Childhood Adult Acute Myeloid Leukemia Research Articles

DNMT3A Mutants Are Enriched in NPMc+ AML and Associated with Adverse Outcome in Childhood AML

Background: Somatic mutations in the DNA methyltransferase 3 alphagene (DNMT3A) are common in adult acute myeloid leukemia (AML), but are rare events in childhood AML (Ho, Ped Blood Cancer, 2011). Missense mutations occur most frequently in the catalytic domain at R882, resulting in loss of enzyme methylation activity and the ability of the enzyme to bind DNA. DNMT3Amutations are associated with adverse outcome in adults, while their impact is understudied in childhood AML. Our objective was to identify the prevalence of DNMT3A -mutated AML across the childhood age spectrum utilizing a large cohort of patients enrolled on pediatric trials and compare findings with adult cohorts. Methods: Pediatric patients treated on two consecutive COG trials with publicly available data were included (n=1607). Mutational profiling was performed by whole genome and transcriptome sequencing methods. Prevalence of DNMT3Amutations, co-occurring mutations, and outcomes (5-year event-free [EFS] and 5-year overall survival [OS]) were analyzed. Results: The prevalence of DNMT3Amutations amongst the entire childhood cohort was 1.2% (n=20/1607). DNMT3A mutations were absent in children <10 years with increased incidence up to 2.6% in patients >10yrs of age. All variants were missense, with the exception of one nonsense mutation and alterations at the 882 residue were the most common (70%). Most importantly, all but 4 DNMT3A mutations occurred in patients with NPM1 mutations (NPMc+; 80%). Conversely, DNMT3A mutations were seen in 9% of all NPM positive patients, demonstrating unique enrichment of this mutation within the NPM1 mutant cohort. Further, R882 mutations were seen in all but a single patient with the dual NPM/DNMT3A variant. It is notable that 11 patients harbored additional FLT3-ITD mutations (triple positive), which we have previously reported to be associated with adverse outcome in adults. In addition to mutations in NPM1 and FLT3, co-occurring variants with DNMT3A were also found in PTPN11 (35%), and WT1, IDH1/2 and MYC each at 10% frequency (Fig.A). Given the recent report of association of NPM1 mutation genotype and association of Type D mutation with adverse outcome (Pigazzi, ASH 2023), we evaluated NPM1 mutation genotype in those with cooperating DNMT3A mutation which demonstrated lack of association of DNMT3A mutations and Type D NPM1 mutations. Current risk classification in COG pediatric AML trials treat NPMc+ patients as favorable risk. Given the prevalence of DNMT3A mutations in NPMc+ patients we inquired whether DNMT3A mutations might modify outcome in this population. The 5 yr EFS estimates for NPMc+; patients with and without DNMT3A was 26.8% vs. 74.8% (p<0.0001; Fig.B) with a corresponding OS estimate at 5yrs of 60.3% vs. 82.2% (p=0.0169). We further concluded that presence or absence of FLT3-ITD does not impact the adverse outcome with similar 5 yr EFS for those with dual NPM1/DNMT3A or triple NPM1/DNMT3A/FLT3-ITD positive AML(20.0% vs. 30.3%respectively). These data are consistent with the adult DNMT3A data previously presented last year (Torabi; ASH 2022; abstract #306) regarding clinical implications of DNMT3A mutation in the setting of cooperating NPM1 mutation. Conclusion: Mutational profiling of children with AML identified DNMT3A mutations in adolescents and young adults and is enriched in those with NPMc+ mutations. As NPMc+ patients are considered to be favorable risk, knowledge of DNMT3A mutations would provide additional information for more precise risk stratification in childhood AML. Children with de novo AML and dual DNMT3A/NPM1 mutations remain at high risk of relapse and adverse outcome regardless of FLT3-ITD status.

CD123 Expression Is Associated With High-Risk Disease Characteristics in Childhood Acute Myeloid Leukemia: A Report From the Children's Oncology Group.

Increased CD123 surface expression has been associated with high-risk disease characteristics in adult acute myeloid leukemia (AML), but has not been well-characterized in childhood AML. In this study, we defined CD123 expression and associated clinical characteristics in a uniformly treated cohort of pediatric patients with newly diagnosed AML enrolled on the Children's Oncology Group AAML1031 phase III trial (NCT01371981). AML blasts within diagnostic bone marrow specimens (n = 1,040) were prospectively analyzed for CD123 protein expression by multidimensional flow cytometry immunophenotyping at a central clinical laboratory. Patients were stratified as low-risk or high-risk on the basis of (1) leukemia-associated cytogenetic and molecular alterations and (2) end-of-induction measurable residual disease levels. The study population was divided into CD123 expression-based quartiles (n = 260 each) for analysis. Those with highest CD123 expression (quartile 4 [Q4]) had higher prevalence of high-risk KMT2A rearrangements and FLT3-ITD mutations (P < .001 for both) and lower prevalence of low-risk t(8;21), inv(16), and CEBPA mutations (P < .001 for all). Patients in lower CD123 expression quartiles (Q1-3) had similar relapse risk, event-free survival, and overall survival. Conversely, Q4 patients had a significantly higher relapse risk (53% v 39%, P < .001), lower event-free survival (49% v 69%, P < .001), and lower overall survival (32% v 50%, P < .001) in comparison with Q1-3 patients. CD123 maintained independent significance for outcomes when all known contemporary high-risk cytogenetic and molecular markers were incorporated into multivariable Cox regression analysis. CD123 is strongly associated with disease-relevant cytogenetic and molecular alterations in childhood AML. CD123 is a critical biomarker and promising immunotherapeutic target for children with relapsed or refractory AML, given its prevalent expression and enrichment in patients with high-risk genetic alterations and inferior clinical outcomes with conventional therapy.

