Pediatric Acute Myeloid Leukemia Patients Research Articles

Tumor-suppressing effects of miR-381-3p in pediatric acute myeloid leukemia via ROCK1 downregulation.

MicroRNA (miR)-381-3p is the newly discovered tumor-associated miRNA, which is frequently associated with diverse human malignancies; but, it is still unknown about its effect on acute myeloid leukemia (AML) in children. This work focused on exploring miR-381-3p's effect on childhood AML and identifying the possible mechanisms facilitating new treatment development. Using qRT-PCR analysis, miR-381-3p expression remarkably reduced in pediatric AML patients and AML cell lines (HL-60 and U937). Following transfection of miR-381-3p mimic or inhibitor into HL-60 and U937 cells, we conducted MTT assay to evaluate cell proliferation, flow cytometry (FCM) to measured cell apoptosis and cell cycle, whereas Transwell assays to detect cell invasion and migration. Our results demonstrated that miR-381-3p overexpression remarkably repressed cell growth, invasion and migration; additionally, miR-381-3p overexpression resulted in arrest of cell cycle and enhanced cell apoptosis. In contrast, miR-381-3p knockdown led to an opposite effect. Moreover, we predicted miR-381's target gene and validated it by luciferase reporter assay and TargetScan, separately. We identified miR-381-3p's binding site in ROCK1 3'-UTR. As revealed by Western-blot (WB) assay, miR-381-3p overexpression notably suppressed ROCK1 level. Moreover, restoring ROCK1 expression abolished miR-381-3p's inhibition on cell proliferation, invasion and migration. Data in this work indicated the role of miR-381-3p as the tumor suppressor within pediatric AML by targeting ROCK1. Therefore, miR-381-3p might serve as a potential therapeutic target for the treatment of pediatric AML.

Measurable residual disease study through three different methods can anticipate relapse and guide pre-emptive therapy in childhood acute myeloid leukemia.

Although outcomes of children with acute myeloid leukemia (AML) have improved over the last decades, around one-third of patients relapse. Measurable (or minimal) residual disease (MRD) monitoring may guide therapy adjustments or pre-emptive treatments before overt hematological relapse. In this study, we review 297 bone marrow samples from 20 real-life pediatric AML patients using three MRD monitoring methods: multiparametric flow cytometry (MFC), fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). Patients showed a 3-year overall survival of 73% and a 3-year event-free survival of 68%. Global relapse rate was of 25%. All relapses were preceded by the reappearance of MRD detection by: (1) MFC (p = 0.001), (2) PCR and/or FISH in patients with an identifiable chromosomal translocation (p = 0.03) and/or (3) one log increase of Wilms tumor gene 1 (WT1) expression in two consecutive samples (p = 0.02). The median times from MRD detection to relapse were 26, 111, and 140days for MFC, specific PCR and FISH, and a one log increment of WT1, respectively. MFC, FISH and PCR are complementary methods that can anticipate relapse of childhood AML by weeks to several months. However, in our series, pre-emptive therapies were not able to prevent disease progression. Therefore, more sensitive MRD monitoring methods that further anticipate relapse and more effective pre-emptive therapies are needed.

Identification of Circular RNA Circ_0003256 as a Novel Player in Pediatric Acute Myeloid Leukemia

Aberrant expression of circular RNAs (circRNAs) is tightly associated with the pathogenesis of human cancers, including pediatric acute myeloid leukemia (AML). In this report, we sought to define the precise action of circ_0003256 in the pathogenesis of pediatric AML. Circ_0003256, microRNA (miR)-582-3p, and protein kinase cAMP-activated catalytic subunit beta (PRKACB) were quantified by quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, cycle distribution, and apoptosis were estimated by MTT, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays, respectively. Direct relationships among circ_0003256, miR-582-3p, and PRKACB were verified by a dual-luciferase reporter and RNA pull-down assays. Our data indicated that circ_0003256 was highly expressed in pediatric AML patients and cells. Suppression of circ_0003256 hindered cell proliferation and promoted apoptosis in THP-1 and MV4-11 cells. Mechanistically, circ_0003256 contained functional binding sites for miR-582-3p, and circ_0003256 suppression influenced cell behaviors by upregulating miR-582-3p. MiR-582-3p directly targeted and inhibited PRKACB and the inhibition of PRKACB phenocopied miR-582-3p overexpression in regulating cell functional behaviors. Moreover, circ_0003256 involved the posttranscriptional regulation of PRKACB through miR-582-3p. Our findings identify that suppression of circ_0003256 impedes the malignant behaviors of pediatric AML cells by regulating PRKACB expression by competing for shared miR-582-3p.

P089: Genetic, pharmacogenetics and outcomes in a cohort of pediatric acute myeloid leukemia patients in two reference centers from Colombia

P089: Genetic, pharmacogenetics and outcomes in a cohort of pediatric acute myeloid leukemia patients in two reference centers from Colombia

A novel signature identified by pyroptosis-related lncRNAs clusters predicts prognosis and tumor immune microenvironment for pediatric acute myeloid leukemia

Pyroptosis is a recently discovered programmed cell death that is involved in tumor formation, prognosis, and curative effect. Pyroptosis-related long non-coding ribonucleic acids (PR-lncRNAs) play key roles in tumorigenesis and tumor progression. However, the inherent relationship between PR-lncRNAs and the prognosis of pediatric acute myeloid leukemia (AML) remains unclear. For this reason, the association of PR-lncRNAs with the prognosis and tumor microenvironment features was analyzed using the Therapeutically Applicable Research to Generate Effective Treatment (TARGET) database. We classified three clusters based on PR-lncRNAs expressions and identified 841 differentially expressed PR-lncRNAs related to overall survival time. Seven key lncRNAs were then identified by least absolute shrinkage and selection operator (LASSO)-Cox and multivariate Cox. A signature based on these seven lncRNAs was also established, and the patients were separated into two groups according to their risk score. The high-risk group was characterized by poorer prognosis, lower expression of immune checkpoints, lower microsatellite instability or microsatellite stability (MSI-L/MSS), and lower drug sensitivity. The results demonstrated that PR-lncRNAs have a potential effect on the tumor immune microenvironment, clinicopathological features, and prognosis in pediatric AML. The nomogram and decision curve analysis suggested that the risk score is one of the most accurate of any other and provided a basis for the exploration of the immune microenvironment of the cell pyroptosis-associated subtypes in children with AML and the construction of a prognostic model with seven key PR-lncRNAs, which provides an approach to evaluate the prognosis of pediatric AML patients.

