Childhood Acute Myeloid Leukemia Research Articles

Decitabine as epigenetic priming with CLAG induce improved outcome of relapsed or refractory acute myeloid leukemia in children

BackgroundDecitabine (DAC), a DNA methyltransferase inhibitor, has shown efficacy combined with chemotherapy for relapsed or refractory (R/R) acute myeloid leukemia (AML) in adults, but less is known about its efficacy in children. Accordingly, we conducted a study which involved a priming regimen consisting of DAC with cladribine, cytarabine, and granulocyte-stimulating factor (DAC-CLAG) and compared the efficacy and safety of this regimen with CLAG alone.MethodsA total of 39 R/R AML children who received the CLAG or DAC-CLAG regimen in Shanghai Children’s Hospital were retrospectively enrolled in this non-randomized study. These regimens were studied sequentially over time. Twenty-two patients received CLAG from 2015, while 17 patients were administered epigenetic priming with DAC before CLAG from 2020. Patients were subsequently bridged to stem cell transplantation (SCT) or consolidation chemotherapy. Complete remission (CR) and adverse effects were analyzed by Fisher’s exact test, and survival was analyzed by the Kaplan–Meier method.ResultsDAC-CLAG conferred a numerically higher CR compared to CLAG (70.59% vs 63.64%; P = 0.740). High CR rates occurred in patients with good cytogenetics (P = 0.029) and prior induction without cladribine (P = 0.099). The 1-year event-free survival (EFS) was 64.71% ± 11.59% and 63.31% ± 10.35% in the DAC-CLAG and CLAG group (P = 0.595), and 1-year overall survival (OS) was 81.45% ± 9.72% and 77.01% ± 9.04%, respectively (P = 0.265). The 1-year OS and EFS after SCT were higher in the DAC-CLAG than in the CLAG cohort (100% vs 92.31% ± 7.39%, P = 0.072; 92.31% ± 7.39% vs 85.71% ± 9.35%, P = 0.158). Univariate analysis revealed that a good prognosis included good cytogenetics (P = 0.002), non-complex karyotype (P = 0.056), CR on reinduction (P < 0.0001), and bridging to SCT (P = 0.0007). Use of a hypomethylating agent (P = 0.049) and bridging to SCT (P = 0.011) were independent prognostic factors. Grade 3/4 hematologic toxicity and infection were the main adverse events.ConclusionsDAC prior to the CLAG regimen improved remission in pediatric R/R AML, and was feasible and well tolerated. CLAG ± DAC as a salvage therapy prior to SCT induced improved survival.

Infection-related mortality and infection control practices in childhood acute myeloid leukemia in a limited resource setting: Experience with the Indonesian national protocol.

In resource-limited settings, addressing infections remains a substantial challenge in the management of children with Acute Myeloid Leukemia (AML). In Indonesia, infection-related mortality (IRM) is thought to be high compared to high-income countries. However, there has been no previous study of infection profile and IRM in Indonesian patients with AML. This study aimed to describe infections and IRM in children with AML treated according to the Indonesian National AML protocol and to describe the implementation of infection control practices in resource-limited settings. This retrospective observational study used secondary data from the medical records of pediatric patients with AML treated with the National Protocol at Dr. Sardjito Hospital, Yogyakarta, Indonesia, from April 2012 to September 2018. Essential patient characteristics, time of IRM, and cause of death were recorded, and infection control practices were observed. Data were analyzed using descriptive statistics. 113 patients with AML were treated with the National protocol, and 83 met the inclusion criteria. Infections occurred in 69 (83%) patients with a total of 123 episodes (mean 1.8/patient). Death was seen in 48 (58%) patients, with 19 (23%) IRM. The majority of infections were in the gastrointestinal tract (n = 51, 30.5%), sepsis (n = 29, 17%), and respiratory tract (n = 28, 17%). Infections mostly occurred during the first induction (41%). There were 90 (73%) episodes of clinically documented infection and 33 (27%) episodes of microbiologically documented infection. The positivity rate of blood cultures was only 27%. The majority of bacteria detected were gram-negative (n = 25, 69%), and among them were Klebsiella pneumonia (19%) and Escherichia coli (19%). Candida albicans was detected in 1 (2%) culture. Suboptimal infection prevention and control were found in the clinical practice. Infections and infection-related mortality in children with AML treated using the National protocol were frequent, mainly occurring during the first induction phase. Compliance with infection prevention and control measures needs improvement. Urgent attention is required for better supportive care, including isolation rooms, antibiotics, and antifungals. The predominance of Gram-negative bacterial infections highlights the necessity for further research into effective prophylaxis. Enhanced healthcare and nursing professional vigilance and tailored antibiotic strategies are vital. Improving compliance and ensuring adequate supportive care resources are essential, emphasizing nursing's pivotal role. Further research is crucial to drive advancements in infection control strategies.

