Pediatric Acute Myeloid Leukemia Patients Research Articles

Effects of total body irradiation on hematopoietic cell transplantation outcomes in pediatric acute myeloid leukemia with prior central nervous system involvement

BackgroundThe implications of previous central nervous system (CNS) involvement in children with acute myeloid leukemia (AML) undergoing hematopoietic cell transplantation (HCT) remain inadequately understood. Patients with CNS disease require more upfront CNS directed intrathecal therapy, but little is known about whether transplant conditioning regimens should be intensified or if previous CNS involvement impacts post HCT outcomes. While total body irradiation (TBI) remains standard for pediatric acute lymphoblastic leukemia myeloablative conditioning, it has been largely replaced with chemotherapy-only myeloablation in pediatric AML, primarily due to toxicity and late effects associated with TBI. In the setting of previous CNS involvement, it has been suggested that TBI-based myeloablation may have advantages due to superior CNS tissue penetration and thus decreased rates of AML relapse post HCT. ObjectiveWe analyzed a publicly available dataset derived from the Center of International Blood and Marrow Transplantation Research (CIBMTR) registry to characterize the impact of TBI in HCT preparative regimens in pediatric AML patients with a history of CNS involvement. MethodsThe study dataset was obtained from the CIBMTR data repository. The study cohort included patients aged ≤21 years who underwent initial allogeneic HCT with myeloablative conditioning for de novo AML in the first or second complete remission between 2008 and 2016, who provided consent for research. Patients with mismatched related donor transplant and non-calcineurin inhibitor graft-versus-host disease (GVHD) prophylaxis were excluded. The dataset was further modified by excluding patients with missing disease site data or those with non-CNS extramedullary disease. Patients were categorized as CNS-positive or -negative AML (AML-CNS(+) and AML-CNS(-), respectively) based on the disease status at diagnosis. Cox regression model and Fine Grey methods were employed to delineate the effects of TBI and CNS disease on key HCT outcomes. ResultsThe study cohort comprised 550 pediatric AML patients, of which 25% (n =136) were AML-CNS(+). CNS involvement was more prevalent in patients aged 0-3 years, patients who were in the second complete remission, and those with a mismatched unrelated donor or umbilical cord blood. AML-CNS(+) patients demonstrated a lower relapse rate (hazard ratio [HR]: 0.50, 95% confidence interval: 0.33-0.76) compared to AML-CNS(-) patients, with comparable disease-free survival (DFS) and overall survival (OS) (p =0.10 and 0.20, respectively) in the two cohorts. The entire TBI treated cohort showed an association with increased risks of Grade 2-4 acute GVHD, bloodstream infections, and endocrine dysfunction. TBI use within the AML-CNS(+) cohort was associated with a lower relapse rate but increased risks of non-relapse mortality and a trend of higher grade 3-4 acute GVHD. ConclusionIn this population-based analysis of pediatric patients with de novo AML undergoing HCT, TBI-based conditioning regimens did not confer an advantage in DFS or OS compared to non-TBI regimens, irrespective of CNS disease status. However, TBI use was associated with increased risks of short- and long-term comorbidities. These findings underscore the need for careful consideration of TBI in pediatric AML.

Integration of measurable residual disease by WT1 gene expression and flow cytometry identifies pediatric patients with high risk of relapse in acute myeloid leukemia.

Molecular testing plays a pivotal role in monitoring measurable residual disease (MRD) in acute myeloid leukemia (AML), aiding in the refinement of risk stratification and treatment guidance. Wilms tumor gene 1 (WT1) is frequently upregulated in pediatric AML and serves as a potential molecular marker for MRD. This study aimed to evaluate WT1 predictive value as an MRD marker and its impact on disease prognosis. Quantification of WT1 expression levels was analyzed using the standardized European Leukemia Network real-time quantitative polymerase chain reaction assay (qRT-PCR) among a cohort of 146 pediatric AML patients. Post-induction I and intensification I, MRD response by WT1 was assessed. Patients achieving a ≥2 log reduction in WT1MRD were categorized as good responders, while those failing to reach this threshold were classified as poor responders. At diagnosis, WT1 overexpression was observed in 112 out of 146 (76.7%) patients. Significantly high levels were found in patients with M4- FAB subtype (p=0.018) and core binding fusion transcript (CBF) (RUNX1::RUNX1T1, p=0.018, CBFB::MYH11, p=0.016). Following induction treatment, good responders exhibited a reduced risk of relapse (2-year cumulative incidence of relapse [CIR] 7.9% vs 33.2%, p=0.008). Conversely, poor responders' post-intensification I showed significantly lower overall survival (OS) (51% vs 93.2%, p<0.001), event-free survival (EFS) (33.3% vs 82.6%, p<0.001), and higher CIR (66.6% vs 10.6%, p<0.001) at 24 months compared to good responders. Even after adjusting for potential confounders, it remained an independent adverse prognostic factor for OS (p=0.04) and EFS (p=0.008). High concordance rates between WT1-based MRD response and molecular MRD were observed in CBF patients. Furthermore, failure to achieve either a 3-log reduction by RT-PCR or a 2-log reduction by WT1 indicated a high risk of relapse. Combining MFC-based and WT1-based MRD results among the intermediate-risk group identified patients with unfavorable prognosis (positive predictive value [PPV] 100%, negative predictive value [NPV] 85%, and accuracy 87.5%). WT1MRD response post-intensification I serves as an independent prognostic factor for survival in pediatric AML. Integration of WT1 and MFC-based MRD results enhances the reliability of MRD-based prognostic stratification, particularly in patients lacking specific leukemic markers, thereby influencing treatment strategies.

CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Cytogenetic abnormalities predict survival after allogeneic hematopoietic stem cell transplantation for pediatric acute myeloid leukemia: a PDWP/EBMT study.

Poor-risk (PR) cytogenetic/molecular abnormalities generally direct pediatric patients with acute myeloid leukemia (AML) to allogeneic hematopoietic stem cell transplant (HSCT). We assessed the predictive value of cytogenetic risk classification at diagnosis with respect to post-HSCT outcomes in pediatric patients. Patients younger than 18 years at the time of their first allogeneic HSCT for AML in CR1 between 2005 and 2022 who were reported to the European Society for Blood and Marrow Transplantation registry were subgrouped into four categories. Of the 845 pediatric patients included in this study, 36% had an 11q23 abnormality, 24% had monosomy 7/del7q or monosomy 5/del5q, 24% had a complex or monosomal karyotype, and 16% had other PR cytogenetic abnormalities. In a multivariable model, 11q23 (hazard ratio [HR] = 0.66, P = 0.03) and other PR cytogenetic abnormalities (HR = 0.55, P = 0.02) were associated with significantly better overall survival when compared with monosomy 7/del7q or monosomy 5/del5q. Patients with other PR cytogenetic abnormalities had a lower risk of disease relapse after HSCT (HR = 0.49, P = 0.01) and, hence, better leukemia-free survival (HR = 0.55, P = 0.01). Therefore, we conclude that PR cytogenetic abnormalities at diagnosis predict overall survival after HSCT for AML in pediatric patients.

Early Detection of Molecular Residual Disease and Risk Stratification for Children with Acute Myeloid Leukemia via Circulating Tumor DNA.

Patient-tailored minimal residual disease (MRD) monitoring based on circulating tumor DNA (ctDNA) sequencing of leukemia-specific mutations enables early detection of relapse for pre-emptive treatment, but its utilization in pediatric acute myelogenous leukemia (AML) is scarce. Thus, we aim to examine the role of ctDNA as a prognostic biomarker in monitoring response to the treatment of pediatric AML. A prospective longitudinal study with 50 children with AML was launched, and sequential bone marrow (BM) and matched plasma samples were collected. The concordance of mutations by next-generation sequencing-based BM-DNA and ctDNA was evaluated. In addition, progression-free survival (PFS) and overall survival (OS) were estimated. In 195 sample pairs from 50 patients, the concordance of leukemia-specific mutations between ctDNA and BM-DNA was 92.8%. Patients with undetectable ctDNA were linked to improved OS and PFS versus detectable ctDNA in the last sampling (both P < 0.001). Patients who cleared their ctDNA post three cycles of treatment had similar PFS compared with persistently negative ctDNA (P = 0.728). In addition, patients with >3 log reduction but without clearance in ctDNA were associated with an improved PFS as were patients with ctDNA clearance (P = 0.564). Thus, ctDNA-based MRD monitoring appears to be a promising option to complement the overall assessment of pediatric patients with AML, wherein patients with continuous ctDNA negativity have the option for treatment de-escalation in subsequent therapy. Importantly, patients with >3 log reduction but without clearance in ctDNA may not require an aggressive treatment plan due to improved survival, but this needs further study to delineate.

Development and validation of a promising 5-gene prognostic model for pediatric acute myeloid leukemia

