Pediatric Acute Myeloid Leukemia Patients Research Articles

Cryptanshinone extract of Salvia miltiorrhiza stimulates pediatric acute myeloid leukemia stem cell apoptosis and the anti-inflammatory mechanism via accelerating microRNA-211-5p to supress Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway activation.

This study was designed to explore cryptanshinone (CPT) extract of Salvia miltiorrhiza stimulating pediatric acute myeloid leukemia (AML) stem cell (LSC) apoptosis and anti-inflammatory mechanism via accelerating microRNA (miR)-211-5p to restrain Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway activation. Obtaining blood samples from pediatric acute myeloid leukemia patients and healthy volunteers and detecting miR-211-5p and JAK2 were performed. Purchase of the human AML cell line KG1a was conducted, and sorting of KG1a cells was to gain LSC. Test of miR-211-5p and JAK2, the phosphorylation of JAK2/STAT3 was implemented. Pretreatment of LSCs was with CPT. Variation of miR-211-5p and JAK2 in LSCs was via plasmid transfection to explore their actions in cell advancement with apoptosis and inflammation. Identification of the targeting of miR-211-5p with JAK2 was implemented. In results: MiR-211-5p was declined in endometrial cancer, while JAK2 was elevated; CPT was available to boost LSC apoptosis and restrain the inflammation; elevated miR-211-5p or repressive JAK2 was available to strengthen the acceleration of CPT on LSCs apoptosis and the repression of inflammation; MiR-211-5p targeted JAK2; augmented JAK2 was available to turn around the action of elevated miR-211-5p. We conclude that CPT extract of Salvia miltiorrhiza stimulated pediatric LSC apoptosis and restrained the inflammation via accelerating microRNA (miR)-211-5p to suppress JAK2/STAT3 pathway activation.

Effects of circ-SFMBT2 on Proliferation, Migration and Invasion of Acute Myeloid Leukemia Cells by Regulating miR-491-5p/HOXA9 Axis

To explore the effects and molecular mechanism of circ-SFMBT2 on the proliferation, migration and invasion of acute myeloid leukemia (AML) cells. Bone marrow samples from 35 pediatric AML patients and 35 healthy controls in Henan Provincial Children's Hospital from April 2015 to April 2017 and human bone marrow stromal cell lines (HS-5) and AML cell lines (HL-60, THP-1, U-937 and Kasumi-1) were collected. The expressions of circ-SFMBT2, miR-491-5p and homeobox A9 (HOXA9) in bone marrow samples and cells were detected by RT-qPCR and Western blot. The Pearson method was used to analyze the correlation of circ-SFMBT2, miR-491-5p and HOXA9 mRNA expression levels in bone marrow samples of AML patients. HL-60 cells were cultured in vitro and divided into 5 groups: Control, si-NC, si-circ-SFMBT2, si-circ-SFMBT2+anti-NC and si-circ-SFMBT2+anti-miR-491-5p, HL-60 cells were transfected with si-NC, si-circ-SFMBT2, anti-NC, and miR-491-5p inhibitor with Lipofectamine™ 3000. RT-qPCR and Western blot were performed to detect the expression levels of circ-SFMBT2, miR-491-5p and HOXA9 in cells of each group. The proliferation activity of HL-60 cells in each group was detected by CCK-8 assay at 24, 48 and 72 h after transfection, respectively. The apoptosis rate was detected by flow cytometry. The migration and invasion abilities of cells were detected by Transwell assay. The regulatory roles of circ-SFMBT2, miR-491-5p and HOXA9 in AML cells were verified by dual-luciferase reporter gene assay, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) experiments. The expression levels of circ-SFMBT2 and HOXA9 mRNA were increased in bone marrow samples and cell lines (HL-60, THP-1, U-937 and Kasumi-1) of children with AML (P <0.001), while the expression level of miR-491-5p was significantly decreased (P <0.001). Pearson correlation analysis showed that the expression levels of circ-SFMBT2 and miR-491-5p in bone marrow samples of AML children were negatively correlated (r =-0.905), miR-491-5p was also negatively correlated with HOXA9 mRNA (r =-0.930), while the expression levels of HOXA9 mRNA and circ-SFMBT2 was positively correlated (r =0.911). The overall survival rate of AML children with high expression of circ-SFMBT2 was significantly decreased than those with low expression of circ-SFMBT2 (P <0.05). Silencing of circ-SFMBT2 could greatly up-regulate the expression of miR-491-5p, decrease the expression of HOXA9, inhibit the proliferation, migration and invasion of AML cells, and promote cell apoptosis (P <0.05). Down-regulation of miR-491-5p expression greatly attenuated the inhibitory effects of circ-SFMBT2 silencing on cell proliferation, migration and invasion (P <0.05). Dual-luciferase reporter gene assay, RNA pull-down and RIP experiments confirmed that circ-SFMBT2 could target miR-491-5p and negatively regulate the expression of miR-491-5p in AML, and HOXA9 was the target of miR-491-5p. Silencing of circ-SFMBT2 may inhibit the proliferation, migration and invasion of AML cells by regulating the miR-491-5p/HOXA9 axis.

A DNA Methylation Signature Predicts Clinical Outcome in Pediatric AML Patients

Pediatric Acute Myeloid Leukemia (AML) is a heterogenous disease with a dismal outcome. Recently, transcriptomic-based leukemic stemness scores have highlighted the importance of DNA methyltransferase 3B in pediatric AML prognosis, which invites a deeper investigation of leukemic stem cells through the scope of their DNA methylome. Other studies have shown the prognostic impact of genome-wide methylation burden (PMID: 29928480, 33080932). As the World Health Organization has recognized methylation analysis as the most apt technology for determining lineage and cell population origins of tumors (PMID: 34921008), it is befitting to evaluate the DNA methylome as a prognostic tool in hematopoietic malignancies. Thus, we hypothesize that a DNA methylation-based signature has the potential to enhance the prognostic precision in pediatric AML. In this study, we applied machine learning approaches to DNA methylation data from 924 pediatric AML patients (only including data from samples with diagnostic bone marrow specimens) from three Children's Oncology Group clinical trials: AAML1031, AAML0531, and AAML03P1, available through GSE190931, GSE124413 and GDC_TARGET-AML, respectively. Clinical endpoints available were defined as :i) Minimal residual disease after induction 1 (MRD1): positive MRD1 if ≥ 1 leukemic cell per 1,000 mononuclear bone marrow cells (≥ 0.1%) determined by flow cytometry; ii) event-free survival (EFS) defined as the time from study enrollment to induction failure, relapse, secondary malignancy, death, or study withdrawal for any reason, with event-free patients censored on last follow-up; iii) overall survival (OS) defined as the time from study enrollment to death, with living patients censored on the date of last follow-up. Processing of the raw data followed best practices in the literature using SeSAMe (PMID: 30085201, 27924034). To independently validate the findings derived from the discovery cohort, we processed in parallel meDNA array data from bone marrow specimens at diagnosis from 201 AML patients treated on the multi-site clinical trials AML02 and AML08 (NCT00136084 and NCT00703820). As step 1, we performed Epigenome-Wide Association Study with Cox Proportional Hazards Regression (Cox-PH EWAS) to identify CpGs with methylation levels most predictive of OS, controlling for risk group categories. At p-value threshold of 1.0e-6, 167 CpGs were associated with OS, which were chosen as candidate CpGs for step 2 analysis using LASSO-based Cox-PH model, with 1,000 iterations of 10-fold cross-validation. 35 CpGs were consistently selected as non-zero coefficients in at least 95% of the models and were used to define the CpG based signature. Patients were further categorized into either Low MethylScoreAML Px (n=563, 60%) or High MethylScoreAML Px (n=361, 40%) groups using recursive partitioning statistics. In the discovery cohort, patients with High MethylScoreAML Px demonstrated a markedly poorer probability of OS (HR 3.91, p&amp;lt;0.0001) and EFS (HR 2.61, &amp;lt;0.0001) in comparison to the low MethylScore. These results were further tested in the validation cohort (n=201) for OS (HR 2.45, p=0.0006) and EFS (HR 2.15, p=0.0004). Notably, in subgroup analyses for both discovery and validation, MethylScoreAML Px remained statistically significant in multivariable models for EFS and OS with adjustments for MRD1 status, risk group, FLT3 status, white blood cell count at diagnosis, and patient age, indicating it as a robust, independent model supplementing currently known prognostic factors. In conclusion, this study unveils MethylScoreAML Px, a methylation-based prognostic risk score that robustly predicts clinical outcome in pediatric AML patients. To our knowledge, we are the first group to describe a Cox-PH-EWAS analysis in pediatric AML, as well as the first to devise and validate a prognostic risk score in &amp;gt;1000 patients from multiple pediatric-only trials. Future directions of research are focused on testing MethylScore in fresh peripheral blood samples using long read, real-time Oxford Nanopore sequencing technology, and comparing it with other risk scores.