Personalization of Induction Therapy for Pediatric AML to Ara-C+Dauno+ Etoposide (ADE) or Clofarabine+Ara-C According to a Polygenic Ara-C SNP Score- ACS10

Acute myeloid leukemia (AML) is a heterogenous disease with overall suboptimal outcome. Although chemotherapy regimens that include cytarabine (ara-C) induce remission in a majority of pediatric AML patients, approximately 30% relapse and subsequently have very dismal outcome. Ara-C is a prodrug requiring activation to ara-CTP which induces leukemic cell death. Thus, inter-patient variation in each patient's ability to activate ara-C is a significant contributor to clinical outcomes. We recently performed a comprehensive pharmacogenomics evaluation of SNPs in ara-C metabolism pathway genes and developed a polygenic ara-C 10-SNP genotype (ACS10) score based on association with leukemic intracellular ara-CTP levels and clinical outcome (Elsayed et al, 2022, JCO1). Our results demonstrated that patients with low ACS10 score had poor outcomes when given standard induction including low-dose ara-C, daunorubicin and etoposide (ADE); however their outcomes were much better by augmenting therapy with either an increased dose of ara-C or the addition of gemtuzumab ozogamicin1. Conversely, patients with a high ACS10 score fared worse when given high-dose ara-C than low ara-C dose-based ADE induction (LDAC). These results suggest that pediatric AML patients may benefit from pharmacogenetic personalization of ADE therapy according to their ACS10 scores. Additionally, these results motivate research regarding the utility of ACS10 for pharmacogenetic personalization of other therapies for pediatric AML. Clofarabine is another nucleoside analog that inhibits both DNA polymerase as well as ribonucleotide reductase, thus enhancing the activity of ara-C. Recent data from the AML08 clinical trial [NCT00703820] has shown promising results with use of clofarabine with cytarabine during remission induction in pediatric AML patients.2 Therefore, we evaluated the association of ACS10 score with outcomes among 91 patients randomly assigned standard ADE induction on the AML02 clinical trial (LDAC arm) [NCT00136084] and 117 patients randomly assigned an induction of clofarabine and ara-C (Clo+Ara-C arm) on the AML08 trial [NCT00703820] (Figure 1A shows the study design). 5-year event-free survival (EFS) and overall survival (OS) of patients treated with ADE improved with increasing ACS10 score; however, for patients treated with Clo+Ara-C, improvement with increasing ACS10 score was not observed (OS shown in Figure 1A). This differential outcome association pattern suggests that thresholding the ACS10 score at 0 could be used to classify patients into low (<=0) and high (>0) ACS10 groups. Patients within low ACS10 score group had a better outcome with Clo+AraC whereas the high ACS10 score patient group fared better with ADE. In particular, there is significant improvement in EFS and OS within the low-ACS10 score patients when treated with Clo+Ara-C as compared to ADE (Clo+AraC vs. ADE; EFS HR=0.45, 95% CI: 0.23-0.82; p=0.01; OS HR=0.45, 95% CI: 0.19-0.93, p=0.028; Figure 1B). In contrast, patients with a high ACS10 score had worse EFS and OS with Clo+AraC induction than with ADE induction (EFS HR= 1.84, 95% CI: 0.99-3.41, p=0.05; OS HR= 2.40, 95%CI: 1.13-5.13; p=0.02, Figure 1B). These results are unlikely to suffer from a strong historical comparison bias because there are not statistically significant differences in the outcomes of the randomly assigned therapies of the AML02 and AML08 trials as well as outcome by the clinical trial (EFS/OS of each arm of the two trials as well as between AML02 and AML08 trials; p=0.8, data not shown). In summary, consistent with our recent report, these results show that for patients with Low-ACS10 genetic score prognosis is poor with standard ADE induction. However, therapy augmentation options such as Clo+AraC, ADE with high-dose Ara-C, and ADE+gemtuzumab may improve the prognosis. This calls for an action on using preemptive genotyping for selection of the most effective induction 1 chemotherapy regimen to achieve maximum clinical benefit. References. 1. Elsayed AH, et al: Polygenic Ara-C Response Score Identifies Pediatric Patients With Acute Myeloid Leukemia in Need of Chemotherapy Augmentation. JCO 2022, PMID:34990262 2. Rubnitz JE, et al: Clofarabine Can Replace Anthracyclines and Etoposide in Remission Induction Therapy for Childhood Acute Myeloid Leukemia: The AML08 Multicenter, Randomized Phase III Trial. JCO 2019, PMID:3124652 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immuno-AML, a Novel Immunogenomic Classifier Predicting Chemotherapy Response in Pediatric Patients with AML

Introduction Acute myeloid leukemia (AML) represents 20% of pediatric leukemias. Despite therapeutic improvements, ∼30% of children die due to therapy-related toxicity and relapse. In solid tumors, the density and functional orientation of the tumor microenvironment (TME) influence prognosis and response to chemotherapy and immunotherapy. Defining immunologic features associated with unfavorable response to standard treatment in pediatric AML might improve stratification systems that can be used for experimental interventions and personalized approaches. In this study, we investigated the potential mechanisms and effect of immune response and evasion in bone marrow (BM) of AML pediatric patients through an integrative gene expression analysis. Methods Thirty-two BM aspirates were collected from pediatric patients with non-promyelocytic AML at diagnosis in 3 different hospitals in Qatar, Pakistan, and Italy. Patients were assigned to 2 prognosis-related groups: patients who maintained a complete remission (CR) after first line of chemotherapy (sustained CR) vs patients who progressed/relapsed and/or obtained CR after hematopoietic stem cells transplantation (HSCT) (non-sustained CR). BM mRNA samples were analyzed with RNA sequencing (RNA-Seq) and/or NanoString's PanCancer IO 360 Gene Expression research panel. In particular, mRNA of 25 samples (Cohort 1, discovery set) were analyzed by NanoString's PanCancer IO 360. Subsequently, a subset of samples from Cohort 1 (n=19), was analyzed by RNA-Seq for technical validation. Cohort 1 results were then used to build a predictive classifier that was tested on Cohort 2 samples (n=7, validation set), analyzed with RNA-Seq. Results Gene expression analysis of 25 samples (Cohort 1, sustained CR, n=6; non-sustained CR, n=19) using NanoStringassay revealed 67 significant differentially expressed genes (DEGs; p < 0.05, LIMMA) among sustained CR vs non-sustained CR groups. Patients with sustained CR displayed coherent overexpression of immune genes related to IFN activation (IFIT3, IFITM1, IFI27, STAT1, GBP1), cytotoxic function (PRF1, GNLY), antigen presentation and B cell functions (TAP1, CD19), and T cell infiltration (CD3D, CD8A), consistent with a favorable T helper 1 (Th1)-cytotoxic polarization of the TME. This pattern was not observed in non-sustained CR patients, which overexpressed genes consistent with macrophage-myeloid suppression (CD68, TREM1) and intrinsic oncogene signaling linked with T cell exclusion and immune suppression (WNT5A, WNT-β- catenin pathway, and EFGR) (Fig1 A,B). This 67-gene signature (67GenSig) was technically validated using an orthogonal platform (RNA-Seq) on a subset of 19 patients (sustained CR, n=5; non-sustained CR, n=14) for which sufficient RNA-Seq data was available (65 out of 67 transcripts were available for analysis, 2 transcripts were filtered out after QC). Unsupervised PCA (Fig1 C) based on 67GenSig clearly segregated samples according to outcome, confirming the previous NanoString results. Next, we sought to identify a predictive classifier to precisely discriminate sustained CR from non-sustained CR AML pediatric patients using a minimal number of genes. By applying a Random Forest-based feature selection on 67GenSig transcripts of RNA-Seq data from Cohort 1 samples, we developed a 13-gene classifier (Immuno-AML) that was tested in a new independent dataset (Fig1 D; Cohort 2, n=7: sustained CR, n=4; non-sustained CR, n=3). Overall, genes included in the Immuno-AML predictor substantiated a positive Th1/cytotoxic vs myeloid polarization. The Immuno-AML predictor was able to classify patients in the validation cohort (Cohort 2) in "sustained CR" and "non-sustained CR" prognostic groups with a very good performance achieving 85.71% accuracy, 100% specificity, and 75% sensitivity. Conclusion Our data suggest that response to conventional therapy in AML pediatric patients is conditional to the presence of a Th1 polarization of the TME while is prevented by myeloid-mediated immune suppression and intrinsic oncogenic signaling. The optimized predictive signature (Immuno-AML) might be tested and/or refined in larger cohort to develop a clinical-grade predictive biomarker. It might help to provide a rational guidance for risk stratification criteria and personalized treatment decision paths, implying the possibility of combining immunotherapy drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Cibersortx Signature Predicts Outcome in Pediatric AML Patients

