive hundred and three lncRNAs related to 19 CRGs in AML samples from the TCGA database were obtained, and 21 differentially expressed lncRNAs were identified based on the 2-year overall survival (OS) outcomes of AML patients. A 4-cuproptosis-related lncRNA signature for survival was constructed by LASSO Cox regression. High-risk AML patients exhibited worse outcomes. Univariate and multivariate Cox regression analyses demonstrated the independent prognostic value of the model. ROC, nomogram, and calibration curves analyses revealed the predictive power of the signature. KEGG pathway and ssGSEA analyses showed that the high-risk group had higher immune activities. Lastly, AML patients from different risk groups showed differential responses to various agents.ConclusionA cuproptosis-related lncRNA signature was established to predict the prognosis and inform on potential therapeutic strategies for AML patients.