Relapse-Enriched Gene Expression Signature of Prognostic Relevance in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the abnormal proliferation and accumulation of immature myeloid cells. While standard induction therapy achieves remission in a substantial proportion of AML patients, relapse rates remain high, particularly in children (approximately 40%) and adults (around 70%). Despite advancements in AML treatment strategies, the management of relapsed and refractory disease remains significant clinical challenge, resulting in dismal outcomes with a three-year survival rate below 10%. Therefore, there is an urgent need to identify predictive markers for relapse and refractory AML to improve treatment outcomes. To address this urgent need, we evaluated differential gene expression in relapse vs diagnosis and its impact on outcomes. RNA sequencing data from bone marrow samples were obtained from pediatric AML patients enrolled in four Children's Oncology Group clinical trials: AAML0531 (NCT00372593), AAML1031 (NCT01371981), AAML03P19 (NCT00070174), and CCG2961 (NCT00003790) through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Gene expression data was batch corrected using Combat. RNA-seq data from 1441 patients available at https://portal.gdc.cancer.gov/projects/TARGET-AML were included in the current analysis of which 31 had RNA-seq data from both diagnosis and relapse and 1420 had RNA-seq data from only from diagnosis. In step 1, we identified genes differentially expressed at relapse using RNA-seq data from 31 matched diagnosis and relapse samples using wilcoxon signed rank test. At p-values &amp;lt;0.005, 201 genes were differentially expressed, with 147 upregulated and 54 downregulated at relapse compared to diagnosis (Figure 1). To further establish clinical relevance of these genes, we evaluated their association with event-free survival (EFS) in the 1420 patients with RNA-seq data at diagnosis (N=1420) using Cox proportional hazard model, identifying 47 genes significantly associated with EFS (p&amp;lt;0.05). Of these, 9 were pseudogenes, 4 had lower expression at relapse and 34 were expressed at higher levels in the relapse samples. We evaluated these 34 genes using a LASSO penalized Cox proportional hazards model, with over 1000 iterations of 10-fold cross-validation. Genes passing more than 95% of the iterations were further selected to build a relapse signature which constituted 23 genes.Notably, this signature was comprised of several important genes including TCF3, a transcription factor with known mutations in some B-cell acute lymphoblastic leukemia (B-ALL) cases; BIRC5 (Survivin), an apoptosis inhibitor whose overexpression has been linked to therapeutic resistance across cancers; PAX5, a key gene for B-cell differentiation that has identified aberrations B-ALL; BCL7A, a member of the BCL gene family associated with lymphoid malignancies, as B-cell lymphomas; and PARP1, known for its role in DNA repair and the recent therapeutic implications of its inhibitors in BRCA-mutated solid tumors. Patients were further stratified into high and low relapse risk groups based on relapse signature scores using recursive partitioning. Patients with high relapse signature score had inferior EFS and OS as compared to those with low signature score (EFS, HR: 2.25, 95% CI 0.25-0.99, p&amp;lt;0.001); OS, (HR 2.35,95% CI 0.40-0.99, p&amp;lt;0.001), Figure 1). Multivariable Cox regression analysis further revealed that the relapse score remained a significant predictor of patient outcomes after adjusting for established risk factors including risk group, race, white blood cell count, and age at diagnosis (EFS: HR 1.64, 95% CI, 1.40-1.92, p&amp;lt;0.001 and OS: HR 1.62, 95% CI 1.33-1.97, p&amp;lt;0.001). To validate the signature, we evaluated it in an independent cohort of 147 pediatric AML patients treated on St Jude AML08 clinical trial with RNA seq data available. Consistent with the discovery cohort, patients with high score had worse EFS (HR: 1.83, p=0.045) and tended to have lower OS (HR: 1.71, p=0.12). These results indicate the relapse-enriched gene expression signature can effectively risk-stratify newly diagnosed pediatric AML patients. Ongoing studies are focused on validating this signature in other independent cohorts, as well as integrating it with cytogenetic features and other previously reported signatures of relevance such as LSC to establish its prognostic utility.

Dysregulation of Immune Regulators in AML Bone Marrow Promotes Dysfunction of CD8 + T Cells

Background: Acute myeloid leukemia (AML) originates in the bone marrow, evading immune recognition and destruction. The bone marrow microenvironment (BME), comprising diverse immune cells and regulators, profoundly influences AML development. However, the impact of immune regulators in the BME, particularly on T-cell anti-AML immunity, remains inadequately understood. Therefore, evaluating the impact of immune regulators in the AML bone marrow microenvironment on T cell dysfunction is of paramount importance and necessity. M ethod : Bone marrow blood samples were obtained from pediatric AML patients at diagnosis and age-matched healthy individuals considered as hematopoietic stem cell transplantation (HSCT) donors after written informed consent. By centrifuging at 2000-3000rpm for 10min, we obtained the supernatant as plasma and stored it in a -80°C refrigerator. After using proteomics to identify the expression of 180 immune regulatory factors in plasma samples, we performed GO and KEGG functional enrichment analysis. After density gradient centrifugation, we isolated bone marrow mononuclear cells (BMMNCs) and immediately identified frequencies of T cell populations and expression levels of functional markers by flow cytometry when they were fresh. Moreover, the co-culture model of CD8 +T cells and THP-1 cell line was used to evaluate the anti-AML effector function of T cells. CD8 +T cells in BMMNCs were sorted by FACS and then subjected to whole transcriptome sequencing and in vitro experiments. After using the TRRUST and KnockTF databases for transcription factor prediction analysis, we selected phospho-flow cytometry to detect and verify the association between the level of phosphorylation of transcription factors and the function of CD8 +T cells. The data were analyzed using R software. Results: Using proteomics techniques, we uncovered a substantial presence of immune-suppressive regulatory factors in AML bone marrow plasma. Interestingly, the canonical NF-κB pathway, mediated by immune regulators and vital for immune regulation in BME, displayed significant downregulation, implying T-cell dysfunction. In our investigation, we identified dysfunctional CD8 +T cells in the primary AML bone marrow, characterized by heightened expression of co-inhibitory receptors and pro-dysfunctional transcription factors, as well as significantly decreased anti-AML effector function. Transcriptome sequencing further linked AML CD8 +T cell dysfunction to reduced transcriptional activity of NF-κB p65 in the canonical NF-κB signaling pathway. Intriguingly, in vitro inhibition of NF-κB p65 and its phosphorylation led to elevated expression of exhaustion markers, such as PD-1, LAG-3, and TIM-3 on primary CD8 +T cells. Most notably, the stimulation of the canonical NF-κB signaling pathway effectively restored activation and effector function-related pathways in dysfunctional AML CD8 +T cells. Conclusion: Immune regulators in the bone marrow microenvironment play a vital role in maintaining dysfunctional AML CD8 +T cells. These findings provide a novel strategy to investigate the potential mechanisms and interventions for T-cell dysfunction.