Acute Myeloid Leukemia (AML) is a devastating disease associated with high morbidity and mortality. The role of supportive immune cells such as NK cells and mast cells in AML tumorigenesis and relapse has been increasingly scrutinized. However studies to evaluate impact of varying cell type fraction on the clinical outcome are lacking. Recently, a software tool CIBERSORTx (Cell-type identification by estimating relative subsets of RNA transcripts) has unveiled opportunities to use bulk gene expression data for cell type fraction estimation. CIBERSORTx uses a reference matrix of cell-specific expression and mathematical deconvolution to estimate cell-type abundances from bulk tissue transcriptomes. The leukocyte signature matrix (LM22), which consists of 22 hematopoietic lineages, is a well-characterized reference matrix based on the expression profiles of 547 genes. In this study, we leveraged the CIBERSORTx algorithm to develop a cell type score (CiberScore) using the gene-expression data obtained from diagnostic bone marrow specimens (using Affymetrix U133A microarray) from pediatric AML patients treated on the multi-site AML02 clinical trial (NCT00136084). 163 patients with both gene expression and outcome data were included and clinical endpoints 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 using 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. In a step-wise approach, we first deconvoluted the bulk gene-expression data through CIBERSORTx using the LM22 as a reference panel to derive the percent cell fraction of 22 distinct hematopoietic cell lineages across all patients. We evaluated the inter-patient differences in cell fraction against clinical endpoints defined above and observed significant association between survival endpoint and cell type fractions of mast cells resting and NK cells activated. We further utilized a machine-learning approach and performed 1,000 iterations of 10-fold cross-validation of Cox proportional hazard regression modeling with LASSO penalty using OS as a clinical endpoint. Cell-type fractions passing at least in 800 of 1000 models (9 hematopoietic cell types Figure 1a) were selected to create a CiberScore equation by multiplying cell fraction with average coefficient obtained from 1000 models. Patients were further classified into two groups: Low-CiberScore (n=122, Quartile 1-3) and High-CiberScore (n=41, Quartile 4). No significant difference by CiberScore groups was observed for patient characteristics such as age, initial risk group status, FLT3-ITD or cytogenetics. With respect to clinical outcome patients within High-Ciberscore group had greater MRD1 positivity (p=0.010) and poor EFS (HR 2.96, p<0.0001) and OS (HR 4.78, <0.0001) as compared to patients within Low-Ciberscore group. Kaplan-Meier survival curves for OS and EFS by CiberScore are shown in Figure 1b. In multivariable analysis, CiberScore remained significantly associated with EFS and OS after adjusting for induction 1-MRD1 status, risk-group, FLT3-status, WBC-count at diagnosis and age, implying it to be an independent prognostic factor. In conclusion, in this study we leveraged CIBERSORTx to generate cell type fraction using bulk gene-expression data from pediatric AML patients and further utilized machine learning algorithms to develop a prognostic model based on 9 leukocyte lineages. CiberScore remained significant in independent RNA seq cohorts from NCI TARGET pediatric AML repository. Ongoing studies are focused on further validation of this signature in other cohorts as well as comparing the similarities and differences in adult vs. pediatric AML with respect to the cell fraction differences of prognostic relevance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anagrelide Represents a Potential Therapeutic Therapy for Acute Myeloid Leukemia Cells Based on Phosphodiesterase 3A Expression