Pediatric acute promyelocytic leukemia and Fanconi anemia: Case report and literature review.

Acute promyelocytic leukemia (APL) represents 5%-10% of childhood acute myeloid leukemia (AML) and is the most curable subtype of AML. Fanconi anemia (FA) is one of the most common inherited bone marrow failure syndromes caused by biallelic pathogenic variants (PV) in specific DNA-repair genes. Biallelic PVs in FANCD1/BRCA2 (FA-D1) account for 3% of FA and are associated with early-onset leukemia and a high risk of solid tumors. We report a 4 year-old boy from non-consanguineous parents diagnosed with standard risk APL. This child had café-au-lait spots and an extra thumb remnant. Genomic sequencing revealed two PV in FANCD1/BRCA2 confirming a diagnosis of FA-D1. Chromosomal breakage studies were compatible with FA. Each parent carried one variant and had no personal history of cancer. Morphological then molecular remissions were achieved with all-trans retinoic acid and Arsenic trioxide. This patient underwent haploidentical stem cell transplant. In addition to our patient, a literature search revealed four additional patients with APL/FA, with a total of three patients with FA-D1. This raises the possibility of an association between such rare disorders. Practical management of APL in the setting of FA-D1 is discussed with an overview of current evidence and knowledge gaps.

A 4-gene prognostic index for enhancing acute myeloid leukaemia survival prediction.

Despite advancements in utilizing genetic markers to enhance acute myeloid leukaemia (AML) outcome prediction, significant disease heterogeneity persists, hindering clinical management. To refine survival predictions, we assessed the transcriptome of non-acute promyelocytic leukaemia chemotherapy-treated AML patients from five cohorts (n = 975). This led to the identification of a 4-gene prognostic index (4-PI) comprising CYP2E1, DHCR7, IL2RA and SQLE. The 4-PI effectively stratified patients into risk categories, with the high 4-PI group exhibiting TP53 mutations and cholesterol biosynthesis signatures. Single-cell RNA sequencing revealed enrichment for leukaemia stem cell signatures in high 4-PI cells. Validation across three cohorts (n = 671), including one with childhood AML, demonstrated the reproducibility and clinical utility of the 4-PI, even using cost-effective techniques like real-time quantitative polymerase chain reaction. Comparative analysis with 56 established prognostic indexes revealed the superior performance of the 4-PI, highlighting its potential to enhance AML risk stratification. Finally, the 4-PI demonstrated to be potential marker to reclassified patients from the intermediate ELN2017 category to the adverse category. In conclusion, the 4-PI emerges as a robust and straightforward prognostic tool to improve survival prediction in AML patients.

Coagulopathy in pediatric acute promyelocytic leukemia in Bangladesh: A single-center, prospective study

BackgroundAcute promyelocytic leukemia (APL) is one of the most curable subtypes of acute myeloid leukemia in childhood. It usually presents with a characteristic coagulopathy. The aim of the study was to determine the extent and outcome of this coagulation disorder. MethodsThis prospective observational study was conducted at Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, and included PML:RAR-α (Promyelocytic Leukemia-Retinoic Acid Receptor-α) positive APL cases. At presentation, all patients were assessed with coagulation parameters and followed up clinically and hematologically while treated with ATRA (all-trans-retinoic acid)-based chemotherapy. The presence of disseminated intravascular coagulation (DIC) was determined using the DIC scoring system of the International Society on Thrombosis and Hemostasis (ISTH). ResultsAmong 20 APL cases, the mean DIC score was 5.75 ± 0.6. DIC was detected in 90% (n = 18/20) of the patients. The incidence of fatal thrombo-hemorrhagic complications was 15% (n = 3/20). Though hemorrhagic complications are common, thrombosis may also occur in pediatric APL.