Pre-Transplant Levels of Multiparametric Flow-Cytometry Measurable Residual Disease (MFC-MRD) Predict Outcomes in Children with Acute Myeloid Leukemia Given Allogeneic Hematopoietic Stem Cell Transplantation

Background: Measurable residual disease assessed by multiparametric flow-cytometry (MFC-MRD) is largely used in trials for childhood de novo acute myeloid leukemia (AML) in the early phase of treatment, to stratify patients into different risk groups and to tailor treatment. Despite this, unlike acute lymphoblastic leukemia, the impact of MFC-MRD before allogeneic hematopoietic stem cell transplantation (HSCT) on patients' outcomes has yet to be established. The aim of this study was to evaluate the prognostic role of MFC-MRD before HSCT in a cohort of children with AML treated in centers belonging to Associazione Italiana Ematologia/Oncologia Pediatrica (AIEOP) cooperative group. Patients and Methods: We analyzed MFC-MRD in 208 pediatric patients affected by de novo AML who underwent a first allo-HSCT after a myeloablative conditioning regimen in an AIEOP Centre between 12/2011 and 12/2021. Assessment of MFC-MRD was performed on BM sample within 60 days from the transplantation date. Analysis of 5-10 color-panel MFC-MRD was centrally performed at the Laboratory of Diagnosis and Research, University of Padova, according to standardized operating procedures previously described (Buldini et al., BJH 2017). We defined MFC-MRD positivity as the presence of at least 50 leukemic events of 500,000 acquired nucleated cells. Five-year overall (OS) and event-free survival (EFS) were estimated using Kaplan and Meier method and differences between groups were tested using the log-tank test; the cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated using the Fine&amp;Gray method to take into account the respective competitive risks. Results: Median age at HSCT of these 208 patients was 7.8 years (range 0.4-23.3); 168 patients (80.8%) were transplanted in CR1, 30 (14.4%) in CR2 and 10 (4.8%) in CR3 or more advanced disease. Forty-eight patients (23.1%) were transplanted from a matched family donor, 85 (40.9%) from an unrelated donor (59 fully-matched and 26 mismatched), while 75 (36%) from a partially matched family donor. The source of stem cells was bone marrow in 117 patients (56.2%), peripheral blood in 81 (39%) and cord blood in 10 (4.8%). Median time from MRD evaluation and HSCT was 22 days (range, 6-57). Overall,168 out of 208 (78.9%) patients were MFC-MRD negative in the pre-HSCT BM, while 44/208 (21.1%) showed a positive MFC-MRD. Among those, MFC-MRD was &amp;lt; 0.1% in 12/44 (27%) patients, 0.1-&amp;lt;1% in 16 patients (36.5%) and ≥ 1% in 16 patients (36.5%). The 5-year OS and EFS of the whole cohort were 79.2% and 65.7%, respectively, while CIR and NRM were 24.7% and 8.6%. Notably, patients with negative MFC-MRD had a significantly better 5-yr OS and EFS (Fig.A) as compared to those with any MFC-MRD positivity (OS 86.3% vs 50.2%; EFS 71.6% vs 42.4%, p &amp;lt;0.0001 in both analysis). CIR significantly differed between patients with MFC-MRD negative and positive (21.1% vs 43.9%, p=0.001) (Fig.B). We also observed a higher NRM in patients with MFC-MRD positivity (13.6%) as compared to those with negative MFC-MRD (7.2%), although this was not statistically significant (p=0.1). Disease status (CR1 vs CR2 vs other CR) did also influence EFS (69.9% vs 55.6% vs 23.3%, p&amp;lt;0.001), OS (83.2% vs 66.6% vs 40.6%, p=0.02) and CIR (21.1% vs 37.7% vs 66.6%, p&amp;lt;0.001), while other variables (including year of HSCT, type of donor and stem cell source employed) did not. In a multivariable model for OS, EFS and CIR, including MFC-MRD, disease status and year of HSCT, MFC-MRD (pos vs neg) remained statistically significant (HR for OS 3.5, p=0.0004; HR for EFS 2.38, p=0.003; HR for CIR 2.14, p=0.02). In addition, advanced disease phase was an independent risk factor for CIR (HR 1.81, p=0.008). Conclusion: These data document the independent prognostic value of MFC-MRD before HSCT in a large cohort of pediatric AML patients, using a centralized evaluation by 5-10 color MFC. Patients with negative MFC-MRD before HSCT have a significantly lower risk of treatment failure. In light of these results, we recommend monitoring MFC-MRD before HSCT in pediatric AML patients as a critical tool for therapy-intervention decision before or after HSCT (e.g., additional treatment for improving leukemia control or less intensive/shorter GvHD prophylaxis) with the aim to improve patients' outcomes.

ACS10- Cytarabine Pharmacogenomics Score Impacts Survival in Pediatric AML Patients Treated on AAML1031 Trial and Associates with Outcome Differences in Black AML Patients

Cytarabine (ara-C) has been used to treat acute myeloid leukemia (AML) for decades. While ara-C based regimens successfully induce remission in the majority of patients, significant number of patients experience relapse. Although advances in hematopoietic stem cell transplantation (HSCT) and supportive care regimens have improved over the years, it is imperative that personalized methods to optimize pharmacological intervention and improve outcomes are needed. To identify genetic predictors of ara-C response, we recently reported an ara-C pharmacogenomics score (ACS10) consisting of 10 SNPs in nine ara-C pathway genes (PMID: 34990262). In pediatric patients treated on AML02 and AAML0531 trials, low ACS10 score (&amp;lt;0) was associated with poor survival as compared to patients with high score (&amp;gt;0). Given these promising results, we sought to investigate the ACS10 score in a large cohort of children treated on Children's Oncology Group clinical trial AAML1031 (NCT01371981). AAML1031 trial enrolled 1,097 newly diagnosed patients from 0 to 29.5 years of age that received either conventional AML treatment (ara-C, daunorubicin and etoposide (ADE): arm A) or conventional treatment with the addition of bortezomib (ADE+bortezomib: arm B). Overall, no benefit for adding bortezomib was observed (PMID: 32029509). The current study included 717 patients with both DNA specimens and clinical outcome data available. ACS10 SNPs were genotyped using Taqman assays and ACS10 score was calculated for each patient as per previous report (PMID: 34990262). Overall, for the 717 patients included in this study, 249 patients had low ACS10score (&amp;lt; 0) and 468 patients had a high ACS10 score (&amp;gt;0). When combining patients treated on arm A and arm B, ACS10 score was not associated with overall survival (OS). However, patients with low ACS10 scores had poorer event-free survival (EFS) than those with high ACS10 scores, although this difference was not statistically significant (HR: 0.827, 95% CI (0.669-1.022), P=0.078). Patients with high ACS10 scores in arm A had improved EFS (HR: 0.689, 95% CI (0.50-0.94), P=0.023, Fig1), while OS did not show significant differences (P=0.378). Excluding the patients who received HSCT, showed increased EFS for patients with high ACS10 scores as compared to low ACS10 scores when treated with standard chemotherapy (HR: 0.628, 95% CI (0.445-0.887), P=0.008 Fig 1). In contrast, a significant difference was not observed within the standard ADE+bortezomib arm (arm B). At the whole cohort level, black patients (N=84) had reduced OS (HR: 1.47, 95% CI (1.02-2.13), P=0.041) as compared to white patients, while there was no significant decrease in EFS (HR: 1.22, 95% CI (0.89-1.66), P=0.206) (N=522). Within arm A, black patients had significantly poorer outcome as compared to white patients (EFS, HR: 1.718, 95% CI (1.099-2.686), P=0.018 Fig1; OS HR: 1.80, 95% CI (1.05-3.10), P=0.032). Difference in outcome by race in arm B was not significant implying addition of bortezomib might have some benefit to black patients. Subsequent comparison by treatment arm showed a non-significant but better outcome in black patients when treated on arm B (Black patients: EFS, arm A vs. arm B: HR:0.66, 95% CI (0.37-1.17), P=0.154). Consistent with our previous investigation, low ACS10 score group was more abundant in black patients as compared to white patients (69% of black patients vs. 28% of white patients had low ACS10 score, P&amp;lt;0.001). These results are particularly salient given trends showing poor outcome in black as compared to white AML patients. Despite AAML1031 being one of the largest pediatric AML cohorts, the small number of black patients limited our ability to do further subset analyses by scores and arms (only 10 of 39 black patients treated on arm A had high ACS10 score). In conclusion, we demonstrate the utility of the PGx-based ACS10 score in a large cohort of newly diagnosed pediatric AML patients when treated with standard ADE induction regimen. Consistent with previous observation, black patients with low ACS10 score had inferior outcome in standard arm, however such an association was not observed for the bortezomib arm. Higher abundance of the low ACS10 SNP scores in black patients warrants further in-depth evaluation. Our future and ongoing studies include compiling data from multiple clinical trials to increase the sample size as well as expanding investigations within Hispanic/Latino AML patients