FLT3 ITD and NUP98 translocations Cooperate to Induce Type I Interferon Signaling That Impedes Hematopoietic Progenitor Differentiation

FLT3-Internal Tandem Duplication (FLT3 ITD) mutations occur at similar frequencies in both childhood and adult acute myeloid leukemia (AML) patients, but they cooperate with different initiating mutations at different ages. In childhood AML, FLT3 ITD often cooperates with translocations that generate fusion proteins, such as NUP98 translocations (NUP98-t). In adult AML, FLT3 ITD often cooperates with mutations that inactivate RUNX1, DNMT3A, TET2 and other genes that regulate self-renewal and myeloid differentiation. This raises the question of whether FLT3 ITD induces similar, or distinct, changes in gene expression and cell fate when it arises in the setting of pediatric and adult driver mutations. To answer this question, we evaluated pre-leukemic changes in hematopoiesis and gene expression in neonatal and juvenile mice that carried pediatric-like FLT3 ITD/NUP98-HOXD13 and adult-like FLT3 ITD/Runx1 DEL/+ mutation profiles. Both NUP98-HOXD13 (NHD13) and Runx1 DEL/+ severely depleted phenotypic hematopoietic stem cells (HSCs) when coupled with FLT3 ITD. However, FLT3 ITD/NHD13 cooperated to enhancer ectopic self-renewal capacity in lineage committed hematopoietic progenitor cells (HPCs) whereas FLT3 ITD/Runx1 DEL/+ did not. FLT3 ITD/NHD13 and FLT3 ITD/Runx1 DEL/+ both induced changes in gene expression and enhancer activation that were not seen with either mutation alone. These cooperative changes were almost entirely non-overlapping. The most notable cooperative change in FLT3 ITD/NHD13 HPCs was activation of a type I Interferon target gene signature. By contrast, this signature was actively repressed in FLT3 ITD/Runx1 DEL/+ HPCs due to mir155 activation. Thus, FLT3 ITD/NHD13 and FLT3 ITD/Runx1 DEL/+ mutations have largely non-overlapping, cooperative effects on hematopoietic cell fate and gene expression.Interferon signaling sustained the FLT3 ITD/NHD13 HPC population by impeding differentiation into more committed myeloid progenitors. When we deleted the type I interferon receptor (Ifnar) on a FLT3 ITD/NHD13 background, the HPC population was virtually eliminated, yet the pre-granulocyte monocyte (pGM) and granulocyte-monocyte progenitor (GMP) populations expanded. HPC proliferation and death did not change in the absence of Ifnar. These findings suggest that type I interferon impedes HPC differentiation into pGMs during the neonatal and juvenile stages of life. Of note, ectopic interferon was insufficient to enhancer HPC numbers in setting of a sole NHD13 mutation. Thus, interferon is necessary to sustain FLT3 ITD/NHD13 HPCs, but not sufficient, and other signaling pathways downstream of FLT3 ITD must cooperate with NHD13 in parallel to drive HPC expansion. To our surprise, Ifnar deletion did not reduce repopulating activity in FLT3 ITD/NHD13 bone marrow - as measured by limiting dilution transplants - it simply shifted repopulating activity to more committed pGMs. Our raise two important conceptual points. First, we show that different initiating mutations can elicit completely different transcriptional responses downstream of FLT3 ITD. Precision medicine strategies must account for these differences as effective therapies for adult FLT3 ITD AML may not yield similar success in the pediatric population. Second, type I interferon shapes pre-leukemic hematopoiesis in the setting of FLT3 ITD/NUP98-t mutations. In doing so, the mutant cells may hijack a program that we recently identified as promoting normal perinatal HPC expansion. DisclosuresNo relevant conflicts of interest to declare.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

High EVI1 Expression Predict Adverse Outcome in Children with Acute Myeloid Leukemia: A Multicenter, Nonrandomized Clinical Study in China