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. PDE3A is a well-characterized cyclic nucleotide phosphodiesterase (PDE) that hydrolyzes cAMP, cGMP, and cUMP. Recent studies have demonstrated that PDE3A was upregulated in several solid tumors of patients including breast cancer, ovarian carcinoma, melanoma, which correlated with poor prognosis. PDE3A activates inflammatory pathways that mediate cancer cell stemness by suppressing the cAMP/PKA pathway. Targeting PDE3A with chemicals suppressed cancer cells by inducing cell apoptosis and reducing metastasis. PDE3A inhibitor Anagrelide (ANA) is an antithrombotic and platelet reducing agent used to treat the thrombocythemia associated with myeloproliferative diseases. This study aims to explore the relationship between PDE3A expression and AML prognosis, and to investigate the feasibility and mechanism of PDE3A as a therapeutic target for AML. To explore the relationship between PDE3A expression and the survival time of patients, a total of 141 pediatric AML patients were recruited in this retrospective study from October 2013 to September 2021 in the Children's Hospital of Soochow University. The follow-up period was until March 2022. The transcriptomic expression level of PDE3A in newly diagnosed bone marrow samples was obtained according to the results of RNA sequencing. Patients were divided into two groups based on median PDE3A expression: PDE3Ahigh group (n= 70) and PDE3Alow group(n=71). Results showed AML patients in PDE3Ahigh group had significantly lower 5-year overall survival (OS) (71.4% vs 92.9%, P = 0.0010), event free survival (52.1% vs 84.6%, P = 0.0150) and relapse free survival (73.2% vs 91.4%, P = 0.0325) compared to patients in PDE3Alow group. Next, to evaluate PDE3A expression level in AML cell lines, we used RT-PCR and Western Blot to compare the difference of PDE3A expression in HEL, Kasumi-1, MV4-11, NB4, K562, HL60 and MEG-01. Among these cell lines, HEL has significantly higher PDE3A expression compared to others at both transcriptional and protein levels. We found that ANA specifically inhibited HEL growth in a concentration dependent manner after 72 hours of ANA treatment. ANA reached half of the inhibitory concentration on HEL at 0.9μM. Interestingly, ANA had no inhibitory effects on other tested AML cell lines which expressed low level of PDE3A. Thus, we choose HEL as PDE3Ahigh AML cell model and treatment with 0.9μM ANA for 72 hours for further experiments. By flow cytometry, we found ANA interrupted cell cycle by G1 arrest in HEL, but did not induce cell apoptosis. In consistent with these results, the expression of cell cycle-related proteins including CDK2, CDK4, CDK6, CDK9 and C-MYC were significantly down-regulated in a time-dependent manner after ANA treatment in HEL compared to control group. To explore the effect of ANA on PDE3A high expression AML in vivo, we established the preclinical model of AML by injecting 2 million luciferase overexpressed HEL cells into NSG mice via tail vein. Fourteen days later, the mice were treated with vehicle or 5mg/kg ANA treatment by gavage daily for 15 days as the therapeutic model. More interestingly, in vivo imaging assay indicated that ANA treatment reduced leukemia burden compared with the vehicle group. The anti-leukemia effects of ANA significantly prolonged survival time compared to vehicle (median survival time, 32.5 vs 24.5 days, P = 0.0091). In conclusion, our clinical data suggested that high expression of PDE3A in newly diagnosed pediatric AML patients was associated with poor clinical survival. Besides, our in vitro and in vivo data indicated that ANA targeting PDE3A suppressed progression of AML by interrupting cell cycle, providing a promising approach to eradicate AML with high level of PDE3A expression.

Specific Cytogenetic Abnormalities at Diagnosis Predict Survival after Hematopoietic Cell Transplant in Poor-Risk Pediatric Acute Myeloid Leukemia: A PDWP/EBMT Study

Introduction: Cytogenetic abnormalities are an important prognostic factor for predicting overall survival (OS) and relapse incidence (RI) in children with acute myeloid leukemia (AML). Although allogeneic hematopoietic cell transplant (HCT) is generally recommended for patients with AML in first complete remission (CR1) and poor-risk (PR) cytogenetics, whether these cytogenetic and molecular features retain their prognostic value after HCT is unknown. Additionally, PR AML is a heterogeneous category that includes several distinct cytogenetic abnormalities, some of which might have a worse prognosis than others. In this study, we evaluated whether the cytogenetic risk classification at diagnosis of AML was predictive of post-HCT outcomes in pediatric patients. Methods: All patients <18 years of age at the time of HCT reported to the European Society for Blood and Marrow Transplantation (EBMT) registry, who had received their first allogeneic HCT for AML in CR1 between 2005 and 2020, and who had their karyotype evaluation at diagnosis available were included in this analysis. Patients were subgrouped as (a) monosomy 7/del7q or monosomy 5/del5q, (b) 11q23 abnormality excluding t(9;11), (c) complex or monosomal karyotype, or (d) other. A complex karyotype was defined as one with three or more structural abnormalities, and a monosomal karyotype was defined as a monosomy with one or more structural abnormalities, excluding WHO-designated recurring translocations or inversions based on 2017 ELN recommendations. Other cytogenetic abnormality subgroup included t(6;9), t(3;5), t(9;22), t(8;16), inv(3) or t(3;3), t(16;21), abn(11p15), and del(12p) or abn(12p13). Cytogenetic subgroup, age at HCT, whether HCT was from a female donor to a male recipient, patient/donor CMV status, time from AML diagnosis to HCT, and year of HCT were included as predictors for OS, RI and non-relapse mortality (NRM) in the multivariate analysis. Results: We included 744 pediatric patients (42.2% female; median age at HCT: 8.6 years [range: 0.3-18 years]) from 139 participating centers in the study. Median follow-up after HCT was 4.4 years. Thirty-seven percent had an 11q23 abnormality, 24% had monosomy 7/del7q or monosomy 5/del5q, 24% had a complex or monosomal karyotype, and 15% met the criteria for other PR cytogenetic abnormalities. Only 7% had secondary AML. Pre-HCT minimal residual disease (MRD) data were available for 346 patients, 86% of whom were MRD negative at the time of HCT. Patients received grafts from a matched related donor (27%), a mismatched related donor (11%), an unrelated donor (46%), or a cord blood donor (16%). Of the total patients, 55% received bone marrow, 29% received peripheral blood-derived stem cells and the rest (16%) received cord blood as the stem cell source. Myeloablative conditioning was used in 97% of the patients. The OS and leukemia free survival for the entire cohort was 76% and 70% respectively at 2 years. In a multivariate model, 11q23 (hazard ratio [HR] = 0.59, P = 0.01) and other poor-risk cytogenetic abnormalities (HR = 0.49, P < 0.01) were associated with significantly better OS when compared with monosomy 7/del7q or monosomy 5/del5q. The "other PR cytogenetic abnormalities" category was also associated with a lower risk of disease relapse after HCT (HR = 0.4, P = 0.01). Receiving an HCT from an unrelated donor, as compared with a matched related donor, was associated with lower RI (HR = 0.58, P = 0.03). Additionally, older age at HCT (12-18 years) was associated with the highest risk of NRM (HR = 1.95, P = 0.02) and with worse OS (HR = 1.71, P < 0.01) than seen in younger patients (aged 4-12 years). Conclusion: PR cytogenetic abnormalities at diagnosis remain predictive of OS after HCT for AML in pediatric patients. Monosomy 7/del7q or monosomy 5/del5q confer a poor prognosis even after HCT, whereas 11q23 abnormality and other PR cytogenetic abnormalities predict a favorable outcome after HCT. Our data also suggest that an HCT from an unrelated donor may offer greater protection against relapse than an HCT from a matched related donor, but this requires further validation. Efforts for improving conditioning regimens and transplant techniques should focus on reducing NRM and reducing RI in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Profiling of Acute Myeloid Leukemia Identified ARMH1, a Novel Protein Associated with Proliferation, Migration, and Drug Resistance