Cardiotoxicity Related Mortality Among Children with Acute Myeloid Leukemia in Low Middle Income Countries: Is It Treatment or Sepsis Related?

Background: Heart disease and cancer are two major causes of morbidity and mortality worldwide, accounting for at least 70% of the medical reasons for mortality across the globe. Modern treatment strategies led to improvements in cancer survival, however, these gains might be offset by the potential negative effect of cancer therapy on cardiovascular health. Over the last few decades, cooperative oncology group trials for pediatric acute myeloid leukemia (AML) have improved overall survival (OS) to nearly 65% by combining intensive chemotherapy with improved supportive care. Despite their efficacy, anthracyclines are linked to an increased risk of cardiotoxicity. Objectives:The study aimed to describe the incidence of cardiotoxicity among pediatric patients with acute myeloid leukemia, assess the effect of cardiotoxicity on treatment outcome in terms of overall survival (OS) and event free survival (EFS) and to determine the different risk factors (Age, gender, body mass index, cumulative doses of anthracyclines and sepsis) that may contribute to cardiotoxicity. Patient and Methods: A retrospective study, conducted at the National Cancer Institute (NCI), Cairo University, Egypt.,that included all pediatric patients ≤ 18 years old diagnosed and treated as Acute Myeloid Leukemia (AML) during the period from the first of January 2015 to the end of December 2019. Retrospective analysis of the different features of cardiotoxicity associated with the treatment of AML. Results : The study included 142 patients with a median age of 8.5 years old and M/F ratio 1.2:1.Out of the total study population, Eighty four patients (59.2%) developed cardiotoixicity in which 59.5% were acute onset cardiotoxicity and 40.5 % were chronic early cardiotoxicity and there was no detected cases of chronic late cardiotoxicity.Cardiac dysfunction was detected in 65/142 (45.8%) patients at time of death in association with sepsis. The cumulative incidence of cardiotoxicity was higher in the presence of blood stream infection with p value (p&amp;lt;0.001).55/84 (65.5%) patients with cardiotoxicity showed progressive cardiac dysfunction and they all died due to sepsis that was associated with cardiac dysfunction. At the end of the study the OS and EFS were 16.2 % and the median survival time was 1.9 month.The cumulative OS,EFS and the median survival time were reduced in the presence of acute cardiotoxicity with statistical significant p-value (p&amp;lt;0.001). Conclusion: The incidence of cardiotoxicity is high among pediatric AML patients and the most important risk factor for cardiotoxcity is blood stream infection. The Cumulative OS, EFS and median survival time are reduced in patients with acute cardiotoxicity which represents a major challenge that warrants more attention to the field of cardio-oncology.

Immunological Footprints of Pediatric Acute Myeloid Leukemia in Local Bone Marrow T Cells

Introduction: Despite notable advancements, childhood acute myeloid leukemia (AML) remains associated with one of the poorest prognoses among pediatric malignancies. Conventional chemotherapies carry significant toxicity, underscoring the urgent need for novel e.g. immunotherapy-based approaches. Despite AML being previously considered non-immunogenic due to its low mutational burden, the complex interplay between leukemic blasts and T cells within the bone marrow in pediatric patients remains largely unexplored. This study investigates the AML-induced footprint on local bone marrow T cells (bmT cells) representing tumor-infiltrating lymphocytes, with the aim to reveal functional points of action for future immunotherapy. Methods: Cryopreserved bone marrow samples from both pediatric AML patients (n=29) and age-matched healthy bone marrow donors (HD, n=9) were analyzed. Multicolor flow cytometry quantified surface expression of inhibitory signaling and activation receptors, as well as T cell differentiation stages. RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) were performed of sorted CD8 + bmT cells. Bulk RNA and ATAC-Seq data was visualized by principal component analysis (PCA). Another PCA was performed on the combined dataset of RNA- and ATAC-Seq. Differential analysis using DESeq2 (fitType “parametric”, testType “Wald”) identified 135 differentially expressed genes in CD8 + bmT cells between HD and AML patients based on fold change (≥1/≤-1) and statistical criteria (s-value&amp;lt;0.05). Results: Based on CD45RO, CD62L and CD95 surface expression, late differentiation stages of bmT cells were enriched in AML. Namely, the frequency of naïve bmT cells was reduced (33.9% vs. 57.5%, mean values, p=0.0018), while the frequency of effector memory bmT cells was increased in AML patients (21.5% vs. 9.4%, p=0.0010). This could be validated by RNA-Seq of sorted CD8 + bmT cells which documented higher expression of cytotoxic effector T cell genes such as Perforin, Granulysin and Granzyme B. In addition, our analysis also revealed higher expression of functional relevant genes associated with T cell cytotoxity (ADGRG1), effector function (BATF/TPX2) or exhaustion (SLAMF7) in AML patients. Next, surface expression of receptors for inhibitory signaling and activation was analyzed. Several of these receptors have also been described as immune checkpoints (ICP). Analyzing differential expression between HD and AML patients showed pronounced upregulation of inhibitory markers (TIM-3: 7.1% vs. 1.7%, p&amp;lt;0.0001, CD39: 7.7% vs. 2.0%, p=0.0103, LAG-3: 8.0% vs. 2.9%; CTLA-4: 3.9% vs. 1.6%, p=0.0024; PD-1: 34.8% vs. 21.3%, p=0.0016). Since CD8 + bmT cells showed the most promising effects in the flow cytometry analysis, this subpopulation of bmT cells was further characterized by RNA- and ATAC-Seq. Here, clustering of HD and AML patients' CD8 + bmT cells was observed in PCA. Interestingly, PCA also revealed differential clustering of CD8 + bmT cells from AML primary and relapse samples. Confirming the observed differences in phenotype of bmT cells in AML patients, we identified distinct bmT cell populations specifically induced in AML patients compared to HD, based on phenotype and differential ICP expression patterns. Through tSNE analysis, we discovered a CD8 + effector memory T cell population with a TIM-3 +PD-1 +2B4 + phenotype, comprising 2.3% of all bmT cells in AML samples compared to only 0.03% in HD. Conclusion: This study reveals significant alterations in the activation and inhibition potential of bmT cells in pediatric AML compared to HD, as confirmed by comprehensive analysis of the epigenome, transcriptome and surfaceome. We confirm the observed differences through the identification of a T cell population with a typically exhausted phenotype specific for AML patients when compared to HD, implying persistent antigen exposure and potential direct or indirect interactions with leukemic blasts. Although prospective clinical trials will have to confirm functional relevance in patient cohorts, this study provides crucial insights into the immune landscape of pediatric AML, underscoring the potential for targeted immunotherapeutic interventions.

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) &amp;gt;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 &amp;lt;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.