Backgroud and purpose In clinical studies in adult acute myeloid leukemia (AML), high ecotropic virus integration-1 (EVI1) gene expression has shown to be an independent prognostic factor. However, the predictive value of high EVI1 expression (EVI1+) in children with AML is little known. The purpose of this study was to investigate frequency and prognostic significance of high EVI1 expression in children with AML. Patients and methods Total of 403 newly diagnosed childhood AML patients from 7 centers of south of China from November 2014 to May 2020 were included in the study. A diagnostic assay detecting multiple EVI1 splice variants was developed to determine the relative EVI1 expression by single real-time quantitative polymerase chain reaction in the 403 newly diagnosed pediatirc patients with AML younger than 14 years. Patients were treated with C-HUANAN-AML15 protocol. In the C-HUANAN-AML15 protocol, tandem 2 courses of FLAG-IDA regimen, or sequential DAE(3+5+10) and DAE(3+5+8) were applied as induction chemotherapy. One course of Homoharringtonine (substitution of Amsacrine in MRC-AML 15 protocol)/ Cytarabine/ Etoposide and one course of Mitoxantrone/ Cytarabine in consolidation chemotherapy were uniform in both groups. 75 patients selected hematopoietic stem cell transplantation (HSCT) in first CR. The median follow-up period was 20 (0.8 to 67.7) months up to July 2020. The similarity of clinical data was analyzed by the chi square test and COX proportional hazard function model. Complete remission (CR) rates, event-free survival (EFS) and overall survival (OS) were compared by Log-Rank chi square test. Results EVI1+ was found in 36/403 (8.9%) of children with de novo AML. The clinical features of the patients with EVI1+(n=36) compared with EVI1-(n=367) AML were summarized in Table 1. No significant differences in sex, white blood cell count, and age were noted between EVI1+and EVI1- AML. When studying the relationship between EVI1+ and conventional classification criteria such as morphology (FAB classification) and cytogenetic data, EVI1+ was predominantly found in FAB M7, t(9;11) and other t(v;11q23), as well as complex karyotype, but mutually exclusive with t(8;21), inv(16)/t(16;16), CEBPA, NPM1 or C-KIT mutations. Furthermore, EVI1+ was significantly associated with unfavoralbe cytogenetic risk. Twenty-five patients (22 cases in the EVI1-group and 3 cases in the EVI1+ group) who gave up treatment or transferred to other hospital when treatment was not completed were excluded, and the remaining 378 AML children with standard treatment were included in the survival analysis. Univariate analysis showed that initial white blood cell count (≥50×109/L), RUNX1-RUNX1T1 fusion gene, FLT3-ITD mutation and EVI1+ affected 5-year EFS (P=0.000, 0.001, 0.016, 0.011). Multivariate prognostic analysis with COX proportional hazard function model for EFS identified EVI1+ as an independent prognosis factor (HR0.531, 95%CI 0.298-0.945, P=0.032). Patients with EVI1+ AML (n=12) who received HSCT in first CR had better 5-year EFS, but the difference was not statistically significant (46.103% vs. 56.250%, P=0.33). Conclusion It could be concluded that EVI1+ can be found in ~10% of pediatric AML in China. It is predominantly found in intermediate to unfavorable cytogenetic subtypes and predicts adverse outcome. EVI1 screening at diagnosis should be included in risk stratification of pediatric AML. Whether pediatric patients with EVI1+ AML can benefit from HSCT in first CR needs further reasearch. Disclosures No relevant conflicts of interest to declare.

DNMT3A Mutation inChinese Childhood Acute Myeloid Leukemia

PurposeDNMT3A mutation has been found in approximately 20% of adult acute myeloid leukemia (AML) patients and in 0~1.4% of children with AML, and the hotspots of mutations are mainly located in the catalytic methyltransferase domain, thereinto, mutation R882 accounts for 60%. Although the prognosis of adult patients with DNMT3A mutations is dismal, the prognosis of DNMT3A mutation in childhood AML is still unclear. Here, we tried to determine the incidence and prognostic significance of DNMT3A mutations in Chinese childhood AML.Methods We detected the mutations in DNMT3A exon 23 by PCR and direct sequencing in 342 children with AML (0~16 years old) from January in 2005 to June in 2013, treated on BCH-2003 AML protocol. The correlation of DNMT3A mutations with clinical characteristics, fusion genes, other molecular anomalies (FLT3-ITD, NPM1, C-KIT and WT1 mutations), and treatment outcome were analyzed.ResultsDNMT3A mutations were detected in 4 out of 342 (1.2%) patients. The mutations in coding sequences included S892S, V912A, R885G, and Q886R. Furthermore, there were two intronic mutations (c.2598-15C>T and c.2739+55A>C) found in one patient. No association of DNMT3A mutations with common clinical features was found. Two patients with DNMT3A mutations died of relapse or complications during treatment, one patient gave up treatment due to introduction failure in day33. Only one patient achieved continuous complete remission.Conclusions DNMT3A mutations were rare in Chinese children with AML. The mutation positions were different from the hotspots reported in adult AML. DNMT3A mutations may have adverse impact on prognosis of children with AML. DisclosuresNo relevant conflicts of interest to declare.