Risk classification in pediatric acute myeloid leukemia (P-AML) is crucial for personalizing treatments. Thus, we aimed to establish a risk-stratification tool for P-AML patients and eventually guide individual treatment. A total of 256 P-AML patients with accredited mRNA-seq data from the TARGET database were divided into training and internal validation datasets. A gene-expression-based prognostic score was constructed for overall survival (OS), by using univariate Cox analysis, LASSO regression analysis, Kaplan–Meier (K-M) survival, and multivariate Cox analysis. A P-AML-5G prognostic score bioinformatically derived from expression levels of 5 genes (ZNF775, RNFT1, CRNDE, COL23A1, and TTC38), clustered P-AML patients in training dataset into high-risk group (above optimal cut-off) with shorter OS, and low-risk group (below optimal cut-off) with longer OS (p < 0.0001). Meanwhile, similar results were obtained in internal validation dataset (p = 0.005), combination dataset (p < 0.001), two treatment sub-groups (p < 0.05), intermediate-risk group defined with the Children's Oncology Group (COG) (p < 0.05) and an external Japanese P-AML dataset (p = 0.005). The model was further validated in the COG study AAML1031(p = 0.001), and based on transcriptomic analysis of 943 pediatric patients and 70 normal bone marrow samples from this dataset, two genes in the model demonstrated significant differential expression between the groups [all log2(foldchange) > 3, p < 0.001]. Independent of other prognostic factors, the P-AML-5G groups presented the highest concordance-index values in training dataset, chemo-therapy only treatment subgroups of the training and internal validation datasets, and whole genome-sequencing subgroup of the combined dataset, outperforming two Children's Oncology Group (COG) risk stratification systems, 2022 European LeukemiaNet (ELN) risk classification tool and two leukemic stem cell expression-based models. The 5-gene prognostic model generated by a single assay can further refine the current COG risk stratification system that relies on numerous tests and may have the potential for the risk judgment and identification of the high-risk pediatric AML patients receiving chemo-therapy only treatment.

Clinico-Hematological Profile of Acute Myeloid Leukemia: Experience From a Tertiary Care Cancer Center in North India.

Complete diagnosis of acute myeloid leukemia (AML) requires knowledge of clinical information combined with morphologic evaluation, immunophenotyping, karyotyping, and molecular genetic testing. The study intends to evaluate the demographic profile, clinical workup, and investigation, including flow cytometric immunophenotyping, in adult and pediatric age groups of AML. This is a retrospective study of AML patients treated between January 2017 and December 2021. Clinical and demographic characteristicsand investigation findings were recorded from case files and the hematology database. A total of 896 cases of AML were registered during the given period, of which 819 cases were de-novo AML. Among those 819 cases, more than two-thirds of cases, i.e., 78.9% (N = 646), received induction chemotherapy. A significantly higher male-to-female ratio was observed (1.5:1). The median age was 22 years. The median time for diagnosis was three days and the median time for treatment intervention was four days. There were significant differences in the Eastern Cooperative Oncology Group (ECOG) performance status scores between pediatric and adult AML patients. Pediatric AML patients presented with better ECOG performance scores (ECOG performance scores 0 and 1) than adult patients (74.76% vs. 43.14%, p < 0.001). Further comparing adult vs. pediatric AML patients, normal karyotype (60.56% vs. 31.93%, p < 0.001) and NPM1 (22.25% vs. 6.72%, p < 0.001)and FLT3-ITD mutations (20.28% vs. 7.98%, p<0.001) were more common in the adult group, whereas AML-ETO (40.76% vs. 16.34%, p < 0.001) was more common in the pediatric group. The study highlights the presenting age is lower than global figures. The median time for initial diagnosis and the start of treatment is within the acceptable norms. Normal karyotype andNPM1and FLT3 mutations were common in adult AML patients, whereas AML-ETO was more common in the pediatric cohort. These findings will help plan prospective studies and see the correlation with treatment outcomes. The laboratory workup practice currently complies with the standard guidelines at our center.

Cognitive-behavioral stress management relieves anxiety, depression, and post-traumatic stress disorder in parents of pediatric acute myeloid leukemia patients: a randomized, controlled study

ABSTRACT Objectives Cognitive-behavioral stress management (CBSM) is an effective psychological intervention to relieve psychological and symptomatic distress. This study aimed to investigate the effect of CBSM in anxiety, depression, and post-traumatic stress disorder (PTSD) in parents of pediatric acute myeloid leukemia (AML) patients. Methods Totally, 56 pediatric AML patients and 100 parents were randomized into the CBSM group (28 patients and 49 parents) and the normal control (NC) group (28 patients and 51 parents) to receive corresponding interventions for 10 weeks. The questionnaire scores were assessed at month M0, M1, M3, and M6. Results In parents of pediatric AML patients, self-rating anxiety scale score at M1 (p = 0.034), M3 (p = 0.010), and M6 (p = 0.003), as well as anxiety at M3 (p = 0.036) and M6 (p = 0.012) were decreased in the CBSM group versus the NC group. Self-rating depression scale score at M3 (p = 0.022) and M6 (p = 0.002), as well as depression at M6 (p = 0.019) were declined in the CBSM group versus the NC group. Symptom checklist-90 (a psychotic status questionnaire) score at M3 (p = 0.031) and M6 (p = 0.019) were declined in the CBSM group versus the NC group. Regarding PTSD, the impact of the events scale-revised score at M3 (p = 0.044) and M6 (p = 0.010) were decreased in the CBSM group versus the NC group. By subgroup analyses CBSM (versus NC) improved all outcomes in parents with anxiety at M0 and depression at M0 (all p < 0.050), but could not affect the outcomes in parents without anxiety or depression at M0 (all p > 0.050). Conclusion CBSM reduces anxiety, depression, and PTSD in parents of pediatric AML patients.

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.

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

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.

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.

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

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