Despite advancements in treatments, half of pediatric acute myeloid leukemia (AML) patients relapse (Chen, J. & Glasser, C.L., 2020), making it pertinent to identify novel biomarkers that can be used for better stratification of patients post end of induction (EOI) therapy as well as to develop targeted therapies that could lead to higher clinical remission (CR) rates. Single-cell RNA sequencing (scRNAseq) analysis has revolutionized cancer research by identifying novel cell types, genes, and pathways. Our group used scRNAseq analysis of pediatric AML bone marrow (BM) samples collected at diagnosis (Dx) and EOI to identify the upregulation of multiple genes including a novel gene, Armadillo like helical domain containing 1 (ARMH1)/C1orf228, in cancerous blast cells (Thomas, B.E. et al., 2020). ARMH1 has not been previously associated with AML. Also, ARMH1 expression was observed to be significantly higher at Dx in samples from patients with relapse compared to samples from patients with CR (P<2.2e-16). Independent validation for ARMH1 blast-association using single cell and bulk transcriptome profiling revealed that ARMH1 expression was significantly higher in scRNAseq data of AML Dx blast cells compared to EOI non-blast cells (fold change, FC=1.8, P<2.2e-31) as well as in bulk RNAseq data of Dx samples with high blast% (>90%) compared to EOI samples (FC=1.8, P=.003) (Ulukaya, G. et al., 2021). Further analysis on other leukemias like T cell-acute lymphocytic leukemia (T-ALL) and mixed phenotypic acute leukemia (MPAL) also revealed higher expression of ARMH1 in the blast cells as compared to non-blast cells. Survival analysis on bulk RNAseq data using the survival genie platform (Dwivedi, B. et al., 2022) revealed that higher expression of ARMH1 was significantly associated with poor overall survival (OS) in both AML (P=.003) (Ulukaya, G. et al., 2021) and T-ALL (P=.014) datasets. Comparative gene expression analysis using the the Pediatric Single Cell Cancer Atlas - PedSCAtlas - tool (Mumme, H.L. et al., 2021), revealed negligible expression of ARMH1 in normal lymphoid, myeloid, and erythroid cell lineages, further confirming the leukemic blasts-specific over-expression of ARMH1. ARMH1 encodes a cytosolic protein of yet unknown function. The malignant blast specificity of ARMH1 suggested a role in leukemic cells' survival/maintenance. To ascertain its functional involvement in cancer cells, ARMH1 was silenced using siRNAs in both normal and AML cell lines. Through in vitro assays, we demonstrate that ARMH1 silencing significantly reduced leukemic cells' capacity to proliferate (P=.0041) and migrate (P=.0001). We also observed slightly increased sensitivity to chemotherapy drug, Cytarabine in the ARMH1 silenced cancer cells compared to control cancer cells in which the gene is fully functional. However, suppression of ARMH1 expression had no effect on cell growth in the normal HEK293t cell line (P=0.913). On the other hand, overexpression of ARMH1 via transient transfection of ARMH1 gene into the AML cell line, made the cells more resistant to Cytarabine and significantly increased both migration (P=.041) and cell proliferation (P=.032). In conclusion, we demonstrate for the first time that ARMH1, a still poorly understood protein, contributes to the increased proliferation and migration of malignant AML cells. With specific overexpression in blast cells of several pediatric leukemias and the newly discovered roles in proliferation, migration, and chemotherapy resistance, ARMH1 may be a good candidate for targeted treatment of pediatric leukemias.

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD33 rs12459419 SNP Regulated the Total CD33 Expression Level

Background: Gemutuzumab ozogamicin (GO) is a molecularly targeted drug which combines a humanized anti-CD33 antibody (p67.6) with calicheamicin. GO has differed therapeutic efficacy in cases of acute myeloid leukemia (AML). Single nucleotide polymorphisms (SNPs) in the CD33 gene have been investigated as one of the reasons for this difference. SNPs (CC, CT, and TT types) in rs12459419 have been reported to be associated with clinical outcomes in pediatric AML patients treated with GO-containing chemotherapy. On the other hand, there are reports of poor association in adult AML patients, and the significance of rs12459419 SNPs in remains to be clarified. CD33 is composed of two extracellular domains, IgV and IgC. p67.6, anti-CD33 antibodies, recognize IgV, while HIM3-4 antibodies recognize IgC. rs12459419 SNP is located in the splicing acceptor region of exon 2 of the CD33 gene, when the SNP is T, transcripts that skip exon 2 which encodes the IgV domain, are more likely to occur. The increasing expression of this exon skipping product may be accompanied by a decreasing amount of wild-type CD33 (CD33FL) and an increasing amount of IgV domain-deficient CD33 (CD33ΔE2). GO only recognizes CD33FL, but not CD33ΔE2. We used CRISPR/Cas9 genome editing system to generate cell lines with specifically modified CD33 SNPs to elucidate the expression of CD33FL and CD33ΔE2 by CD33 SNPs and its effect on sensitivity to GO in a uniform genetic background. Methods and Results: First, we analyzed CD33 SNPs in 23 AML patients treated with GO (CC type: 13; CT type; 9: TT type: 1). The outcome of 17 patients, excluding a history of hematopoietic stem cell transplantation before treated with GO, was evaluated. Three-year overall survival rate for the type of CC vs. CT was 70.7% vs. 83.3% (p value = 0.609), and 3-year disease-free survival rate for the type of CC vs. CT was 80.8% vs. 83.3% (p value = 0.895). There was no significant difference in CC and CT. Next, using p67.6 recognizes only CD33FL and HIM3-4 recognizes both CD33FL and CD33ΔE2, we evaluated the expression ratio of CD33FL and CD33ΔE2 in relation with CD33 SNP. We assumed that the expression levels of MFI(median fluorescence intensity) were roughly similar in HIM3-4, but the MFI evaluated by both antibodies tended to be higher in the CT and CC types than in the TT type (MFI for p67.6 of CC, CT and TT was 2675, 1752 and 245, respectively; MFI for HIM3-4 of CC, CT and TT was 699, 629 and 53, respectively). The patient samples differ in many genetic backgrounds besides CD33 SNPs, making accurate evaluation difficult. Genome editing was performed by co-transfection of guide RNAs targeting CD33 SNPs, Cas9 protein and with single-stranded oligonucleotides to the target leukemia cell line. Single cell cloning was performed by limiting dilution to generate single nucleotide altered cells for the CD33 SNP. OCI-AML3 wild-type (TT type) and the obtained CD33 SNP-modified clones (CC type, 4 clones and CT type, 4 clones) were evaluated by flow cytometry with p67.6 and HIM3-4.The results showed that the MFI of both antibodies was higher in the CT and CC types than in the TT, the wild type (MFI for p67.6 of CC, CT and TT was 10760, 13686 and 4801, respectively; MFI for HIM3-4 of CC, CT and TT was 3115, 4067 and 887, respectively). These results showed similar trends to the clinical samples implying that differences in CD33 SNPs affect the amount of total CD33 expression on the cell surface itself. Conclusion: The CD33 rs12459419 SNP regulated the expression level of total CD33 in clinical samples and gene-edited cell lines. There was no obvious difference in the expression levels of total CD33 and CD33FL at the cell surface between the CC and CT types, but it was higher than that of the TT type. The difference in CD33 expression may influence the therapeutic efficacy of GO.