Identification of a Relapse Signature in t(8;21) Pediatric AML

Pediatric acute myeloid leukemia (AML) is a malignancy of the blood in which myeloid progenitor cells are interrupted in their normal course of development and proliferate out of control. Recent studies such as the TARGET initiative, have revealed pediatric AML to be driven by relatively few molecular abnormalities when compared to adult AML. Most frequently occurring in genes directly involved in or regulating transcription. These molecular aberrations are drivers of disease, inform patient risk stratification and are transcriptomically unique from one another. Of these events, RUNX1-RUNX1T1 is the most common, evident in approximately 15% of pediatric AML cases. While RUNX1-RUNX1T1 is a positive outcome prognosticator, 20% of these patients go on to relapse with significantly poorer outcomes. Our objective was to identify a relapse signature that could be identified at the time of diagnosis so that alternative therapeutic strategies can be employed. Here we report the investigation of RNAseq data from 3,022 pediatric AML patient samples and identification of a transcriptomic relapse-signature (TRS) via differential expression analysis, supervised clustering and machine learning modelling. We performed differential gene expression analysis comparing diagnostic t(8;21) patient samples for those who relapsed (R) (n=38)vs. those who did not relapse (NR) (n=111). Analysis was performed using DESeq2 in R 4.3.0. P-values were adjusted with Benjamini-Hochberg correction to control for false discovery rate a significance threshold for padj of &amp;lt; 0.05 was applied. A list of 462 differentially genes was returned. Hierarchical clustering of the 462 differentially expressed genes revealed 4 patient clusters, each with a distinct transcriptomic signature ( Fig 1A). K-means (n=4) also returned similar results to the hierarchical clustering. Kaplan-Meier analysis of the 4 clusters showed a significant difference in patient outcomes both in terms of overall survival (P &amp;lt; 0.001) and event-free survival (P &amp;lt; 0.05) ( Fig 1B). In marked contrast to clusters 1-3, cluster 4 was highly enriched in patients who relapsed at a rate of 86%. Clusters 1, 2 and 3 had relapse rates of 36, 29 and 38 percent respectively. To identify the most relevant genes that could serve as a TRS, we trained a random forest (RF) model using transcript per million (TPM) transformed and length normalized counts for each of the 462 differentially expressed genes at the time of diagnosis between the R and NR cohort; the response variable was patient relapse outcome. Data was split into 70% training and 30% testing cohorts. As the small size of our cohort was a potentially limiting factor, we utilized 5-fold cross validation in the training of our model. Recursive feature selection was applied to the model and was iteratively trained using subsets of features (predictor genes) ranging from 1 to 100. Features were then recursively sorted by importance and an optimal number of 30 predictors were chosen. Application of our model to the testing dataset of 45 patients (R = 12, NR = 33) allowed us to accurately predict the outcome in 40 of 45 patients. This model accurately predicted 33 of 33 NR and 7 of 12 R patients. This model was both specific and sensitive for NR and specific to R but lacked sensitivity as we were only able to identify 58% of relapse patients. Finally, this 30-gene list was then compared to survival analysis on a by-gene basis which yielded a final panel of 16 genes which we call our TRS. These genes were; PRDM8, RARG, CSF1, PDE4B, CCL3, NAB1, WHRN, SGSM2, LPIN3, CHARLIE1B, SATB1, ZNF441, ZNF521, AL451123.1, TSPYL4 and B3GNT5. The RF model was re-trained on this smaller panel and re-tested with similar results. By applying differential expression analysis, K-means clustering and machine learning to RNAseq data from a cohort of 149 pediatric AML patients we successfully identified a relapse-signature using a panel of 16 genes evident at the time of diagnosis and prognostic of patient outcomes. Future efforts will focus on increasing the sensitivity of our model to relapse prediction and further validation on other pediatric t(8;21) cohorts.

A 46 Long Non-Coding RNAs Expression Signature Accurately Predicts Relapse and Acts As an Independent Prognostic Factor in Pediatric Acute Myeloid Leukemia

Risk stratification using cytogenetics, mutations and minimal residual disease (MRD) has allowed to increase the cure rates of pediatric acute myeloid leukemia (AML), reaching now up to 70% in contemporary protocols. Nevertheless, approximately 30% of patients still experience relapse, indicating that there is a need to optimize stratification strategies. Recently, long non-coding RNA (lncRNA) expression has been shown to hold prognostic power in multiple cancer types. Here, we built a 46 relapse-related lncRNAs prognostic signature, named AML lnc46, using 790 pediatric acute myeloid leukemia transcriptomes obtained from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) repository. Bootstrap validation (bootstrap resampling times = 1000) was used for internal validation and showed that the signature performed good in predicting the 1-, 2-, and 3-year relapse-free survival (RFS), with an area under the curve of 0.825, 0.824, and 0.822, respectively. Moreover, external validation using the TARGET repository validated the efficiency of the signature. Most importantly, we demonstrate that AML lnc46 not only is an independent predictor of RFS, but also identifies patients who were assigned a different risk-group by currently-used classification methods. In conclusion, the identified AML lnc46 might, after further prospective validation, provide additional information to guide management of pediatric AML patients. Figure 1. (A) Kaplan-Meier survival curve of relapse-free survival of high-and-low risk AML lnc46 pediatric AML patients, (B) Univariate and multivariate independent Cox regression analyses of known clinical and molecular characteristics and the AML lnc46 signature.

Analysis of Safety and Efficacy of Avapritinib As Targeted Therapy for Pediatric Acute Myeloid Leukemia Patients with KIT Mutation after Transplantation

Objective: To investigate the safety and efficacy of Avapritinib in pediatric acute myeloid leukemia (AML) with KIT mutation after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: The clinical data of 6 AML patients with KITmutation and RUNX1::RUNX1T1 fusion gene positive in Children's Hospital of Soochow University were retrospectively analyzed, including 2 cases of KIT:N822K, 3 cases of KIT: exon17 and 1 case of KIT: Asp816Tyr. After achieving complete remission in morphology, allo-HSCT was performed. The clinical outcomes of prophylactic therapy (2/6) and preemptive intervention (4/6) with Avapritinib after complete stem cell engraftment were observed, and the adverse events during treatment were recorded. Results: Adverse events occurred in 4 of the 6 patients, among whom neutropenia was the most common (4/6), followed by facial edema (2/6) and thrombocytopenia (1/6). 2 patients who received prophylactic treatment achieved morphological complete remission, and minimal residual disease (MRD), fusion gene and KIT mutations were all negative. 4 patients received preemptive intervention because of RUNX1::RUNX1T1 fusion gene turned positive after allo-HSCT, and 1 patient's RUNX1::RUNX1T1 turned negative after single-agent Avapritinib treatment for 2 months. 2 patients did not achieve continuous RUNX1::RUNX1T1 negative after preemptive therapy with Decitabine (DAC) and donor lymph infusion (DLI), and then was treated with Avapritinib, one's RUNX1::RUNX1T1 fusion achieved continuously negative after 1 month treatment of Avapritinib, the other's achieved continuous negative after 7 months treatment of Avapritinib. One patient was treated with Avapritinib after DAC and DLI as preemtive intervention, and RUNX1::RUNX1T1 fusion did not turn negative, but the relative value of RUNX1::RUNX1T1 fusion was lower than before and lived stable for more than 10 months. Conclusions: Avapritinib is safe and effective in the prophylactic and preemptive treatment of AML with KIT mutation after allo-HSCT in children, which provides a clinical drug for the prevention of relapse after transplantation.

