Improved Outcome for ALL by Prolonging Therapy for IKZF1 Deletion and Decreasing Therapy for Other Risk Groups.

PURPOSE: The ALL10 protocol improved outcomes for children with ALL by stratifying and adapting therapy into three minimal residual disease-defined risk groups: standard risk, medium risk (MR), and high risk. IKZF1-deleted (IKZF1del) ALL in the largest MR group still showed poor outcome, in line with protocols worldwide, accounting for a high number of overall relapses. ALL10 showed high toxicity in Down syndrome (DS) and excellent outcome in ETV6::RUNX1 ALL. Poor prednisone responders (PPRs) were treated as high risk in ALL10. In ALL11, we prolonged therapy for IKZF1del from 2 to 3 years. We reduced therapy for DS by omitting anthracyclines completely, for ETV6::RUNX1 in intensification, and for PPR by treatment as MR. METHODS: Eight hundred nineteen patients with ALL (age, 1-18 years) were enrolled on ALL11 and stratified as in ALL10. Results were compared with those in ALL10. RESULTS: The five-year overall survival (OS), event-free survival (EFS), cumulative risk of relapse (CIR), and death in complete remission on ALL11 were 94.2% (SE, 0.9%), 89.0% (1.2), 8.2% (1.1), and 2.3% (0.6), respectively. Prolonged maintenance for IKZF1del MR improved 5-year CIR by 2.2-fold (10.8% v 23.4%; P = .035) and EFS (87.1% v 72.3%; P = .019). Landmark analysis at 2 years from diagnosis showed a 2.9-fold reduction of CIR (25.6%-8.8%; P = .008) and EFS improvement (74.4%-91.2%; P = .007). Reduced therapy did not abrogate 5-year outcome for ETV6::RUNX1 (EFS, 98.3%; OS, 99.4%), DS (EFS, 87.0%; OS, 87.0%), and PPR (EFS, 81.1%; OS, 94.9%). CONCLUSION: Children with IKZF1del ALL seem to benefit from prolonged maintenance therapy. Chemotherapy was successfully reduced for patients with ETV6::RUNX1, DS, and PPR ALL. It has to be noted that these results were obtained in a nonrandomized study using a historical control group.

Integrating copy number data of 64 iAMP21 BCP-ALL patients narrows the common region of amplification to 1.57 Mb.

Background and purpose: Intrachromosomal amplification of chromosome 21 (iAMP21) is a rare subtype of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It is unknown how iAMP21 contributes to leukaemia. The currently known commonly amplified region is 5.1 Mb. Methods: We aimed to narrow down the common region of amplification by using high resolution techniques. Array comparative genomic hybridization (aCGH) was used to determine copy number aberrations, Affymetrix U133 Plus2 expression arrays were used to determine gene expression. Genome-wide expression correlations were evaluated using Globaltest. Results: We narrowed down the common region of amplification by combining copy number data from 12 iAMP21 cases with 52 cases from literature. The combined common region of amplification was 1.57 Mb, located from 36.07 to 37.64 Mb (GRCh38). This region is located telomeric from, but not including, RUNX1, which is the locus commonly used to diagnose iAMP21. This narrow region, which falls inside the Down Syndrome critical region, includes 13 genes of which the expression of eight genes was significantly upregulated compared with 143 non-iAMP21 B-other cases. Among these, transcriptional repressor RIPPLY3 (also known as DSCR6) was the highest overexpressed gene (fold change = 4.2, FDR < 0.001) and most strongly correlated (R = 0.58) with iAMP21-related genome-wide expression changes. Discussion: The more precise definition of the common region of amplification could be beneficial in the diagnosis of iAMP21 based on copy number analysis from DNA sequencing or arrays as well as stimulate functional research into the role of the included genes in iAMP21 biology.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program 'iTHER'.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

Molecular Measurable Residual Disease Assessment before Hematopoietic Stem Cell Transplantation in Pediatric Acute Myeloid Leukemia Patients: A Retrospective Study by the I-BFM Study Group.

Hematopoietic stem cell transplantation (HSCT) is a curative post-remission treatment in patients with acute myeloid leukemia (AML), but relapse after transplant is still a challenging event. In recent year, several studies have investigated the molecular minimal residual disease (qPCR-MRD) as a predictor of relapse, but the lack of standardized protocols, cut-offs, and timepoints, especially in the pediatric setting, has prevented its use in several settings, including before HSCT. Here, we propose the first collaborative retrospective I-BFM-AML study assessing qPCR-MRD values in pretransplant bone marrow samples of 112 patients with a diagnosis of AML harboring t(8;21)(q22; q22)RUNX1::RUNX1T1, or inv(16)(p13q22)CBFB::MYH11, or t(9;11)(p21;q23)KMT2A::MLLT3, or FLT3-ITD genetic markers. We calculated an ROC cut-off of 2.1 × 10-4 that revealed significantly increased OS (83.7% versus 57.1%) and EFS (80.2% versus 52.9%) for those patients with lower qPCR-MRD values. Then, we partitioned patients into three qPCR-MRD groups by combining two different thresholds, 2.1 × 10-4 and one lower cut-off of 1 × 10-2, and stratified patients into low-, intermediate-, and high-risk groups. We found that the 5-year OS (83.7%, 68.6%, and 39.2%, respectively) and relapse-free survival (89.2%, 73.9%, and 67.9%, respectively) were significantly different independent of the genetic lesion, conditioning regimen, donor, and stem cell source. These data support the PCR-based approach playing a clinical relevance in AML transplant management.

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients: Association with MRD Status and Patient Outcome.

For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort­hazard ratio (95% confidence interval) of 2.50 (1−9.66); p = 0.05­together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels.

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients' samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse.

INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Minimal residual disease (MRD) detection in acute lymphoblastic leukaemia based on fusion genes and genomic deletions: towards MRD for all.

Minimal residual disease (MRD) diagnostics are implemented in most clinical protocols for patients with acute lymphoblastic leukaemia (ALL) and are mostly performed using rearranged immunoglobulin (IG) and/or T-cell receptor (TR) gene rearrangements as molecular polymerase chain reaction targets. Unfortunately, in 5-10% of patients no or no sensitive IG/TR targets are available, and patients therefore cannot be stratified appropriately. In the present study, we used fusion genes and genomic deletions as alternative MRD targets in these patients, which retrospectively revealed appropriate MDR stratification in 79% of patients with no (sensitive) IG/TR target, and a different risk group stratification in more than half of the cases.

Unravelling the Sequential Interplay of Mutational Mechanisms during Clonal Evolution in Relapsed Pediatric Acute Lymphoblastic Leukemia.

Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is characterized by clonal heterogeneity. Genomic mutations can increase proliferative potential of leukemic cells and cause treatment resistance. However, mechanisms driving mutagenesis and clonal diversification in ALL are not fully understood. In this proof of principle study, we performed whole genome sequencing of two cases with multiple relapses in order to investigate whether groups of mutations separated in time show distinct mutational signatures. Based on mutation allele frequencies at diagnosis and subsequent relapses, we clustered mutations into groups and performed cluster-specific mutational profile analysis and de novo signature extraction. In patient 1, who experienced two relapses, the analysis unraveled a continuous interplay of aberrant activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity. The associated signatures SBS2 and SBS13 were present already at diagnosis, and although emerging mutations were lost in later relapses, the process remained active throughout disease evolution. Patient 2 had three relapses. We identified episodic mutational processes at diagnosis and first relapse leading to mutations resembling ultraviolet light-driven DNA damage, and thiopurine-associated damage at first relapse. In conclusion, our data shows that investigation of mutational processes in clusters separated in time may aid in understanding the mutational mechanisms and discovery of underlying causes.

Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.

Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation.

Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

AML Subtype Is a Major Determinant of the Association between Prognostic Gene Expression Signatures and Their Clinical Significance.

Relapse in acute myeloid leukemia (AML) may result from variable genetic origins or convergence on common biological processes. Exploiting the specificity and sensitivity of regulatory DNA, we analyze patient samples of multiple clinical outcomes covering various AML molecular subtypes. We uncover regulatory variation among patients translating into a transcriptional signature that predicts relapse risk. In addition, we find clusters of coexpressed genes within this signature selectively link to relapse risk in distinct patient subgroups defined by molecular subtype or AML maturation. Analyzing these gene clusters and the AML subtypes separately enhances their prognostic value substantially and provides insight in the mechanisms underlying relapse risk across the distinct patient subgroups. We propose that prognostic gene expression signatures in AML are valid only within patient subgroups and do not transcend these subgroups.

Epigenetic drug screen identifies the histone deacetylase inhibitor NSC3852 as a potential novel drug for the treatment of pediatric acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment-related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed. PROCEDURE: To identify potential novel therapeutic targets for myeloid disorders in children, including DS-AMKL and non-DS-AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre-)leukemic pediatric patient cells. RESULTS: Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan-histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation. CONCLUSION: NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS-AMKL.

Prognostic impact of t(16;21)(p11;q22) and t(16;21)(q24;q22) in pediatric AML: a retrospective study by the I-BFM Study Group.

To study the prognostic relevance of rare genetic aberrations in acute myeloid leukemia (AML), such as t(16;21), international collaboration is required. Two different types of t(16;21) translocations can be distinguished: t(16;21)(p11;q22), resulting in the FUS-ERG fusion gene; and t(16;21)(q24;q22), resulting in RUNX1-core binding factor (CBFA2T3). We collected data on clinical and biological characteristics of 54 pediatric AML cases with t(16;21) rearrangements from 14 international collaborative study groups participating in the international Berlin-Frankfurt-Münster (I-BFM) AML study group. The AML-BFM cohort diagnosed between 1997 and 2013 was used as a reference cohort. RUNX1-CBFA2T3 (n = 23) had significantly lower median white blood cell count (12.5 × 109/L, P = .03) compared with the reference cohort. FUS-ERG rearranged AML (n = 31) had no predominant French-American-British (FAB) type, whereas 76% of RUNX1-CBFA2T3 had an M1/M2 FAB type (M1, M2), significantly different from the reference cohort (P = .004). Four-year event-free survival (EFS) of patients with FUS-ERG was 7% (standard error [SE] = 5%), significantly lower compared with the reference cohort (51%, SE = 1%, P < .001). Four-year EFS of RUNX1-CBFA2T3 was 77% (SE = 8%, P = .06), significantly higher compared with the reference cohort. Cumulative incidence of relapse was 74% (SE = 8%) in FUS-ERG, 0% (SE = 0%) in RUNX1-CBFA2T3, compared with 32% (SE = 1%) in the reference cohort (P < .001). Multivariate analysis identified both FUS-ERG and RUNX1-CBFA2T3 as independent risk factors with hazard ratios of 1.9 (P < .0001) and 0.3 (P = .025), respectively. These results describe 2 clinically relevant distinct subtypes of pediatric AML. Similarly to other core-binding factor AMLs, patients with RUNX1-CBFA2T3 rearranged AML may benefit from stratification in the standard risk treatment, whereas patients with FUS-ERG rearranged AML should be considered high-risk.