Abstract 3954: Increased mosaic chromosomal alterations among survivors of childhood acute myeloid leukemia

Abstract Background: We previously characterized point mutations of clonal hematopoiesis (CH) including single nucleotide variants or small insertion/deletions residing in a set of cancer-related genes (e.g., DNMT3A and TET2), and found that CH point mutations occurred at a much higher prevalence in childhood cancer survivors (CCS) than in age-sex-race matched community controls. However, mosaic chromosomal alterations (mCAs), another form of CH with megabase-scale structural alterations (copy gain, copy loss and copy-neutral loss of heterozygosity), have not been examined. Methods: Detection of mCAs was performed on whole-genome sequencing (WGS, 30X) data from 5-year CCS with the Mosaic Chromosomal Alterations (MoChA) caller which utilizes phased genotypes, coverage, and B allele frequency (BAF) at heterozygous sites (HETS). We implemented the pipeline established by the TOPMed consortium to detect low mutant cell fractions (CF ≥1%). Variant filtration criteria: minor allele frequency &amp;lt;1%, the read depth of either allele &amp;lt;5, and only one variant retained in each 1Kb window. Called mCA filtration criteria: &amp;lt;100 HETS; lod score &amp;lt;5; relative coverage &amp;gt;2.9 or BAF deviation &amp;gt;0.16 and relative coverage &amp;gt;2.5. Autosomal mCAs (CF ≥1%, HETS &amp;gt;100 and length &amp;gt;1Mb) retained. Multivariable Cox models evaluated the association between mCAs and specific late-effects including all-cause mortality, obesity, myocardial infarction (MI) or cardiomyopathy (CMP); multivariable logistic models evaluated the association between mCAs and childhood cancer diagnoses. All models were adjusted for age, sex and therapeutic exposures. Results: In our evaluation of 4,330 CCS (median age at blood draw: 26.8 years) and 433 controls (33.6 years) with WGS data from the St. Jude Lifetime Cohort, the prevalence of mCAs was slightly higher among CCS than community controls (4.83% vs. 4.16%). Among CCS, mCAs were associated with therapeutic exposures (alkylating agents: odds ratio [OR]=1.48, 95% CI=1.09-2.00; antimetabolites: 1.53, 1.02-2.31; corticosteroids: 0.60, 0.40-0.91). In contrast to recent mCA studies in the general population, we observed no co-occurrence with CH point mutations, and no associations with late-effects including all-cause mortality, obesity, MI or CMP. Interestingly, mCAs were significantly associated with the primary diagnosis of acute myeloid leukemia (AML, OR=2.86, 95% CI=1.63-5.01). Furthermore, this finding was replicated in 2898 5-year survivors from the Childhood Cancer Survivor Study (OR=3.77, 95% CI=1.94-7.33). Conclusion: mCAs were not associated with age-related morbidities and mortality among CCS. However, we identified a striking association between mCAs and previous diagnoses of AML independent of treatment exposures. If validated among AML patients at the time of diagnosis, mCAs, which reflect the underlying genome instability, may serve as a diagnostic biomarker for AML. Citation Format: Xijun Zhang, Na Qin, Qian Dong, Cheng Chen, John Easton, Heather Mulder, Xiang Chen, Kyla Shelton, Cindy Im, Yutaka Yasui, Greg Armstrong, Kirsten K. Ness, Melissa M. Hudson, Paul Auer, Jinghui Zhang, Zhaoming Wang. Increased mosaic chromosomal alterations among survivors of childhood acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3954.

Abstract P02: Uncovering Tailored Therapies for Acute Myeloid Leukemia in Children with Down Syndrome

Abstract P02: Uncovering Tailored Therapies for Acute Myeloid Leukemia in Children with Down Syndrome

UBTF tandem duplications in pediatric myelodysplastic syndrome and acute myeloid leukemia: implications for clinical screening and diagnosis.

Recent genomic studies in adult and pediatric acute myeloid leukemia (AML) demonstrated recurrent in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF). These alterations, which account for ~4.3% of AMLs in childhood and about 3% in adult AMLs under 60, are subtype-defining and associated with poor outcomes. Here, we provide a comprehensive investigation into the clinicopathological features of UBTF-TD myeloid neoplasms in childhood, including 89 unique pediatric AML and 6 myelodysplastic syndrome (MDS) cases harboring a tandem duplication in exon 13 of UBTF. We demonstrate that UBTF-TD myeloid tumors are associated with dysplastic features, low bone marrow blast infiltration, and low white blood cell count. Furthermore, using bulk and single-cell analyses, we confirm that UBTF-TD is an early and clonal event associated with a distinct transcriptional profile, whereas the acquisition of FLT3 or WT1 mutations is associated with more stem celllike programs. Lastly, we report rare duplications within exon 9 of UBTF that phenocopy exon 13 duplications, expanding the spectrum of UBTF alterations in pediatric myeloid tumors. Collectively, we comprehensively characterize pediatric AML and MDS with UBTF-TD and highlight key clinical and pathologic features that distinguish this new entity from other molecular subtypes of AML.

Nivolumab Plus 5-Azacitidine in Pediatric Relapsed/Refractory Acute Myeloid Leukemia (AML): Phase I/II Trial Results from the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia &amp; Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3–4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

Inherent genome instability underlies trisomy 21-associated myeloid malignancies.

Constitutional trisomy 21 (T21) is a state of aneuploidy associated with high incidence of childhood acute myeloid leukemia (AML). T21-associated AML is preceded by transient abnormal myelopoiesis (TAM), which is triggered by truncating mutations in GATA1 generating a short GATA1 isoform (GATA1s). T21-associated AML emerges due to secondary mutations in hematopoietic clones bearing GATA1s. Since aneuploidy generally impairs cellular fitness, the paradoxically elevated risk of myeloid malignancy in T21 is not fully understood. We hypothesized that individuals with T21 bear inherent genome instability in hematopoietic lineages that promotes leukemogenic mutations driving the genesis of TAM and AML. We found that individuals with T21 show increased chromosomal copy number variations (CNVs) compared to euploid individuals, suggesting that genome instability could be underlying predisposition to TAM and AML. Acquisition of GATA1s enforces myeloid skewing and maintenance of the hematopoietic progenitor state independently of T21; however, GATA1s in T21 hematopoietic progenitor cells (HPCs) further augments genome instability. Increased dosage of the chromosome 21 (chr21) gene DYRK1A impairs homology-directed DNA repair as a mechanism of elevated mutagenesis. These results posit a model wherein inherent genome instability in T21 drives myeloid malignancy in concert with GATA1s mutations.

Retracted: The Role of Gender-Related Immune Genes in Childhood Acute Myeloid Leukemia.

[This retracts the article DOI: 10.1155/2022/3235238.].

Exposure to persistent organic pollutants in newborn dried blood spots and childhood acute myeloid leukemia

Acute myeloid leukemia (AML) is a rare malignancy representing 15–20% of all leukemia diagnoses among children. Maternal exposure to persistent organic pollutants is suggestive of increased risk for childhood AML based on existing evidence. We aimed to evaluate the relationship between persistent organic pollutants and childhood AML using newborn dried bloodspots (DBS) from the Michigan BioTrust for Health. We obtained data on AML cases diagnosed prior to 15 years of age (n = 130) and controls (n = 130) matched to cases on week of birth from the Michigan Department of Health and Human Services. We quantified levels of dichlorodiphenyldichloroethylene (p,p’-DDE), hexachlorobenzene (HCB), and polybrominated diphenyl ether congener 47 (BDE-47) in newborn DBS. We also evaluated other organochlorine pesticides, polychlorinated biphenyls, polybrominated biphenyl congener 153, and polybrominated diphenyl ethers, though these were not further evaluated as >60% of observations were above the limit of detection for these chemicals. To evaluate the association between each chemical and AML, we used multivariable conditional logistic regression. In our multivariable model of HCB adjusted for month of birth, maternal age at delivery, and area poverty, we observed no association with AML (Odds Ratio [OR] per interquartile range increase: 1.17, 95% CI: 0.80, 1.69). For p,p’-DDE, ORs were significantly lower for those exposed to the highest tertile of p,’p-DDE (≥0.29 pg/mL, OR: 0.32, 95% CI: 0.11, 0.95) compared to the first tertile (<0.09 pg/mL). We observed no statistically significant associations between HCB and BDE-47 and AML. We observed a reduced odds of exposure to p,’p-DDE and an increased, though imprecise, odds of exposure to HCB among AML cases compared to controls. Future studies would benefit from a larger sample of AML patients and pooling newborn DBS across multiple states to allow for additional variability in exposures and evaluation of AML subtypes, which may have differing etiology.

Acute myelomonoblastic leukemia in children, the particularities of extramedullary infiltration based on a clinical case

Childhood acute myeloidleukemia (AML) represents approximately 5% of childhood malignancies and 15% to 25% of pediatric acute leukemia. The extramedullary manifestation of infiltrating clonal blast populations in a variety of organs and tissues in acute myeloid leukemia (AML) is defined as a distinct entity in the 2016 WHO classification of myeloid neoplasms, myeloid sarcoma The rate of complete remission after induction chemotherapy was lower in patients with extramedullary infiltration, overall survival and event-free survival were not different between patients with and without extramedullary infiltration. Purpose of the study: Presentation of a clinical case of ALM type M4 with the presence of an extramedullary infiltrate in the tissue of the central nervous system in children. Materials and methods: The clinical, paraclinical, imaging data of the patient were studied in parallel with the specialized literature on similar cases. Results: Boy, 5 years old, goes to the doctor with accusations of bilateral palpebral edema, bilateral temporal region edema and bilateral exophthalmos , more pronounced on the left. In the general analysis of the blood: - Hemoglobin - 89 g/l , Leukocytes - 7.2 x 10^9/l; unsegmented neutrophil - 1%; segmented - 18%; lymphocytes - 44%; monocytes - 20%; blast cells - 16%; Platelets - 70 x 10^9/l;VSH - 44 mm/h. During the physical examination: the peripheral lymph nodes are palpable. No signs of central nervous system damage. It is supposed the diagnosis: Acute myeloid leukemia, sarcoma, or neuroblastoma. A medullary puncture: moderately cellular marrow was determined, with blastcells 15.2%. Morphologically, it is more similar to acute myelo-monoblastic leukemia FAB-M4. Computerized Tomography-multiple, hyperdense pathological foci, with localization with inand bilateral extraorbital, associated with exophthalmos. The formation from the sphenoid sinus extends to the nasopharynx , through the pterygopalatine fossa, with narrowing of the lumen and mass effect on the palatine tonsils. Immunophenotyping: CD45-/+ SS low 8% population, CD36+ 6%CD64+ monoblasts 8%, Promonocytes /monocytes – (CD64+/CD14+) 25%.He underwent treatment according to the AML BFM 2004 program with numerous complications, andcranial radiotherapy. At the moment, the child is in morphological remission. Conclusions. The differential diagnosis of hyperdense intracranial masses may include neuroblastoma, meningioma, lymphoma, metastases, or sarcomas. Correct and early diagnosis and initiation of specific treatment are essential to increase the survival rate in cases of acute leukemia in children.