Molecular Profiling of High-Risk Pediatric Acute Myeloid Leukemia

Background: Several landmark genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of adult acute myeloid leukemia (AML) and directly impacted clinical care. However, very little is known about the mutational landscape of pediatric AML, a distinct entity that shares few genetic and clinical characteristics with adult AML. To investigate potential drivers of high-risk pediatric AML, comprehensive genomic profiling was performed on high-risk AML samples as part of a prospective clinical next-generation sequencing program.Methods:Samples obtained from patients with known high-risk features at diagnosis or with refractory or relapsed AML were selected for molecular profiling. Comprehensive testing included whole-exome sequencing (WES) of matched tumor (bone marrow or chloroma tissue) and normal tissue (peripheral blood or buccal swab) samples and transcriptome analysis (RNAseq). Targeted sequencing of 467 cancer-associated genes was used when tumor tissue was limited. Sequencing was performed on Illumina's HiSeq 2500 with 150X and 500X average coverage for WES and targeted sequencing, respectively. Variants were filtered to select alterations in cancer-related genes or genes relevant for patient care.Results:Fifteen patients with AML (mean age 7.7 yrs; range 0.75-19 yrs) met high-risk criteria (high-risk features at diagnosis = 4, relapsed disease = 8, refractory disease = 3) and were selected for profiling. WES and RNAseq were performed on 11 samples, WES only on 3 samples and targeted DNA sequencing on 1 sample. The median number of variants was 60 (range 14- 5950) per case. After filtering, 54 mutations were identified in 35 genes with a mean of 3.6 mutated genes per patient sample (range 0-14); two samples only carried a fusion gene with no other genetic alterations. At least one driver genetic alteration was detected in each patient sample. Thirteen samples carried mutations in at least one gene known to be altered in AML (e.g. IDH1, WT1, TP53, NRAS) (mean, 2; range, 1-6) and 5 samples carried novel mutations in 15 genes not previously implicated in AML (e.g. CARD9, CHD9, Axin1). Mutations in 11 AML related genes were detected in more than one sample including NRAS in 4, TP53 in 3 and KRAS, PTPN11 PHF6, JAK3 in 2 samples each; genes not previously implicated in AML were only mutated in single patients. Of note, mutations in genes encoding members of the RAS pathway occurred in 60% of cases (9/15 samples). RNAseq identified gene fusions in 7/11 samples (63%). Four fusions involving KMT2A and core binding factor genes were also detected by FISH while three fusions were detected by RNAseq only: NUP98-NSD1 in two patients and CBFA2T3-GLIS2 in one patient. Samples carrying driver gene fusions had the lowest number of mutated genes (0-1) compared to samples lacking a gene fusion (1-5 mutated genes), with one exception of a patient with history of infant ALL who later developed KMT2A-AFF1AML with the highest number of mutated genes (n=14). There was no correlation between the number of mutated genes and age, clinical characteristics, initial risk classification at diagnosis or intensity of therapy prior to sequencing.Conclusion:Our study provides an initial overview of the genetic alterations that characterize high-risk, chemo-resistant pediatric AML. Analysis of the data highlights the overall low genetic complexity of high-risk AML despite the aggressive clinical behavior and exposure to intense chemotherapy, including stem cell transplant. Of interest, similar to adult AML, we found that mutations leading to aberrant activation of the RAS pathway were also very frequent in our cohort of pediatric high-risk AML, while genes typically mutated early in the process of leukemogenesis in adult AML, such as NPM1, DNMT3A, FLT3, IDH1, IDH2 were not affected. Such findings suggest that distinct, age-specific mechanisms of leukemogenesis might exist. Furthermore, our data also highlights the important role of RNA sequencing in complementing current standard diagnostic tools, allowing the identification of driver fusion genes in samples for which no other driver event is detected. Larger studies, preferably including diagnostic samples and utilizing broader approaches, are needed to better understand the mechanisms responsible for the initiation and progression of childhood AML. DisclosuresNo relevant conflicts of interest to declare.

Fetal and neonatal hematopoietic progenitors are functionally and transcriptionally resistant to Flt3-ITD mutations.

The FLT3 Internal Tandem Duplication (FLT3ITD) mutation is common in adult acute myeloid leukemia (AML) but rare in early childhood AML. It is not clear why this difference occurs. Here we show that Flt3ITD and cooperating Flt3ITD/Runx1 mutations cause hematopoietic stem cell depletion and myeloid progenitor expansion during adult but not fetal stages of murine development. In adult progenitors, FLT3ITD simultaneously induces self-renewal and myeloid commitment programs via STAT5-dependent and STAT5-independent mechanisms, respectively. While FLT3ITD can activate STAT5 signal transduction prior to birth, this signaling does not alter gene expression until hematopoietic progenitors transition from fetal to adult transcriptional states. Cooperative interactions between Flt3ITD and Runx1 mutations are also blunted in fetal/neonatal progenitors. Fetal/neonatal progenitors may therefore be protected from leukemic transformation because they are not competent to express FLT3ITD target genes. Changes in the transcriptional states of developing hematopoietic progenitors may generally shape the mutation spectra of human leukemias.

Monosomal karyotype is not a predictor of dismal outcome in childhood de novo acute myeloid leukemia

Monosomal karyotype (MK) is known as a far end of the unfavorable cytogenetics in adult acute myeloid leukemia (AML), while available data in childhood AML is scarce. In this study, we investigated the prevalence and prognostic value of MK with retrospectively analyzed 119 patients newly diagnosed with childhood de novo AML. Ten patients (8.4%) revealed to have MK. All MK-positive (MK(+)) AML were associated with complex cytogenetic abnormalities and belonged to the cytogenetic adverse-risk group. Nine of MK(+) patients (90%) achieved complete remission. The event-free survival (EFS) and overall survival (OS) of MK(+) adverse group were comparable to the ESF and OS of MK-negative non-adverse group (EFS 60.0±15.5% vs 59.0±5.1%, P=0.925; OS 70.0±14.5% vs 58.1±5.3%, P=0.696). In multivariate analysis, MK was not an independent adverse prognostic factor for EFS (hazard ratio 0.45, 95% C.I. 0.13–1.50, P=0.194). In addition, 7 of 9 MK(+) patients who received allogeneic hematopoietic stem cell transplantation (HSCT) survived event-free, with a median follow-up of 64 months. In conclusion, MK did not act as an adverse prognostic factor in childhood de novo AML. Allogeneic HSCT might have contributed to the excellent outcome of MK(+) childhood de novo AML.

CSF3R Mutations Represent a Novel Therapeutic Target in Pediatric AML with a High Degree of Overlap with CEBPA Mutations: a Report from COG AAML0531 and COG/NCI Target AML Initiative

Activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) are present in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) Despite the high frequency of these mutations in CNL, they are quite rare in adult acute myeloid leukemia (AML), in which only a single CSF3R mutated case was found in the TCGA AML analysis (0.5%). We have previously demonstrated significant variation in genomic variants between pediatric and adult malignancies, thus prevalence of genomic variants identified in adults need be fully characterized in children. As part of COG/NCI TARGET AML initiative we interrogated the genomic makeup of 200 cases of childhood AML (discovery cohort) using whole genome sequencing and performed subsequent frequency determination of the variants in 787 unselected cases from COG AAML0531 (validation cohort). Somatic variants in CSF3R were initially found to be recurrent in the discovery cohort and underwent frequency determination in the validation cohort to establish their prevalence and correlations with clinical characteristics and outcome.Frequency determination of CSF3R mutation in 787 pediatric patients with available CSF3R data from AAML0531 identified 16 distinct CSF3R mutations in 28 patients (3.6%). Somatic mutations in CSF3R identified in pediatric AML included known oncogenic variants mutations such as T618I and T615A, previously identified in adult CNL studies as well as novel truncations of the CSF3R cytoplasmic domain (Q749X, Y767fs, Y787X and P819/820fs), and missense mutations (E149D, A208V, R223Q, E405K, A431V, and Q516K). Interestingly, although CSF3R truncations usually occur along with a T618I or T615A mutation in CNL/aCML, these two mutation categories were mutually exclusive in pediatric AML.Initial correlation of all CSF3R variants with demographic and clinical/laboratory parameters determined that CSF3R variants were less prevalent in younger patients (age 0-2, p=0.039), with significantly higher association with t(8;21) (32% vs. 14%, p=0.012) and CEBPA mutations (35% vs. 5%, p<0.001). Cumulatively, 18/28 patients with CSF3R mutations (64%) had either CBF translocations or CEBPA mutations and as a result, CSF3R mutation was highly associated with favorable risk (p=0.02) and inversely associated with standard risk disease (p=0.007). Actuarial overall survival at 5 years for patients with and without CSF3R mutations was 91% vs. 64%, respectively (p<0.001). In order to determine the oncogenic potential of the newly discovered variants, all untested variants (N=12) were cloned and expressed in Ba/F3 cells in order to determine whether these variants can bestow cytokine independence to these cells. Of the 16 total CSF3R variants identified, 8 variants present in 18 patients exhibit oncogenic capacity (T615A, T618I, T640N, Q749X, Y767fs, S783fs, Y787X and F819fs). All of the novel variants that exhibited oncogenic potential were truncating mutations.Compared to non-mutated cases, transforming CSF3R variants had a significant association with CEBPA mutations (44% vs. 5%, p<0.001), and led to significant association of CSF3R with favorable risk disease (67% vs. 39%, p=0.019). Actuarial overall survival at 5 years for those with and without transforming CSF3R mutations was 87% vs. 64%, (p=0.047).CSF3R mutations define a distinct molecular subset of pediatric AML, which could be therapeutically targeted in the future using kinase inhibitors such as ruxolitinib. The oncogenic CSF3R mutations found in pediatric AML are either the same point mutations or similar truncation mutations as seen in CNL, suggesting that other cooperating genomic alterations may be important in directing these distinct diseases. Interestingly, we found that the majority of pediatric AML patients with CSF3R mutation have either a core binding factor alteration (such as t(8;21)) or a mutation in CEBPA. The enrichment of CEBPA mutations with CSF3R mutations is particularly striking, as CEBPA mutations are ~9 fold more frequent in patients with transforming CSF3R mutations than those without. Understanding the role of cooperating genomic alteration in CSF3R-driven myeloid malignancies will be the subject of future work.The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. DisclosuresRadich:Novartis: Consultancy, Research Funding; Incyte: Consultancy; Gilliad: Consultancy; Ariad: Consultancy.

Prevalence and prognostic value of IDH1 and IDH2 mutations in childhood AML: a study of the AML–BFM and DCOG study groups

Mutations in the NADP(+)-dependent isocitrate dehydrogenase genes 1 and 2 (IDH1 and IDH2) have recently been found in adult acute myeloid leukemia (AML) patients with a prevalence rising up to 33%. To investigate the frequency of IDH1/2 mutations in pediatric AML, we characterized the mutational hotspot (exon 4) of these genes in diagnostic samples from 460 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 R132 n=8; IDH2 R140 n=10) and the minor allele of single-nucleotide polymorphism (SNP) rs11554137 in 47 children (10.2%). IDH mutations were associated with an intermediate age (P=0.008), FAB M1/M2 (P=0.013) and nucleophosmin1 mutations (P=0.001). In univariate analysis, IDH(mutated) compared with IDH(wildtype) patients showed a significantly improved overall survival (OS; P=0.032) but not event-free survival (EFS; P=0.14). However, multivariate analysis did not show independent prognostic significance. Children with at least one minor allele of IDH1 SNP rs11554137 had similar EFS (P=0.27) and OS (P=0.62) compared with major allele patients. Gene expression profiles of 12 IDH(mutated) were compared with 201 IDH(wildtype) patients to identify differentially expressed genes and pathways. Although only a small number of discriminating genes were identified, analysis revealed a deregulated tryptophan metabolism, and a significant downregulation of KYNU expression in IDH(mutated) cases.

RUNX3 Expression Is an Independent Prognostic Factor in Cytogenetically Abnormal Adult Acute Myeloid Leukemia (AML) Patients with Wild-Type FLT3.

AbstractAbstract 1662▪RUNX3 is a member of Runt-related domain (RUNX) transcription factor family, which regulates cell proliferation and differentiation. Recent studies have suggested a role of RUNX3 in hematopoiesis. Our group previously showed that RUNX3 expression was repressed by fusion protein RUNX1-ETO and CBFβ-MYH11 in core-binding factor acute myeloid leukemia (CBF-AML) and had prognostic implication in childhood AML patients (Cheng et al, 2008). However, the prognostic value of RUNX3 in adult AML remains hitherto unaddressed. To investigate the prognostic value of RUNX3 expression in adult AML patients, we measured RUNX3 mRNA levels in the diagnostic bone marrow samples from 60 adult patients with de novo AML by real time quantitative PCR (RQ-PCR). Mutation status of KIT, FMS-like tyrosine kinase 3 (FLT3), nucleophosmin (NPM1) and CCAAT/enhancer-binding protein α (CEBPA) were assessed by direct sequencing. The adult AML patients (n=60) were dichotomized at median RUNX3 level and divided into high and low expression groups. There was no significant difference in age, sex, white blood cell (WBC) count, frequency of KIT, FLT3, NPM1 and CEBPA mutations between the two groups. Concordant with our previous observation, the low RUNX3 expression group was significantly associated with CBF-AML characterized by t(8;21) and inv(16) (p=0.002). However, in contrast to our previous findings in childhood AML, we observed significant association of survival with RUNX3 levels only when patients were stratified according to their cytogenetics and FLT3 mutation status but not when the whole patient cohort was analyzed or when the patients were stratified by age, sex, WBC count or other genetic aberrations (KIT, NPM1 or CEBPA mutation). In each cytogenetically normal and abnormal subgroup, AML patients were dichotomized at median RUNX3 into high and low expression groups. Among adult AML patients with abnormal karyotypes (n=36), high RUNX3 expression tended to have worse event-free survival (EFS) (high vs low= 50% vs 67%, p=0.213) and overall survival (OS) (61% vs 78%, p=0.233). Because potential relationship between FLT3 mutation status and RUNX3 expression was suggested in the study by Lacayo et al (2004), analysis was performed with further stratification according to the FLT3 status. Among cytogenetically abnormal patients with wild-type FLT3, high RUNX3 expression was significantly associated with poorer OS (53% vs 80%, p=0.048) and tended to have worse EFS (53% vs 73%, p=0.077). In multivariate analysis, RUNX3 expression was an independent prognostic factor for both EFS (p=0.026) and OS (p=0.021) along with age (p=0.013 and 0.004 respectively) and WBC count (p<0.0005 and 0.001 respectively) in this patient subgroup. The group of patients with mutant FLT3 was too small for analysis. In contrast to the finding in cytogenetically abnormal patients, there was a trend towards poorer EFS (high vs low = 50% vs 20%, p=0.066) and OS (50% vs 30%, p=0.118) for low RUNX3 expression in adult AML patients with a normal karyotype (n=20). However, no significant association was observed after stratification with FLT3 status. In conclusion, our findings suggested that unlike in childhood AML patients, the prognostic impact of RUNX3 in adult AML patients was dependent on cytogenetic status and that high RUNX3 expression was an independent adverse prognostic factor in cytogenetically abnormal adult AML patients with wild-type FLT3. A larger prospective study is currently underway to confirm the prognostic value of RUNX3 in adult AML. Disclosures:No relevant conflicts of interest to declare.

Incidence of FLT3 and nucleophosmin gene mutations in childhood acute myeloid leukemia: Serbian experience and the review of the literature

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene (internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain, FLT3/D835) as well as the nucleophosmin (NPM1) gene are the most common abnormalities in adult acute myeloid leukemia (AML). Their significance in pediatric AML is still unclear. In this study we evaluated the frequency of FLT3 and NPM1 mutations in childhood AML. We also examined clinical features and outcome of these patients. FLT3 and NPM1 mutations were analysed in 42 and 37 childhood AML patients, respectively, using polymerase chain reaction (PCR) and direct sequencing. FLT3 mutations were detected in 4/42 patients (9.5%). The frequencies of FLT3/ITD and FLT3/D835 were the same, 2/42 (4.7%). NMP1 mutations were found in 1/37 patients (2.7%). FLT3 gene mutations were correlated with induction failure. Here we report the results of the study of FLT3 and NPM1 gene mutations in childhood AML patients in Serbia. Low frequencies of these molecular markers point out that these abnormalities are rare in this cohort of patients. Comparative study of data on NPM1 mutations in childhood AML revealed that various NPM1 gene mutation types are associated with childhood AML. Our findings as well as previously reported data, contributes to a hypothesis of different biology and etiology of adult and childhood AML. More extensive studies of NPM1 and FLT3 mutations in childhood AML are needed to determine their biological and clinical importance.

Clinical relevance of Wilms tumor 1 gene mutations in childhood acute myeloid leukemia

AbstractWilms tumor 1 (WT1) mutations have recently been identified in approximately 10% of adult acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) and are associated with poor outcome. Using array-based comparative genome hybridization in pediatric CN-AML samples, we detected a WT1 deletion in one sample. The other WT1 allele was mutated. This prompted us to further investigate the role of WT1 aberrations in childhood AML. Mutations were found in 35 of 298 (12%) diagnostic pediatric AML samples. In 19 of 35 (54%) samples, more than one WT1 aberration was found: 15 samples had 2 different mutations, 2 had a homozygous mutation, and 2 had a mutation plus a WT1 deletion. WT1 mutations clustered significantly in the CN-AML subgroup (22%; P < .001) and were associated with FLT3/ITD (43 vs 17%; P < .001). WT1 mutations conferred an independent poor prognostic significance (WT1 mutated vs wild-type patients: 5-year probability of overall survival [pOS] 35% vs 66%, P = .002; probability of event-free survival 22% vs 46%, P < .001; and cumulative incidence of relapse or regression 70% vs 44%, P < .001). Patients with both a WT1 mutation and a FLT3/ITD had a dismal prognosis (5-year pOS 21%). WT1 mutations occur at a significant rate in childhood AML and are a novel independent poor prognostic marker.

Twenty years of follow‐up among survivors of childhood and young adult acute myeloid leukemia

Limited data exist on the comprehensive assessment of late medical and social effects experienced by survivors of childhood and young adult acute myeloid leukemia (AML). This analysis included 272 5-year AML survivors who participated in the Childhood Cancer Survivor Study (CCSS). All patients were diagnosed at age < or =21 years between the years 1970 and 1986, and none underwent stem cell transplantation. Rates of survival, relapse, and late outcomes were analyzed. The average follow-up was 20.5 years (range, 5-33 years). The overall survival rate was 97% at 10 years (95% confidence interval [95%CI], 94%-98%) and 94% at 20 years (95% CI, 90%-96%). Six survivors reported 8 recurrences. The cumulative incidence of recurrent AML was 6.6% at 10 years (95% CI, 3.7%-9.6%) and 8.6% at 20 years (95% CI, 5.1%-12.1%). Ten subsequent malignant neoplasms (SMN) were reported, including 4 with a history of radiation therapy, for a 20-year cumulative incidence of 1.7% (95% CI, 0.02%-3.4%). Six cardiac events were reported, for a 20-year cumulative incidence 4.7% (95% CI, 2.1%-7.3%). Half of the survivors reported a chronic medical condition and, compared with siblings, were at increased risk for severe or life-threatening chronic medical conditions (16% vs 5.8%; P < .001). Among those aged > or =25 years, the age-adjusted marriage rates were similar among survivors and the general United States population (57% for both) and lower compared with siblings (67%; P < .01). Survivors' college graduation rates were lower compared with siblings but higher than the general population (40% vs 52% vs 34%, respectively; P < .01). Employment rates were similar between survivors, siblings, and the general population (93%, 97.6%, and 95.8%, respectively). Long-term survival from childhood AML > or =5-years after diagnosis was favorable. Late-occurring medical events remained a concern with socioeconomic achievement lower than expected within the individual family unit, although it was not different from the general United States population.

Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: A study of the Japanese childhood AML Cooperative Study Group

Mixed-lineage leukemia (MLL)-partial tandem duplication (PTD) is associated with poor prognosis in adult acute myeloid leukemia (AML), but its relationship to pediatric AML is unknown. One hundred fifty-eight newly diagnosed AML patients, including 13 FAB-M3 and 10 Down syndrome (DS) patients, who were treated on the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed for MLL-PTD, as well as internal tandem duplication (ITD) and the kinase domain mutation (D835Mt) in the FLT3 gene. We found MLL-PTD in 21 (13.3%) of 158 AML patients, but not in FAB-M3 or DS patients. The differences between patients with and without MLL-PTD were significant for 3-year overall survival (OS) (56.3% vs. 83.2%, P = 0.018), disease-free survival (DFS) (41.7% vs. 69.6%, P = 0.010), and relapse rate (RR) (54.3% vs. 27.6%, P = 0.0085) of 135 AML patients excluding the FAB-M3 and DS patients. Furthermore, ITD and D835Mt in the FLT3 gene were found in 17 (12.6%) and 8 (5.9%) of these 135 patients, respectively. The differences between patients with FLT3-ITD and the wild-type allele were significant for 3-year OS (35.3% and 84.3%, P < 0.0000001), DFS (40.0% and 66.9%, P < 0.003), and RR (52.4% and 30.3%, P < 0.005). Coduplication of both genes was found in only 3 (1.9%) patients. AML patients with FLT3-ITD, but not D835Mt, showed a poor prognosis. AML patients with MLL-PTD were also correlated with poor prognosis in this study.

The incidence and clinical significance of nucleophosmin mutations in childhood AML

AbstractFrameshift mutations in exon 12 of the nucleophosmin gene (NPM1) result in aberrant cytoplasmic localization of the NPM protein (NPMc+) and occur in 25% to 35% of adult acute myeloid leukemia (AML). In adults with AML, NPMc+ has been associated with normal karyotype, FLT3/ITD mutations, high remission induction rates, and improved survival (particularly in patients lacking FLT3/ITD). NPMc+ has not been well characterized in childhood AML. This study examines the incidence and clinical significance of NPMc+ in 295 children with newly diagnosed AML treated on a large cooperative group clinical trial (POG-9421). We find that NPMc+ is relatively uncommon in childhood AML (23 of 295 patients, 8%); and is significantly associated with FLT3/ITD mutations (P = .046), female sex (P = .029), older age (P = .047), and normal cytogenetics (P < .001). There is a favorable impact of NPMc+ on survival in children lacking FLT3/ITD (5-year EFS, 69% vs 35%; hazard ratio, 0.39; P = .051), which is similar in magnitude to the favorable impact of t(8;21) and inv(16). We conclude that NPMc+ is relatively rare in childhood AML, particularly in younger children. NPMc+ does not abrogate the negative prognostic influence of FLT3/ITD mutations, but may contribute to risk stratification in children who lack FLT3/ITD mutations by identifying a group with superior prognosis.

Twenty Years of Follow-Up among Survivors of Childhood and Young Adult Acute Myeloid Leukemia (AML): A Report from the Childhood Cancer Survivor Study (CCSS).

Survival rates for childhood AML continue to improve. Limited data exist on the comprehensive assessment of late medical and social effects experienced by these aggressively treated individuals. This analysis includes 272 5-yr survivors of AML participating in the CCSS diagnosed ≤ age 21, between 1970–1986. AML survivors who received a blood or marrow transplant are excluded from the current analysis. Comparisons among treatment groups and to a sibling control group are age and gender adjusted. Survivors (55% female) were on average 7 yrs old at dx and 28 yrs at follow-up, 56% received chemotherapy only, 34% chemotherapy/RT. Average follow-up was 22.6 yrs (16.1–33.5). Among these 5-yr survivors, overall survival was 97% (95% CI, 94–98%) at 10 yrs and 94% (95% CI, 92–97%) at 20 yrs. Six individuals reported 8 recurrences a mean of 11.1 yrs (5.6–16.6) from diagnosis, 2 died from relapse, 1 from congestive heart failure (CHF), and 1 from a myocardial infarction (MI). All cause standardized mortality ratio was 4.7 (95% CI 2.8–7.5). Cumulative incidence of recurrent AML at 10 and 20 years was 1.8% (95% CI 0.2–3.4%) and 3.7% (95% CI 1.4–5.9%), respectively. Eight survivors reported 10 subsequent malignant neoplasms (SMN) (5 breast cancers, 3 CNS tumors, 1 ovarian tumor, 1 salivary gland tumor) a mean of 18.7 yrs (9.2–26.4) from diagnosis, 3 with a history of RT exposure. SMN cumulative incidence at 20 yrs was 1.6% (95% CI 0.02–3.3%) with a standardized incidence ratio of 3.2 (95% CI 1.4–6.0). Six cardiac events (5 CHF and 1 MI) occurred a mean of 16.4 yrs (12.8–18.4) from diagnosis, 20-yr cumulative incidence 2.4% (95% CI 0.5–4.4%). Among those aged 25 yrs or more, marriage rates were similar among AML survivors and the general U.S. population (59%), but lower compared to siblings (69%) (p<0.01). College graduation rates were lower among survivors compared to siblings, but higher than in the general population (42% vs. 52% vs. 34%, respectively, p<0.01). No significant difference in graduation rates was found among those treated with or without CNS directed RT (p=0.9). All survivors and siblings were employed (100%) and most had health insurance (93% vs. 90%). Long-term survival from childhood AML greater than 5 yrs from diagnosis is favorable. Late occurring medical events remain concerning. Marriage and education rates may be lower than expected within individual family units; yet not significantly different from the U.S. population.

CEBPα mutations in childhood acute myeloid leukemia

CEBPalpha: mutations have been described in adult acute myeloid leukemia (AML) and conferred a favorable prognosis. However, CEBPalpha mutation has not been reported in children. We investigated 117 children with de novo AML using DNA PCR assay followed by sequencing for each PCR product. CEBPalpha mutations were detected in seven patients, four had FAB M2, two M1 and one M4. CEBPalpha mutations only occurred in patients with intermediate cytogenetics and not in 56 children with AML1-ETO, CBFbeta-MYH11, PML-RARalpha or MLL rearrangements. Five patients had mutations occurred in both N-terminal part and basic-leucine zipper (bZIP) domain, one had an N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Cloning analysis on five samples carrying more than one mutations demonstrated one homozygous combined mutations and four heterozygous biallelic mutations. Four of seven CEBPalpha mutation(+) patients had cooperating mutations with FLT3-ITD or N-ras mutations compared to 27 in 109 CEBPalpha mutation(-) patients. Our results showed that CEBPalpha mutations occurred in 6% of childhood AML and most exhibited combined mutations in both N-terminal part and bZIP domain.

High Expression Levels of Iaps Predict Poor Overall Survival in Childhood De Novo Acute Myeloid Leukemia.

Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. Using flow cytometry and western blot analysis, we therefore investigated 45 leukemic cell samples of children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins (CD95, Bcl-2, Bax, Bcl-xL, Procaspase-3, XIAP, cIAP-1, Survivin). XIAP (p<0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by FAB morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (p<0.01) and Bcl-xL (p<0.01) expression was lower in patients with favorable than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP {30 (n=10) vs. 41 months (n=34); p<0.05} and Survivin {27 (n=10) vs. 41 months (n=34); p<0.05}. We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics and poor overall survival should be confirmed within prospective pediatric AML trials.