Single-Cell Map of Childhood Acute Myeloid Leukaemia Using Variational Auto-Encoders

Acute myeloid leukaemia (AML) is a haematological cancer in the bone marrow, with accumulation and expansion of immature cells of the myeloid lineage. Standard treatment of childhood AML is chemotherapy, which does not achieve durable remission in many patients. Personalised medicine including immunotherapies against surface antigens can potentially target chemotherapy resistant cells and achieve long-term remission. Identifying AML blasts and suitable targets for AML therapy are hampered by the heterogeneity and complex clonal and phenotypic composition of the cancer, as well as its complex evolution as the disease progresses into relapse. Aberrant expression of surface antigens on AML blasts measured by flow cytometry is an established strategy to monitor measurable residual disease. Although the likelihood to find blasts is increased as more markers are included in a staining, manual interrogation of all possible combinations exceeding 10 markers becomes extremely difficult. We aim to identify malignant cells based on expression of 23 surface markers normally or aberrantly expressed during hematopoiesis using spectral flow cytometry and machine learning. The goal of this high-dimensional approach is to distinguish leukaemia cells from normal myeloid cells with high confidence, and to place the blasts along the normal myeloid developmental trajectory. We built a single-cell AML map from 20 paediatric AML patients enrolled in the Children's Oncology Group Phase III trial, AAML1031. All patients were treated with standard chemotherapy on Arm A and consented to provide tissue for research. Cryopreserved samples were obtained for three time-points: diagnosis, remission, and relapse. We assumed full remission samples to be healthy and used them to train variational auto-encoders with different hyper-parameters in a cross validated manner. These models subsequently learned to encode the expression of healthy cells in a reduced dimension and to reconstruct their expression from this latent space. We selected the top-10 models that best reconstruct the healthy cells and used it to encode and reconstruct cells from non-remission samples. Since the model works well for encoding and reconstructing healthy cells, we expect a good reconstruction of healthy cells in non-remission samples. For malignant cells we expect a poor reconstruction, as their expression pattern differs from healthy cells. Thus, we use the reconstruction error of cells from non-remission samples to classify them as healthy or malignant. We annotated some cells with clear aberrant expression as malignant and shuffled them with remission cells not used for model development. This resulted in multiple synthetic mixtures with known percentages of malignant cells (ranging from 10 to 90% blasts). Using these synthetic mixtures, we evaluated how well we can classify malignant cells based on their mean-squared reconstruction error. We can achieve an average area under the receiving operator curve of 0.9 over all synthetic mixtures (Fig. 1). The latent space of the variational auto-encoder captures the developmental trajectory of myeloid lineage bone marrow cells (Fig. 2), and we can use this to identify where among the developmental trajectory the leukaemia occurs. We aim to use the trajectory assignment to study the time evolution of the disease in terms of the distribution of malignant cells across the myeloid lineage as well as investigate differences between diagnosis and relapse. We expect that this work will reveal patterns of evolution from diagnosis to relapse that will inform the development of novel strategies to predict and prevent relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TRIM13 Is a Novel Cell Cycle Hub Regulated By CHAF1B to Promote Acute Myeloid Leukemogenesis

CHAF1B (Chromatin Assembly Factor 1B) functions as a histone chaperone within the DNA replication machinery. It is almost universally overexpressed in cancers, and we have previously shown that this upregulation is necessary to enforce the differentiation block found in acute myeloid leukemia (AML) through CHAF1B's chromatin binding function. In this study, we found that CHAF1B creates a pro-leukemic state in hematopoietic cells by repressing the expression of the E3 ubiquitin ligase TRIM13. Our data suggest that TRIM13 is a strong promoter of entry into the cell cycle functioning through novel nuclear localization and activating ubiquitination of key cell cycle genes. We propose that repression of TRIM13 by CHAF1B is a critical event for leukemogenesis because unchecked entry into cell cycle leads to loss of self-renewal and eventual exhaustion of the tumor. First, we measured the expression levels of CHAF1B and TRIM13 in several bone marrow aspirates from primary pediatric AML patients using immunohistochemistry. TRIM13 was expressed in healthy marrow and almost undetectable upon leukemic progression. CHAF1B, on the other hand, had low to moderate expression in healthy tissues and substantially higher expression upon leukemic progression and relapse. We found restoration of healthy CHAF1B and TRIM13 expression levels upon resolution of the tumor in matched patient aspirates upon remission. To determine if CHAF1B is capable of directly repressing TRIM13 expression, first we observed CHAF1B occupancy at the TRIM13 promoter by ChIPseq in multiple AML cell lines. Then we found loss of CHAF1B by shRNA resulted in increased TRIM13 transcription, and overexpression of CHAF1B by dox-inducible lentivirus drove TRIM13 transcriptional repression. Overexpression of a CHAF1B chromatin binding deficient mutant (CHAF1BRRAA) was unable to repress TRIM13, suggesting that CHAF1B chromatin binding is necessary for TRIM13 repression. This led us to hypothesize that TRIM13 may be a novel tumor suppressor in AML. To test this hypothesis, we overexpressed TRIM13 in AML cell lines and several patient-derived xenografts (PDXs) spanning many subtypes of AML and observed consistent repression of colony formation, increase in apoptosis, and reduction of leukemic progression in vivo. Conversely, loss of TRIM13 expression by shRNA led to an increase in colony formation in vitro and enhanced leukemogenicity of PDXs and AML cell lines in vivo. To better understand the mechanistic contribution of TRIM13 to myeloid leukemogenesis, we started by identifying potential catalytic targets of TRIM13. Since TRIM13 is a RING-type E3 ubiquitin ligase, we used CRISPR to generate in-frame RING domain deleted alleles of TRIM13 (T13RINGdel) and performed ubiquitin proteomics comparing our T13RINGdel and TRIM13 WT clones. We expected that loss of the catalytic function would lead to stabilization of pro-leukemic proteins as has been shown in solid tumor models. We did not find that, but we did observe a profound reduction in ubiquitination of proteins associated with the cell cycle, including CCNA1, CCNA2, CCNB2, and CDK1. Additionally, the total levels of these cell cycle-promoting proteins was reduced in T13RINGdel cells suggesting the ubiquitination event leads to their stability and activity. We confirmed that our T13RINGdel clones of AML cell lines had reduced entry and progression through cell cycle. Using imaging cytometry, we found a novel nuclear localization of TRIM13 and an expression pattern characterized by increased TRIM13 expression as cell cycle progressed. We confirmed the stabilization/activation relationship between TRIM13 and CCNA1 by restoring CCNA1 expression and confirming loss of the colony formation benefit in T13RINGdel AML cell lines. This study is the first to link CHAF1B, a cell cycle gene, with transcriptional repression of genes that promote cell cycle progression. We believe this relationship is critical for AML cells to support high levels of CHAF1B expression, thereby promoting an immature leukemic state throughout replication, without premature entry into multiple damaging rounds of proliferation.