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

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

UBTF Tandem Duplications in Pediatric MDS and AML: Implications for Clinical Screening and Diagnosis

Recent genomic studies in adult and pediatric acute myeloid leukemia (AML) demonstrated recurrent in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor ( UBTF) (PMID: 35176137, PMID:37085611). UBTF-TDs are subtype-defining genomic alterations associated with poor outcomes that account for ~4.3% of AMLs in childhood and up to 3% in adult AMLs under the age of 60. UBTF-TD AMLs are characterized by HOXA/HOXB dysregulation, trisomy 8 or normal cytogenetics and frequent FLT3-ITD and/or WT1 mutations. A more comprehensive investigation into the clinicopathological features of UBTF-TD AMLs is needed to better understand disease features for appropriate diagnosis and to develop more effective therapies. Here, we present 94 unique pediatric cases harboring a tandem duplication in exon 13 of UBTF with a median size of 60bp (range 21-1173bp). Of these 94 cases, 6 (6.4%) were diagnosed with childhood MDS with increased blasts (WHO), suggesting that like in adults, UBTF-TDs are not only confined to AML. UBTF-TD AMLs are associated with an overall lower median white blood cell (WBC) when compared to other common HOXA/B dysregulated leukemias ( UBTF-TD median = 10.2x10 9/L, NPM1-mutation median = 21.8x10 9/L, NUP98-rearranged ( NUP98r) median = 97x10 9/L). Available immunophenotyping on 32 U BTF-TD tumors demonstrated positivity for HLA-DR (31/31; 100%), CD117 (28/29; 95%), and CD34 (26/30; 86%) with variable positivity for CD7 (17/30; 56%) and CD64 (13/25; 52%). A limited number of cases tested for CD123 (n=11) and CD11c (n=15) were all positive for the respective marker. Furthermore, morphologic assessment of these cases revealed pleomorphic blasts with occasional Auer rods and increased immature myeloid cells with salmon-colored granules, commonly with background multilineage dysplasia and increased erythroid precursors. At the transcriptional level, UBTF-TD leukemias are largely similar to NPM1-mutated and NUP98::NSD1 AMLs with the exception of UBTF-TD cases having marked increase in expression of histone genes (e.g., HIST1H4F and HIST1H2B1) and a subset of posterior HOXB cluster genes (e.g., HOXB9). Further inspection using UMAP (Uniform Manifold Approximation and Projection) of expression profiles across different pediatric AML cohorts revealed two unclassified cases of AML that clustered with the UBTF-TD subtype ( Figure 1). One case had a WT1p.S364* and the other a somatic GATA2 p.R338fs and FLT3-ITD, but did not contain an exon 13 duplication or other defining alterations. Upon closer inspection of the UBTF gene in these cases, we uncovered that each case had an in-frame tandem duplication in exon 9 of UBTF encoding a region between HMG box 2 and 3 with features similar to those in exon 13 ( Figure 2). We therefore postulated that exon 9 TDs ( UBTF-e9) could represent a functionally equivalent subgroup of UBTF-TDalterations. To test this hypothesis, cord-blood CD34+ (cbCD34+) cells were transduced to test the transforming potential of UBTF-e9 duplications. Like with UBTF-e13 duplications, cbCD34+ cells expressing UBTF-e9 duplications displayed increased proliferation compared to UBTF -WT and lentiviral empty vector control in liquid culture; and increased colony counts and replating capacity, increased CD117 expression, and a more blast-like morphology in CFU assays. Collectively, these data suggest that like TDs in exon 13, exon 9 TDs are also sufficient to promote leukemogenic phenotypes. In conclusion, we find that UBTF-TDs occur in both MDS and AML and are associated with lower WBC count, myelodysplasia, and stem cell-related surface marker expression. We have also expanded the spectrum of UBTFalterations in myeloid neoplasms through the identification of rare UBTF duplications in exon 9 that induce a similar transcriptional signature to exon 13 UBTF-TDs. The recent identification of UBTF-TDs in adult and pediatric high-risk myeloid neoplasms has highlighted the importance of understanding and appropriately diagnosing tumors with this genomic alteration, including a new class of alterations that will not be detected by PCR-based screening strategies that specifically target exon 13 of UBTF.

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.

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) &amp;gt;1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&amp;RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p&amp;lt;0.0001 and OS 38% vs. 63%, p&amp;lt;0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

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.

Childhood AML Combined with AML1-ETO Fusion Gene and c-KIT or Other Mutations Had Favorable Survival Treated By the C-Huanan AML 2015 Protocol: Study in Multicenter of South of China

Objective: To analyze the impact of AML1-ETO combined with other gene mutations on survival prognosis in children with acute myeloid leukemia (AML) who received the C-HUANAN AML 2015 protocol. Methods: Total 363 children with AML (non APL) who received chemotherapy only from 9 medical centers in South China from January 2015 to December 2020 were retrospective analyzed. The fusion genes and mutation genes spectrum in the bone marrow samples of these children were detected by the first generation and next generation gene sequencing methods. One hundred and fourteen patients had been detected the AML1-ETO fusion gene positive. All of them were treated with the C-HUANAN AML 2015 protocol including 2 groups in which each group contained 4 courses of chemotherapy(FLAG-IDA1,FLAG-IDA2,HAE MidAC versus DAE1,DAE2,HAE,MidAC). The impact of AML1-ETO fusion gene combined with other gene mutations on the prognosis and survival of children was analyzed. Result: A total of 114 AML patients with AML1-ETO fusion gene were included in this study, with a male to female ratio of 1.48:1 and a median age of 85.5 (7-169) months. Among them, there were 35 cases in the DAE group and 79 cases in the FLAG-IDA group. As of April 2021, with a median follow-up time of 21 months (1m-75ms ), 101 cases survived and 13 cases died. OS and EFS were 87.0 ± 3.4%, respectively, with EFS=85.0 ± 3.6%. There was no significant difference in overall survival (OS) and disease-free survival (EFS) between the DAE and FLAG-IDA group of (OS: 86.0 ± 6.5% vs 87.4 ± 4.0%)（ χ 2=0.000, P=0.995), EFS: 79.0 ± 7.6% vs 87.6 ± 3.9%（ χ 2=0.636, P=0.425). Survival analysis of AML1-ETO combined with c-KIT mutation negative or positive(n=33) showed no difference in prognosis (OS: 85.0 ± 4.2% vs 92.4 ± 5.3%)（ χ 2=0.967, P=0.325), EFS: 82.2 ± 4.5% vs 92.4 ± 5.3%（ χ 2=1.602, P=0.206); There was no difference in the prognosis between FLT3-ITD mutation negative and FLT3-ITD gene positive patients(n=8) (OS: 88.0 ± 3.4% vs 75.0 ± 15.3%)（ χ 2=1.516, P=0.218), EFS: 85.5 ± 3.7% vs 75.0 ± 15.3%（ χ 2=1.128, P=0.288); There was no difference in the prognosis between ASXL1 mutation negative and ASXL1 gene positive patients(n=14) (OS: 85.3 ± 3.8% vs 100.0%)（ χ 2=1.872, P=0.171), EFS: 83.0 ± 4.0% vs 100.0%（ χ 2=2.169, P=0.141); There was no difference in the prognosis between WT1 mutation negative and positive patients (n=34) (OS: 86.9 ± 3.9% vs 87.1 ± 7.0%)（ χ 2=0.156, P=0.693), EFS: 85.6 ± 4.0% vs 84.1 ± 7.3%（ χ 2=0.021, P=0.885); There was no difference in the prognosis between negative and positive NRAS mutations (n=13)(OS: 86.6 ± 3.6% vs 92.3 ± 7.4%)（ χ 2=0.033, P=0.856), EFS: 84.4 ± 3.9% vs 92.3 ± 7.4%（ χ 2=0.118，P=0.731). Conclusion: Combination of c-KIT or FLT3-ITD or ASXL1 or WT1 or NRAS gene mutations with AML1-ETO fusion gene in Childhood AML treated by the C-HUANAN AML protocol did not affected patients'survival.

The Relapse Mechanisms and Genomic Landscape Differ in KMT2A-r Pediatric Leukemia in Relation to Relapse Time

The genomic landscape and mechanisms driving relapse in KMT2A-rearranged ( KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 36 KMT2A-r ALL (n=19) and AML (n=17) patients of which 25 relapsed and 11 remained in remission. Twenty diagnose-relapse-germline trios and 5 multiple relapse samples were analyzed by whole genome (WGS) and whole exome sequencing (WES) and 30 patients longitudinally by using patient-specific mutations identified by WGS/WES, including the KMT2A-r (average coverage 3300X). The mutational burden increased from diagnosis to relapse and relapse evolved through branching evolution. Relapse was seeded by multiple diagnostic clones in 56%, by a single sweeping clone detected at diagnosis in 22%, and by a single sweeping clone not detected at diagnosis in 22%. Notably, the evolutionary patterns correlated to relapse time, where multiple diagnostic clones seeding relapse were connected to an earlier relapse with all very early relapse ALL (3/3, relapse &amp;lt;9 months from diagnosis) and half of the early AML relapse showing this pattern (2/4, relapse &amp;lt;1 year in complete remission, CR1). By contrast, later relapse was connected to a sweeping clone at relapse with 67% of early relapse ALL (&amp;gt;9 months from diagnosis) and 40% of late relapse AML (&amp;gt;1 year in CR1) showing this pattern. Pathway analysis showed that cell cycle genes, glucocorticoid signalling, purine metabolism, mismatch repair, and B-cell differentiation, were enriched in early relapse ALL (83%, 5/6) and included TP53, CREBBP, NT5C2 PMS2, PRPS2, NR3C1, IKZF1, with none of the very early relapse infant ALL harboring such alterations (n=4). Further, TP53 and IKZF1alterationsco-occurred (n=4/4). These results were validated in public data sets of 98 KMT2A-r ALL infants (n=84) and children (n=14) at diagnosis and relapse (n=24) and showed that 50% of early relapse ALL, and none of the 8 very early relapse ALL, had such alterations. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the PMS2 and NR3C1 deletions. In AML, TP53 and CCND3 alterations were maintained, and gain of WT1 was seen in late relapse AML. Signalling mutations were the most common type of mutations at diagnosis (64%) and relapse (56%) and the frequency was similar in patients that remained in remission and in those that relapsed (55% versus 60%). One infant ALL and four AML patients had multiple relapses, allowing us to study how the genetic landscape evolved across consecutive relapses. This showed a stepwise replacement of clones during treatment in agreement with a fitter clone that evolves under chemotherapeutic selective pressure. Longitudinal analysis allowed sensitive detection of residual leukemia cells and showed that the relapse clone could be detected at diagnosis in 64% of patients. Further, infants with &amp;gt;10% of molecularly detectable leukemia cells after induction therapy, had a high risk of a very early relapse. Ultra-deep sequencing allowed detection of the relapse clone up to 4 months before relapse. In 11 of the 30 patients (3 remission and 8 relapse), low-frequency KMT2A-fusion positive leukemic cells were found at remission outside of the MRD time points. Our longitudinal data also provided unique insights into clonal response to treatment by showing that 1) a change in therapy can favour the eradication of one clone and expansion of another, 2) a clone that initially was the most sensitive clone to therapy, was the one that eventually caused relapse, and 3), a diagnostic clone can be undetectable for a long time before expanding to cause relapse, suggesting that molecular monitoring with personal mutations is a powerful tool to follow response to therapy. These results provide new biological insights into the relapse mechanisms in KMT2A-r leukemia. The data shows different clonal evolution patters depending on when in time the patient relapsed, with very early relapse ALL being seeded by multiple diagnostic subclones and a paucity of acquired genetic alterations at relapse. By contrast, early relapse ALL was characterized by a single diagnostic clone seeding relapse by a clonal sweep along with acquired mutations in chemoresistance-associated genes. To validate and extend these findings, we are currently analyzing 11 additional infant relapse samples with WGS.