LAMP5 Is an AML-Restricted Immunotherapeutic Target Enriched in High-Risk Acute Myeloid Leukemia

LAMP5, Lysosomal Associated Membrane Protein Family Member 5, a neuronal protein that plays a role in synaptic plasticity is normally found primarily in the brain, however limited studies have identified it to be expressed in leukemia, particularly KMT2A-rearranged leukemias. Importantly, there is low expression of LAMP5 in normal hematopoiesis making it an ideal potential immunotherapeutic target for both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). As a newly identified aberrant protein in leukemias, there is little data available on the prevalence or clinical implications of LAMP5 expression. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric AML patients to define the prevalence, co-occurring cytomolecular aberrations and clinical outcomes associated with LAMP5 expression. Ribo-depleted RNA seq 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 from BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients, LAMP5 was expressed (positive defined as mRNA transcripts per million (TPM) &amp;gt;2) in 396 patients (28%) with mRNA TPM ranging from 0-884 with similar prevalence found in adult AML (28%; BEAT AML N=126/452, SWOG N= 59/199 TCGA N=50/179), but with significantly higher TPM ranging from 0-3,200. Of the 396 pediatric patients with LAMP5 expression, there was a notable enrichment in KMT2A-r AML (N=203, 51%), which accounts for 59% of the total KMT2A-r cohort. Within the KMT2A-r cohort, there is enrichment in the MLLT1 (N=17/18, 94%), MLLT3 (N=61/93, 66%) and MLLT10 (N=58/69, 84%) fusion groups. Additionally, LAMP5 expression was seen in other high risk fusion subtypes including 100% of patients with KAT6A-CREBBP (N=12/12) and 29% of NUP98 fusions (N=45/154). There is absent LAMP5 expression in normal lymphoid and myeloid cells, but there is moderate expression seen in plasmacytoid dendritic cells, which could provide a therapeutic target for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). Given the association with high-risk AML fusion subtypes, the impact of LAMP5 on clinical outcome in AML pediatric patients was evaluated. High rates of relapse were seen in patients with LAMP5 expression, with a relapse rate of 45% compared to 36% in LAMP5-negative patients. 5 year event-free survival (EFS) was 36% for LAMP5-positive patients compared 45% for LAMP5-negative patients (p=0.0005). Similar poor outcomes were seen with overall survival 54% for LAMP5-positive patients vs. 62% for LAMP5-negative patients (p=0.001). In an analysis of a large cohort of AML patients, we found that LAMP5 expression is highly enriched in many high-risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and NUP98 fusions. Patients with LAMP5 expression have high rates of relapse with significantly worse outcomes compared to patients without LAMP5. Given low levels of LAMP5 in normal hematopoiesis, LAMP5 is an ideal AML-restricted immunotherapy target with broad applicability for many high-risk fusion subtypes of AML. Current efforts to generate a LAMP5 chimeric antigen receptor (CAR) T cell are underway which will be used to evaluate preclinical efficacy in AML via cell- and patient-derived xenograft models with the goal to translate this novel therapy into clinical use to improve the current dismal outcomes for these high-risk patients.

Integrated Analysis of KIT Exon 17 Mutations and Flow-MRD Refines Risk Stratification in Pediatric Acute Myeloid Leukemia with RUNX1:: RUNX1T1

INTRODUCTION: Pediatric acute myeloid leukemia (AML) with RUNX1:: RUNX1T1 is considered a favorable risk group, with a high probability of event-free survival (EFS) in recent clinical trials. However, 20-30% of patients still experience relapse within three years and some patients have poor outcome. Several prognostic factors, including secondary genetic abnormalities and treatment response, have been previously reported. Flow cytometry-based measurable residual disease (flow-MRD) has been suggested as a poor prognostic factor in AML with RUNX1:: RUNX1T1. Additionally, KIT mutations, particularly mutations of exon 17, have been associated with poor EFS in both adult and pediatric patients. However, the combined impact of KIT exon 17 mutations and flow-MRD on prognosis has not been thoroughly investigated. This study aims to evaluate the clinical and genetic features of pediatric patients with RUNX1:: RUNX1T1-positive AML enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-12 trial and determine if integrated analysis of flow-MRD and KIT exon 17 mutations can improve risk stratification. METHOD: A total of 101 pediatric AML patients with RUNX1:: RUNX1T1 were enrolled in the study. The patients were treated in the JPLSG AML-12 trial, and their clinical and genetic characteristics were analyzed. Targeted capture sequencing was performed on bone marrow samples obtained at diagnosis. The custom gene panel was designed for mutation profiling of pediatric AML, including 504 genes. Flow-MRD was centrally monitored at the end of induction 1 (EOI1) and the end of induction 2 but was not used to guide subsequent therapies. Leukemia-associated immunophenotypes were identified in diagnostic specimens and marker combinations that allowed detection of leukemia with a sensitivity of at least 0.1% were applied to subsequent samples. RESULTS: Patients with KIT exon 17 mutations had a higher white blood cell count at diagnosis and a significantly higher rate of MRD positivity (≥0.1%) at EOI1 compared to patients without KIT exon 17 mutations. Immunophenotypic analysis showed no significant differences in CD117 or CD33 expression between patients with and without KIT exon 17 mutations; whereas CD19 expression was observed less frequently and, although not significantly, CD56 expression was observed more frequently in the patients with KIT exon 17 mutations. The genetic landscape analysis revealed that KIT mutations were the most common and tended to coexist with ARID1B mutations, while NRAS mutations were mutually exclusive. The 5-year EFS and overall survival (OS) for the entire cohort were 67.3% and 82.7%, respectively. Negative MRD at EOI1 was associated with significantly superior 5-year EFS and OS (71.6% and 85.9%) compared to positive MRD (12.5% and 37.5%)(p&amp;lt;0.01). Patients with KIT exon 17 mutations had significantly inferior 5-year EFS and OS (46.9% and 62.2%) compared to those without KIT exon 17 mutations (76.8% and 92.4%) (p&amp;lt;0.01). Furthermore, we evaluated a prognostic impact of MRD within the KIT exon 17 mutated and non-mutated cases. As for the non-mutated cases, the 5-year OS in the patients with negative MRD was 92.2%. Among the KIT-mutated cases, the 5-year OS in the patients with negative and positive MRD was 68.0% and 33.3%, respectively. CONCLUSION: Patients with both non-mutated KIT exon 17 and negative MRD have the best prognosis, while positive MRD and KIT exon 17 mutations are associated with poorer outcomes. These findings indicated that integrated analysis of flow-MRD and KIT exon 17 status enables optimal risk assignment strategies in pediatric AML with RUNX1:: RUNX1T1.

Characterization of the Molecular Landscape of Pediatric Acute Myeloid Leukemia: A Retrospective AIEOP AML2013/01 Study

Background. Advances in genomic techniques have recently shed light on leukemia biology, recognizing that pediatric acute myeloid leukemia (AML) is a highly genetically heterogeneous disease. In the last two decades, genetic and cytogenetic abnormalities contributed to the stratification of patients in different classes of risk, where children receive a risk-adapted therapy. In the recently concluded AIEOP-AML2013/01 clinical trial, 371 Italian pediatric AML patients were allocated into three risk groups that were largely based on measurable residual disease at the end of induction therapy and on recurrent molecular markers with a recognized impact on children's outcome. Methods and Results. We screened by RT-PCR, RQ-PCR, capillary electrophoresis and Sanger sequencing 41 genetic markers on 371 RNA and DNA samples, identifying a molecular marker at diagnosis for 247 cases out of the 371 enrolled (67%). As far as gene mutations detected are concerned, we identified 24/371 cases harboring a mutation in exon 12 of NPM1 gene (6.5%), 16/371 a FLT3-ITD mutation (4.3%), and 7 harboring both NPM1 and FLT3-ITD mutations (2%). Moreover, we also found 2 cases having a partial tandem duplication affecting KMT2A gene (2/371, 0.5%), one of them occurring together with a FLT3-ITD mutation. For chromosomal aberrations, we confirmed that t(8;21) RUNX1::RUNX1T1 and inv(16) CBFB::MYH11 are recurrent translocations in pediatric AML, being found in 13.5% (50/371) and 12% (44/371) of cases respectively. Sixty-four (17,3%) AML cases were characterized by a KMT2A fusion with different partner genes, with the t(9;11) KMT2A::MLLT3 being the most recurrent (24/371, 6.5%), followed by t(10;11) KMT2A::MLLT10 (21/371, 5.7%), t(6;11) KMT2A::AFDN (10/371, 2.7%), t(11;19) KMT2A::ELL (6/371, 1.6%). In 14 patients, we found a NUP98 translocations (3.8%), and, in particular, 9 cases harbored the t(5;11) NUP98::NSD1 (2.4%, 5 of them together with a FLT3-ITD mutation), 4 the t(11;12) NUP98::KDM5A (1%) and only one case the t(2;11) NUP98::HOXD13 (0.3%). Other chromosomal aberrations were rarer in our cohort, with t(16;16) CBFA2T3::GLIS2 accounting for 2.4% of cases (9/371), t(6;9) DEK::CAN 1.3% (5/371, co-occurring with FLT3-ITD in 3/5 cases), t(1;22) RBM15::MKL1 1.1% (4/371), t(3;5) NPM1::MLF1 0.5% (2/371, one concomitant with FLT3-ITD), t(16;21) FUS::ERG 0.5% (2/371), t(16;21) RUNX1::CBFA2T3 0.3% (1/371), del(9) SET::NUP214 0.3% (1/371), t(8;16) KAT6A::CREBBP 0.3% (1/371), t(9;22) BCR::ABL1 0.3% (1/371). In addition, we retrospectively evaluated the incidence and prognostic relevance of mutations at specific loci of KIT, FLT3, ASXL1, WT1 and CEBPA genes. As concerns the KIT gene, we identified mutations at exon 8 or 17 in 16/371 AML (4.3%). Of note, all the 16 cases had a concomitant core-binding factor (CBF) rearrangement, and in particular 10/16 the t(8;21) RUNX1::RUNX1T1 translocation and 6/16 the inv(16) CBFB::MYH11, with a relative occurrence of 17% in the CBF-r AML subgroup (16 KIT mutated AML/94 CBF-r) and a slight, but not significant, impact on outcome. As regards exon 13 of ASXL1 gene and the tyrosine-kinase domain of FLT3 gene ( FLT3-TKD), our study confirmed a low prevalence of these mutations, which occurred in 2.5% and 3.4% of AML respectively. On the contrary, WT1 mutations (exons 6-9) occurred in a consistent portion of AML (45 mutated cases, 13.2%)did not affect the outcome of FLT3-ITD mutated AML. Conversely, by screening both at bZIP and NTD domains of the CEBPA gene, we identified 18 double-mutated patients (4.8%), and notably all these patients had no other molecular marker detected at diagnosis, reducing the proportion of negative cases from 33% to 28%. We found that CEBPA mutated cases are characterized by a favorable outcome (EFS 93% vs 58% for CEBPA wt, p=0.008), this finding corroborating the indication to allocate these patients to the standard risk group. Conclusion. Overall, in the prospective AIEOP AML2013/01 trial, we described the frequency of different molecular lesions and improved the genetic landscape of AML. The future use of NGS-based screenings could further optimize the characterization of the genetic landscape of childhood AML, thus refining the risk class patients stratification and modulation of treatment approaches.

Racial and Ethnic Differences in Acuity and Cumulative Frontline Organ Toxicity at the Time of Relapse in Pediatric Acute Myeloid Leukemia

Introduction: Racial and ethnic disparities in survival outcomes exist in pediatric acute myeloid leukemia (AML), but the role of relapse and its potential contribution to outcome disparities is unknown. We sought to examine differences in relapse-free survival (RFS) and clinical characteristics at the time of relapse by race and ethnicity using a real-world multi-institution cohort of pediatric AML patients. Methods: A retrospective cohort including all pediatric (age &amp;lt;19 years) patients treated for AML from 2011-2019 at 13 institutions in the United States was established and is being actively expanded to include diagnoses through 2023. Data collection was accomplished by detailed chart abstraction performed by trained study personnel. We additionally merged resource utilization data from the Pediatric Health Information System to ascertain patient acuity and cumulative organ toxicity. Analyses were restricted to patients identified in the medical record as Hispanic, non-Hispanic Black (NHB), and non-Hispanic White (NHW) to specifically assess historically marginalized patient populations. Other racial/ethnic groups including multi-racial were excluded due to small numbers. Kaplan-Meier survival curves were generated for RFS by race/ethnicity. Adjusted Cox models compared the hazard of relapse by race/ethnicity. Models adjusted for age at diagnosis, sex, initial risk classification, weight category at diagnosis, and acuity at initial presentation. To assess misspecification of outcome, we performed sensitivity analyses restricted to relapses occurring within 1 year of diagnosis (early RFS). Among the patients who relapsed, descriptive frequencies for clinical and treatment characteristics were reported by race/ethnicity. Results: A total of 437 pediatric patients with AML (28% Hispanic, 18% NHB, median follow-up time 3.0 years) were included in the analytic cohort. There were no statistically significant differences in RFS or early RFS by race/ethnicity (Figure 1). However, among relapsed patients, important qualitative differences were noted at the time of relapse (Table 1). Hispanic and NHB patients were older at relapse than NHW patients. Greater proportions of Hispanic and NHB patients (compared to NHW) experienced cardiovascular, respiratory, and renal failure during frontline therapy. Cumulative prevalence of organ failure in 2 or more systems since initial diagnosis was higher for both Hispanic (50%) and NHB (50%) patients compared to NHW patients (29%). Overweight/obesity at relapse was more prevalent among Hispanic (41%) and NHB (50%) compared to NHW (21%) patients. NHB patients were also more likely to present at relapse with higher acuity, defined as requiring ICU-level resources for 2 or more organ systems within 72 hours of admission, compared to NHW patients (75% vs. 57%). Conclusions: Our data suggest a similar risk of relapse by race and ethnicity. However, differences in cumulative frontline organ toxicity and acuity of presentation may lead to differential treatment at the time of relapse and drive subsequent overall survival disparities for Hispanic and NHB patients with pediatric AML. Future Directions: This was a planned interim analysis, and chart abstraction is ongoing to expand this cohort (anticipated N ~1000). We intend to pursue a series of formal mediation analyses regarding the role of relapse, cumulative organ toxicity during frontline treatment, and selection of salvage regimen on disparities in survival outcomes with the expanded cohort.

Intensification of Therapy and Pharmacogenetic Personalization Mitigate Racial Disparities in Pediatric Acute Myeloid Leukemia Outcomes

Introduction Recent reports have highlighted disparities in outcomes for black children, adolescents, and young adults with acute myeloid leukemia (AML) compared to their non-Hispanic white counterparts. An analysis of pediatric AML patients treated from 1996-2013 revealed that black race was associated with inferior event-free survival (EFS) and overall survival (OS) (Conneely 2021). In a study of adolescent and young adult patients with AML, black race was a predictor of poor outcome, particularly among patients 18-29 years old (Larkin 2022). Addressing these outcome disparities and developing treatment strategies to overcome them is crucial. In this study, we evaluated the interactions between outcomes by race and cytarabine pharmacogenomics in pediatric AML patients treated on two multi-institutional clinical trials. Methods Pediatric patients with newly diagnosed AML treated on AML02 (NCT00136084) or AML08 (NCT00703820) were included. Patients received high-dose cytarabine, daunorubicin, and etoposide (HDAC), low-dose cytarabine, daunorubicin, and etoposide (LDAC), or clofarabine and cytarabine (Clo/AraC) as initial therapy. The pharmacogenomics ACS10 scores, derived from combination of 10 single nucleotide polymorphisms, were calculated as reported (Elsayed 2022). Results Overall, 86 patients were black and 359 were white; 68 had other or unknown racial backgrounds and were not included in this analysis. Complete remission rates after two courses of induction therapy did not differ between black and white patients (92.6% vs 95.0%, p=0.63), nor did the rates of MRD negativity after one course of therapy (55.8% vs 55.4%, p=0.85). The EFS rates of black and white patients were nearly identical, with 5-year estimates of 58.3% (95% CI = 48.8%-69.8%) and 58.2% (95% CI = 53.2%-63.6%; p=0.89, Fig 1). Likewise, OS did not differ between black and white patients (63.8%; 95% CI = 54.3%-74.8% vs 69.4%; 95% CI = 64.7%-74.5%; p=0.24). The cumulative incidence (CI) of relapse did not differ by race (26.0% vs 26.1%, p=0.99), nor did the CI of treatment-related mortality (7.0% vs 6.8%, p=0.78). The prevalence of core binding factor (CBF) leukemia was higher in black patients as compared with white patients (30% vs 19%, p=0.04), but we did not find differences in EFS between black and white patients with or without CBF AML. We recently showed that low ACS10 scores were associated with worse outcomes in patients treated with low-dose cytarabine-based induction. In the present study, there was a significant difference in the distribution of ACS10 scores according to race, with low scores occurring in 73% of black patients compared with 30% of white patients (p&amp;lt;0.001). When patients on all treatment regimens were considered, there were no differences in EFS between black and white patients with low (p=0.88) or with high ACS10 scores (p=0.72). However, black patients with low ACS10 scores had significantly better outcomes after treatment with Clo/Ara-C induction compared with LDAC induction (p=0.01 for EFS and p=0.04 for OS, Fig 2). In multivariable model adjusting for age, risk group, and leukocyte count, Clo/Ara-C induction was identified as the best treatment option for black patients with low ACS10 scores (EFS: HR = 0.2, p=0.03). Discussion Our findings contrast with recent reports, as we did not observe significant outcome differences between black and white patients in terms of complete remission rates, EFS, OS, CI of relapse, or CI of treatment-related mortality. The higher prevalence of low ACS10 scores in black patients compared with white patients likely contributes to outcome disparities observed among patients treated with LDAC in the present study and those in the literature, which have included patients who almost exclusively received low-dose cytarabine-based induction therapy. In the present study, black patients with low ACS10 scores had significantly worse outcomes compared to those with high scores when treated with LDAC. However, these differences in outcome were eliminated by the use of HDAC or Clo/Ara-C induction. By contrast, black patients with high ACS10 scores did not appear to benefit from HDAC or Clo/Ara-C induction. Our results suggest that pharmacogenomics differences between black and white patients should be considered when tailoring induction regimens to improve outcomes of black patients and bridge the racial disparity gap in AML treatment.

Spatial Technologies Reveal the Immune Landscape of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is a cancer with a particularly low mutational burden in comparison to other pediatric and adult cancers and therefore, thought to be a poor candidate for T cell-engaging immunotherapies (Gröbner et al., 2018; Pfister et al., 2022). However, little is known about the composition and function of T cells in the bone marrow (BM) of pediatric AML patients. Insight into the frequency and function of BM T cells in children with AML is relevant for naturally occurring AML immune defenses, as well as for T cell-engaging immunotherapies (Koedijk et al., 2021). Here, we performed a multidimensional characterization of the tumor immune microenvironment in children with newly diagnosed AML. We obtained 82 formalin-fixed and paraffin-embedded (FFPE) diagnostic bone biopsies from a representative cohort of pediatric patients with treatment-naïve de novo AML (N=72) and from age- and sex-matched pediatric patients with treatment-naïve early-stage rhabdomyosarcoma without malignant BM infiltration (non-leukemic controls, N=10). We employed immunohistochemistry (IHC) alongside immune-related gene expression profiling (NanoString PanCancer IO 360 TM Panel) and spatial transcriptomics (NanoString GeoMx TM Whole Transcriptome Atlas). Moreover, we acquired an additional dataset of pre-treatment immune-related gene expression profiling obtained from the BM of 30 flotetuzumab-treated (CD3 x CD123 bispecific antibody) refractory/relapsed (R/R) adult AML patients (NCT02152956; Vadakekolathu et al., 2020) for TIDE-based immune deconvolution (Jiang et al., 2018). Using IHC, we identified (a trend towards) a decreased abundance of the overall number of T cells ( P=0.09) and CD8 + T cells ( P=0.011; Panel A) in the BM of pediatric AML patients in comparison to non-leukemic controls, respectively. Notably, the extent of overall T cell and CD8 + T cell infiltration could differ up to 180-fold between individual AML patients. In general, this difference in T cell infiltration was not associated with the abundance of leukemic blasts or patient's cytogenetic alterations. Nevertheless, children with complex karyotype AML had higher numbers of BM T cells compared to children with normal karyotype AML ( P=0.03).Using differential gene expression analysis, we identified genes related to T cell-attracting chemokines, cytotoxicity, and immune checkpoints to be significantly upregulated in T cell-infiltrated- compared to T cell-depleted samples. Moreover, the ratio of anti- to pro-inflammatory macrophages (M2/M1-ratio) was significantly lower in T cell-infiltrated- compared to T cell-depleted pediatric AML samples ( P&amp;lt;0.001). Interestingly, this M2/M1-ratio was also significantly lower in R/R adults that responded to flotetuzumab immunotherapy in comparison to non-responders ( P&amp;lt;0.001). In fact, this M2/M1-ratio outperformed several other known predictors of response to flotetuzumab immunotherapy (AUROC: 0.852 (95% CI: 0.71-0.99), P=0.001; Jiang et al., 2018; Ayers et al., 2017). In addition, IHC revealed that 9 immune-infiltrated samples, including 6 KMT2A-rearranged AML samples, harbored large networks of T- and B cells (representative image of B cell-networks shown in panel B). Using spatial transcriptomics, we dissected the composition of these lymphoid aggregates and revealed localized anti-tumor immunity in the BM of AML. In comparison to tumor areas, these aggregates showed a higher abundance of activated cytotoxic T cells ( P&amp;lt;0.001), memory B cells ( P&amp;lt;0.001), and plasma cells ( P&amp;lt;0.001), suggesting the presence of tertiary lymphoid structure-like (TLS-like) aggregates in the BM of AML. In conclusion, we identified a subset of pediatric AML patients with relatively high T cell infiltration and a relatively low abundance of anti-inflammatory macrophages in the BM at diagnosis. Furthermore, we found that the BM M2/M1-ratio may be informative of response to T-cell engaging immunotherapies in adult AML, which needs to be validated in pediatric AML. Lastly, for the first time, we identified TLS-like aggregates in the BM of AML patients, which have been associated with immunotherapy response in many cancers (Schumacher &amp; Thommen, 2022). Additional studies to further characterize the function and relevance of these lymphoid aggregates are ongoing.

Establishing a novel Fanconi anemia signaling pathway-associated prognostic model and tumor clustering for pediatric acute myeloid leukemia patients.

Considering the connection between the Fanconi anemia (FA) signaling pathway and tumor development, we aim to investigate the links between the FA gene expression and the survival prognosis of acute myeloid leukemia (AML) patients. Our study begins by identifying two distinct clusters of pediatric AML patients. Following the batch matching of the TARGET-AML, TCGA-LAML GSE71014, GSE12417, and GSE37642 cohorts, the samples were divided into a training set and an internal validation set. A Lasso regression modeling analysis was performed to identify five signatures: BRIP1, FANCC, FANCL, MAD2L2, and RFWD3. The AML samples were stratified into high- and low-risk groups by evaluating the risk scores. The AML high-risk patients showed a poorer overall survival prognosis. To predict the survival rates, we developed an FA Nomogram incorporating risk score, gender, age, and French-American-British classification. We further utilized the BEAT-AML cohort for the external validation of FA-associated prognostic models and observed good clinical validity. Additionally, we found a correlation between DNA repair, cell cycle, and peroxide-related metabolic events and FA-related high/low risk or cluster 1/2. In summary, our novel FA-associated prognostic models promise to enhance the prediction of pediatric AML prognosis.