CAR.CD123-NK Cells Have an Equally Effective but Safer Off-Tumor/on-Target Profile As Compared to CARCD123-T Cells for the Treatment of Acute Myeloid Leukaemia

Paediatric Acute Myeloid Leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvement of intensive standard therapy. Leukaemia cells of paediatric AML patients show a high expression of the CD123 antigen, this finding providing the biological basis to target CD123 with Chimeric Antigen Receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumor toxicity, as well as by the long "vein-to-vein" time. In this scenario, we developed an off-the-shelf product based on allogeneic Natural Killer (NK) cells derived from peripheral blood of healthy donors, and engineered to express a second generation CAR.CD123 incorporating 4.1bb as costimulatory domain. CAR.CD123-NK cells showed significant in vitro anti-leukaemia activity not only against CD123+ AML cell lines, but also against CD123+ primary blasts (38,1%±4,8 NT-NK vs 3,6%±3,4 CAR-NK of CD123+ residual leukemic cells after 7-day co-culture; p<0.05). Moreover, the in vitro data were corroborated by an animal model of human AML-bearing immune-deficient mice. In particular, NSG mice were intravenously engrafted with CD123+ THP-1 cells genetically modified to express luciferase (Figure 1A). Mice showing leukemia engraftment were treated on Day 0 and 7 with effector cells (NT-T, CAR.CD123-T, NT-NK or CAR.CD123-NK cells). Bioluminescent imaging was used to measure the AML burden over time (1B). As expected, bioluminescence increased rapidly in mice treated with both controls NT-T and NT-NK cells (1B-C). Notably, CAR.CD123-NK cells induced a kinetics of leukemia control similar to that of CAR.CD123-T cells. These data were also corroborated by the overall survival (OS) analysis of the mice in each of the considered cohorts (1D). Remarkably, no difference was observed in the OS of mice treated with either CAR.CD123-NK cells or CAR.CD123-T cells. Once the anti-leukemia activity of CAR.CD123 NK cells was proved, we moved to evaluate the comparative on-target off-tumor effect of CAR.CD123 T and NK cells. We engrafted human haematopoietic cells from cord blood in an immune-deficient mouse model (hGM-CSF/hIL3 NOG genetically modified to express human GM-CSF and IL-3 cytokines). Ten weeks post-transplantation, mice were infused with either CAR.CD123-T, CAR.CD123-NK or un-modified T or NK cells (Fig. 2A). Five days after injection of effectors cells, all mice infused with CAR.CD123-T cells were dead, developing acute toxicity; by contrast, treatment with CAR.CD123-NK cells was not associated to toxicity as shown by OS data with 100% of the mice surviving at the Day 15, end of experiment (Fig. 2B). Lastly, it has been previously reported that CD123 antigen is expressed on endothelial cells and that these latter are specifically recognized and lysed by CAR.CD123-T cells. In light of these findings, we sought to define the toxicity profile of CAR.CD123 cells in a comprehensive animal model in which we developed a human endothelial tissue. We engrafted the endothelial model in NSG mice, to test the safety profile of effector T and NK cells that have been infused after 2 weeks from the implant of human endothelial cells. Animals were sacrificed to collect the transplanted endothelium (Fig. 2C). Whereas the controls developed a dense network of vessels, as assessed by Haematoxylin/Eosin staining and counts of vessels/mm2 number, we observed a significant reduction of the number of vessels per mm2 in mice receiving CAR.CD123-T cells (p=0,001). In contrast, we did not observe any difference in terms of vessels/mm2 number in mice treated with CAR.CD123-NK cells or NT-NK cells. Overall, our data indicate the feasibility of an innovative ''off-the-shelf'' therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and improved safety profile as compared to CAR.CD123-T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Whole Genome Sequencing for Detection of Prognostically Relevant Genetic Abnormalities in Paediatric Acute Myeloid Leukaemia

Background Genetics is important in the diagnosis of paediatric acute myeloid leukaemia (AML). A number of chromosomal abnormalities and mutations have prognostic significance and timely genetic analysis is essential for risk stratification. The recent Myechild 01 clinical trial for children with AML required testing for >20 different genetic abnormalities to assign newly diagnosed patients into three prognostic genetic risk groups: good, intermediate and poor (Figure A). Throughout the trial a combination of techniques was employed to detect these abnormalities; including chromosomal analysis, FISH for cytogenetically cryptic abnormalities, notably the poor risk fusions: t(5;11)(q35;p15)/NUP98::NSD1, inv(16)(p13q24)/CBFA2T3::GLIS2, t(7;12)(q36;p13)/ETV6::MNX1, as well as molecular testing for NPM1, FLT3-ITD and CEBPA mutations. Here, we present a small retrospective study demonstrating that whole genome sequencing (WGS) has the potential to detect all prognostically significant abnormalities in childhood AML within a single test. Methods WGS was performed on matched diagnostic and remission samples from 25 paediatric AML patients entered to the Myechild 01 treatment trial. Remission samples, minimal residual disease negative by flow cytometry and/or molecular testing, were used as germline controls. For validation, whole transcriptome sequencing (WTS) was performed on diagnostic RNA from each patient in the study. Results Leukaemia associated genetic abnormalities were detected in all 25 patients, including structural variants associated with gene fusions (n=14), single nucleotide variants (SNV) (n=20) and/or copy number abnormalities/aneuploidies (n=17). All gene fusions and mutations were confirmed by WTS. WGS reliably detected all risk stratifying abnormalities identified by standard techniques (Figure B), including 11 patients with gene fusions: RUNX1::RUNX1T1 (n=6), CBFB::MYH11 (n=3), KMT2A::MLLT3 (n=1) and KMT2A::MLLT10 (n=1) and 3 patients with copy number abnormalities: del(5q) (n=2) and del(12p) (n=1) and one patient with an NPM1 mutation. Among 10 patients with no risk stratifying abnormalities detected by standard techniques, WGS identified risk defining genetic features in 3 of them. Two patients could be reclassified as good risk due to the discovery of biallelic CEBPA mutations. Interestingly, one of the CEBPA positive patients presented with a homozygous mutation (VAF=0.97), which WGS revealed to be the consequence of copy number neutral loss of heterozygosity for the long arm of chromosome 19. A single patient could be reclassified as poor risk due to the finding of a rearrangement of MECOM with CDK6. Cytogenetics showed a deletion of 7q as the sole karyotypic abnormality, which was revealed to be an unbalanced der(7)t(3;7)(q21.2;q26.2) translocation by WGS. Over-expression of MECOM was confirmed by WTS. This patient also had a GATA2 mutation. A risk defining abnormality was not detected by either standard testing or WGS in 7 patients, however 5 of them presented with chromosomal abnormalities which were confirmed and further characterised by WGS, including aneuploidies (n=3) and/or structural rearrangements (n=4). Across the cohort, WGS detected a wide range of abnormalities not currently used in the risk stratification algorithm, including the fusions genes: NPM1::MLF1 (n=1) and PICALM::MLLT10 (n=2), which, although not stratifying in Myechild 01, have been reported to be associated with a good and a poor prognosis, respectively. SNV in genes commonly mutated in AML were observed in 19 patients (76%) including NRAS (n=6), KIT (n=3) EZH2 (n=3) and GATA2 (n=2). It has been suggested that the co-occurrence of mutations may be used to refine risk groups in childhood AML, notably the presence of KIT and NRAS mutations in RUNX1::RUNXT1 and CBFB::MYH11 positive leukaemia, respectively. Conclusion This small pilot study has demonstrated the ability of WGS to identify a wide range of genetic abnormalities that are currently used for risk stratification in childhood AML. The full spectrum of genomic events observed here highlights the potential of WGS to refine risk stratification and identify novel genetic changes as therapeutic targets in future trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tracking Clonal Evolution in Pediatric AML

Acute myeloid leukemia (AML) makes up nearly 20% of acute pediatric leukemias and has a long-term survival rate of approximately 70%. A key driver of the low overall survival for children with AML is relapsed disease and less than 40% of the children with relapsed AML will survive past five years. We recently characterized relapsed pediatric AML (pAML) to better understand the spectrum of genomic alterations at disease recurrence (Umeda et al., 2022). However, the genome-wide patterns of genetic evolution from diagnosis to relapse have yet to be characterized for pAML. Thus, there is a gap in knowledge of how alterations at diagnosis evolve during treatment to drive relapse and poor outcome. We performed whole genome sequencing (WGS) and whole transcriptome sequencing (RNA-Seq) on a cohort of 50 pediatric AML patients with paired diagnosis and relapse samples. The median age at diagnosis was 10.7 yrs (range 0.7-20yrs), and the median time between diagnosis and relapse was 334 days (range 93-1,773 days). The main driver group contained a KMT2A fusion event (n=16, 32%). NUP98 fusion made up the second largest group at 14% (n=7). Also within the cohort are newly defined categories, including a BCL11B structural variant (n=1) and patients with UBTF tandem duplication AML (n=4). These features are overall similar to the molecular profiles of pediatric AML (Farrar et al., 2016; Umeda et al., 2022). Approximately 22,500 alterations (including point mutations, indels, and structural variants) were detected by WGS (median coverage 36X). The median number of alterations at diagnosis and relapse per patient was, respectively, 224 (range 118-867) and 411 (range 73-1,909). Most of the cohort noticed more alterations at relapse than diagnosis, but some (n=8) had fewer at relapse. Additionally, there was no trend with the time to relapse and the number of acquired alterations. The most commonly mutated genes regardless of timepoint included FLT3 (n=16, 6 were internal tandem duplications), WT1 (n=18), NRAS (n=16), and SETD2 (n=9). We identified over 11,000 relapse enriched variants (median 169, range 3-1,378), including alterations that had a significantly higher allele frequency (q-value < 0.05) at relapse compared to diagnosis. This included 1,028 relapse enriched variants that alter protein coding genes. FLT3 contained 7, 2, and 7 relapse enriched, relapse depleted, and time point stable alterations, respectively. WT1 contained 6, 2, and 10 relapse enriched, relapse depleted, and time point stable alterations, respectively. Furthermore, the relapse-enriched list included three SETD2 protein-truncating mutations and four AUTS2 structural variants often co-occurring with KMT2A fusions, indicating that these mutations may cooperate with KMT2A fusion and contribute to the disease progression. To increase our understanding of the clonal structure, 36 cases with additional DNA material were re-sequenced with a custom panel covering 6,300 variants, including all coding variants for each case. This led to a validation rate of 97% of all variants and the increased sequencing depth of the panel (median 265X for diagnosis and 274X for relapse) gave greater insight into the changes in clonal structure between diagnosis and relapse time points. Most of the cohort (70%) featured a relapsed enriched clone that was minor at diagnosis and became clonal in relapse. Additional cases (n=10) featured a clone that was undetectable by WGS at diagnosis and after treatment became the dominant clone in relapse. Only one case maintained the same major clone in both diagnosis and relapse. In summary, our data show a comprehensive characterization of changes in the clonal structure of pAML between diagnosis and relapse, revealing relapse-enriched gene alterations which may be driving relapse disease. Farrar, J. E., Schuback, H. L., Ries, R. E., Wai, D., Hampton, O. A., Trevino, L. R., . . . Meshinchi, S. (2016). Genomic Profiling of Pediatric Acute Myeloid Leukemia Reveals a Changing Mutational Landscape from Disease Diagnosis to Relapse. Cancer Res, 76(8), 2197-2205. doi:10.1158/0008-5472.CAN-15-1015 Umeda, M., Ma, J., Huang, B. J., Hagiwara, K., Westover, T., Abdelhamed, S., . . . Klco, J. M. (2022). Integrated genomic analysis identifies UBTF tandem duplications as a recurrent lesion in pediatric acute myeloid leukemia. Blood Cancer Discov. doi:10.1158/2643-3230.BCD-21-0160 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Leukoreductive response to the combination of sorafenib and chemotherapy in hyperleukocytosis of FLT3-ITD mutated pediatric AML.

Twelve to 22% of pediatric acute myeloid leukemia (AML) patients present with hyperleukocytosis, which is one of the main risk factors of early death due to its clinical complications: leukostasis, causing pulmonary or central nervous system injuries, tumor lysis syndrome, and disseminated intravascular coagulation. Sorafenib is a multi-kinase inhibitor that blocks the Fms-Related Tyrosine Kinase 3 receptor (FLT3) in AML patients with a FLT3-internal tandem duplication (FLT3-ITD), leading to a reduction of proliferation. Here we report four de novo diagnosed or relapsed pediatric FLT3-ITD-positive AML patients with hyperleukocytosis, which were treated with sorafenib in combination with cytoreductive chemotherapy prior to the start of the induction phase. We observed a fast reduction of white blood cells in peripheral blood and bone marrow. This resulted in a rapid clinical stabilization of the patients. Adverse side effects-such as dermatologic toxicity, elevation of transaminases and hypertension-occurred but were mild and inductive chemotherapy could be started in parallel or subsequently. This implies sorafenib as a safe and effective treatment option in combination with chemotherapy during cytoreductive prephase for children with this life-threatening condition.

Safe and effective off-the-shelf immunotherapy based on CAR.CD123-NK cells for the treatment of acute myeloid leukaemia

BackgroundPaediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long “vein-to-vein” time. MethodsWe developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123).ResultsCAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells.To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38− stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells.ConclusionsOur data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells.