Pediatric 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.

Characteristics and prognostic impact of IDH mutations in AML: a COG, SWOG, and ECOG analysis

AbstractSomatic mutations in isocitrate dehydrogenase (IDH) genes occur frequently in adult acute myeloid leukemia (AML) and less commonly in pediatric AML. The objective of this study was to describe the prevalence, mutational profile, and prognostic significance of IDH mutations in AML across age. Our cohort included 3141 patients aged between <1 month and 88 years treated on Children’s Cancer Group/Children’s Oncology Group (n = 1872), Southwest Oncology Group (n = 359), Eastern Cooperative Oncology Group (n = 397) trials, and in Beat AML (n = 333) and The Cancer Genome Atlas (n = 180) genomic characterization cohorts. We retrospectively analyzed patients in 4 age groups (age range, n): pediatric (0-17, 1744), adolescent/young adult (18-39, 444), intermediate-age (40-59, 640), older (≥60, 309). IDH mutations (IDHmut) were identified in 9.2% of the total cohort (n = 288; IDH1 [n = 123, 42.7%]; IDH2 [n = 165, 57.3%]) and were strongly correlated with increased age: 3.4% pediatric vs 21% older, P < .001. Outcomes were similar in IDHmut and IDH-wildtype (IDHWT) AML (event-free survival [EFS]: 35.6% vs 40.0%, P = .368; overall survival [OS]: 50.3% vs 55.4%, P = .196). IDH mutations frequently occurred with NPM1 (47.2%), DNMT3A (29.3%), and FLT3-internal tandem duplication (ITD) (22.4%) mutations. Patients with IDHmut AML with NPM1 mutation (IDHmut/NPM1mut) had significantly improved survival compared with the poor outcomes experienced by patients without (IDHmut/NPM1WT) (EFS: 55.1% vs 17.0%, P < .001; OS: 66.5% vs 35.2%, P < .001). DNTM3A or FLT3-ITD mutations in otherwise favorable IDHmut/NPM1mut AML led to inferior outcomes. Age group analysis demonstrated that IDH mutations did not abrogate the favorable prognostic impact of NPM1mut in patients aged <60 years; older patients had poor outcomes regardless of NPM1 status. These trials were registered at www.clinicaltrials.gov as #NCT00070174, #NCT00372593, #NCT01371981, #NCT00049517, and #NCT00085709.

Clinical characteristics and prognosis analysis of patients with de novo ASXL1-mutated AML treated with the C-HUNAN-AML-15 protocol: A multicenter study by the South China Pediatric AML Collaborative Group.

ASXL1 mutation is an independent prognostic factor in adult acute myeloid leukemia (AML), but its effect on the prognosis of pediatric AML is poorly understood. This study aimed to investigate the clinical characteristics and prognostic factors of ASXL1-mutant pediatric AML from a large Chinese multicenter cohort. A total of 584 pediatric patients with newly diagnosed AML from 10 centers in South China were enrolled. The exon 13 of ASXL1 was amplified by polymerase chain reaction (PCR), and then analyzed the mutation status of the locus. (n = 59 for ASXL1-mut group, n = 487 for ASXL1-wt group). ASXL1 mutations were found in 10.81% of all patients with AML. A complex karyotype was significantly less common in the ASXL1-mut AML group than in the ASXL1-wt group (1.7% vs. 11.9%, p = 0.013). Furthermore, TET2 or TP53 mutations were predominantly found in the ASXL1+ group (p = 0.003 and 0.023, respectively). The 5-year overall survival (OS) and event-free survival (EFS) of the total cohort were 76.9% and 69.9%. In ASXL1-mut AML patients, a white blood cell (WBC) count ≥50 × 109 /L had significantly poorer 5-year OS and EFS than a WBC count <50 × 109 /L (78.0% vs. 44.6%, p = 0.001; 74.8% vs. 44.6%, p = 0.003, respectively), while receiving hematopoietic stem cell transplantation (HSCT) had a higher 5-year OS and EFS (84.5% vs. 48.5%, p = 0.024; 79.5% vs. 49.3%, p = 0.047, respectively). In the multivariate Cox regression analysis, patients with high-risk AML undergoing HSCT tended to have a better 5-year OS and EFS than those receiving chemotherapy as a consolidation (HR = 0.168 and 0.260, both p < 0.001), and WBC count ≥50 × 109 /L or failure to achieve complete response after the first course were independent adverse predictors of OS and EFS (HR = 1.784 and 1.870, p = 0.042 and 0.018; HR = 3.242 and 3.235, both p < 0.001). The C-HUANA-AML-15 protocol is a well-tolerated and effective in the treatment of pediatric AML. ASXL1 mutation is not an independent adverse prognosis predictor for survival in AML, whereas ASXL1-mut patients tend to have a poor prognosis if WBC count ≥50 × 109 /L, but they can benefit from HSCT.

Pathogenic TP53 mutations Are Associated with a Poor Prognosis in De Novo AML

Introduction: Mutations in TP53 are the most common mutations in human cancer and occur in almost every cancer subtype. In adult acute myeloid leukemia (AML), TP53 mutations, especially those associated with complex cytogenetics, are associated with a poor prognosis. However, given the rarity of TP53 mutations in pediatric AML, less is known about its prognostic significance. Methods:TP53 variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00002798, NCT00070174, NCT01407757, NCT01371981). Next generation sequencing was retrospectively employed on diagnostic specimens to screen for variants including single nucleotide variants (SNVs) and insertions and deletions (indels). Variants were classified as benign, likely benign, unknown significance, likely pathogenic, and pathogenic using ClinVar and ACMG/AMP guidelines. Results: Of 1790 patients (age: median 10.48 years, range 0.01-29.84 years), 68 (3.8%) had TP53 variants. Sixty-four (94.1%) of these were SNVs, 2 (2.9%) were in-frame indels, and 2 (2.9%) were frameshifts leading to early protein truncation. Twenty-six (38.2%) were considered likely pathogenic or pathogenic (TP53mut) (age: median 8.38 years, range 0.31-17.97 years), 33 (48.5%) considered likely benign or benign, and the remaining 9 (13.2%) were of unknown significance. Thirty-four (53.1%) TP53 SNVs fell within the DNA binding domain, including 100% of TP53mut SNVs (including many occurring at DNA binding sites). We compared disease characteristics and clinical outcomes between patients with TP53mut and patients without any TP53 variant (TP53wt). Patients with non-pathogenic variants were excluded from the analysis. There was a higher prevalence of complex cytogenetics (defined as 3 or more chromosomal aberrations, including at least one structural aberration on karyotype) in TP53mut patients compared to TP53wt patients (55% vs. 18%; p=<.0001). There was no difference in the prevalence of co-occurring contemporarily defined high-risk alterations (TP53mut 30.8% vs. TP53wt 32.1%; p=0.879) but TP53mut patients were less likely to have co-occurring low-risk alterations compared to TP53wt patients (15.4% vs. 41.1%; p=0.008). While there were no differences in complete remission rates (TP53mut 85% vs. TP53wt 77.6%; p=0.426) or their ability to clear minimal residual disease (TP53mut 55% vs. TP53wt 71.6%; p=0.132), TP53mut patients had a lower event-free survival (EFS) compared to TP53wt patients (5yr: 13.3% vs. 46.3%; p <.0001), which was largely driven by a higher relapse risk (RR) (5yr: 76.5% vs. 42.1%; p <.0001). Salvageability following an event was largely unsuccessful for TP53mut patients, including only 1 long-term survivor following an event, which contributed to a lower overall survival (OS) compared to TP53wt patients (5yr: 15.9% vs. 64.7%; p < .0001). Further, EFS (13.3% vs. 26.3%; p <.0001; Figure 1a), RR (5yr: 76.5% vs. 60.9%; p <.0001; Figure 1b) and OS (5yr: 15.9% vs. 42.1%; p <.0001; Figure 1a) were significantly inferior compared to contemporarily defined high-risk patients. When patients with co-occurring high-risk alterations were excluded from analysis, TP53mut patients maintained their poor 5-yr EFS (13.3%), RR (76.5%) and OS (15.9%). Conclusions: In the largest study of TP53 mutations in pediatric patients with de novo AML, we show that pathogenic TP53variants are rare but prognostically significant. TP53mut patients were more likely to have complex cytogenetics and less likely to have co-occurring low-risk alterations. Outcomes for TP53mut patients were significantly inferior compared to contemporarily defined high-risk patients. Finally, these poor outcomes were maintained even when TP53mut patients with co-occurring high-risk lesions were excluded from analysis. Taken together, these data would suggest TP53 pathogenic mutations should be considered high-risk defining lesions in pediatric AML. Since HSCT is the standard of care for relapsed disease, the lack of salvageability following an event may suggest this disease is less alloreactive than other subtypes of AML, but further investigation is needed. Given the dismal outcomes after an event, clinical trials and novel targeted agents should be prioritized for relapsed patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Description of a novel subtype of acute myeloid leukemia defined by recurrent CBFB insertions

Description of a novel subtype of acute myeloid leukemia defined by recurrent CBFB insertions

Monosomal karyotype as an adverse risk factor for inferior survivals in children with acute myeloid leukemia

BackgroundAlthough monosomal karyotype (MK) has been recognized as an adverse risk factor in adult acute myeloid leukemia (AML), its impact on pediatric AML outcomes remains unclear. MethodsClinical characteristics, cytogenetic and molecular profiling, and prognostic impact of MK were analyzed in a cohort of 284 children with AML in a Chinese population. ResultsMK was present in 14 (4.5%) cases of the study cohort. MK-AML showed low prevalence of TP53 mutant and 17p13 aberrations. Children with MK-AML had shorter overall survival (OS) and event-free survival (EFS) compared to those with intermediate-risk features (OS: 26 ± 9.7% vs. 2 ± 1.5%, p < 0.001; EFS: 20 ± 7.4% vs. 1 ± 0.3%, p < 0.001) as well as adverse risk (OS: 15 ± 1.4% vs. 2 ± 1.5%, p = 0.012; EFS: 12 ± 1.6%, p = 0.014). Multivariate analysis demonstrated MK as an independent adverse risk factor for reduced survival (OS: HR 2.09, 95% CI 1.12–3.88, p = 0.02; EFS: HR 5.24, 95% CI 1.55–17.54, p = 0.08). ConclusionsThe results show MK as an independent adverse risk factor for inferior survivals.

Crebbp Alterations Are Associated with a Poor Prognosis in De Novo AML

Introduction: The translocation, t(8;16)(p11;p13), results in the fusion between KAT6A and CREBBP and has been associated with a poor prognosis in both pediatric and adult acute myeloid leukemia (AML). This lesion has therefore been re-classified as high risk on the active Phase 3 Children's Oncology Group (COG) trial for de novo AML, AAML1831 (NCT04293562). Less is known about the prognostic significance of CREBBP sequence variants.Methods: CREBBP variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00003790, NCT00070174, NCT01407757, NCT01371981). Fusions involving CREBBP were prospectively obtained via conventional cytogenetics and retrospectively confirmed via RNAseq. Insertions and deletions (indels) leading to frameshift mutations and single nucleotide variants (SNVs) were retrospectively interrogated via next generation sequencing.Results: Of 2216 patients (age: 0-29.8 years), 55 (2.5%) patients had an alteration involving CREBBP. Sixteen (29%) of these were a fusion involving CREBBP (CREBBP/fus), with KAT6A being the most common translocation partner (n=15) and the remaining translocation involving ANK1. The remaining 39 patients (71%) had a CREBBP mutation (CREBBP/mut), including 19 with an indel (CREBBP/indel) leading to a frameshift mutation and 20 with a SNV (CREBBP/SNV).We compared clinical and biologic characteristics between the three cohorts. CREBBP/fus patients were significantly younger than CREBBP/indel and CREBBP/SNV patients (median ages of 2.6 vs. 7.8 vs. 11.9 years; p=0.027). There was a higher prevalence of t(8;21)/RUNX1-RUNX1T1 in CREBBP/indel patients compared to CREBBP/SNV patients (42.1% vs. 5%; p=0.008). In contrast, CREBBP/SNV patients were more likely to be associated with a normal karyotype (40% vs. 5.3%; p=0.02). There was a similar prevalence of co-occurring high-risk lesions in CREBBP/indel (n=5; CBFA2T3-GLIS2, KMT2A-AFF1, KMT2A-MLLT4, MLLT10-PICALM, NUP98-HOXA9) and CREBBP/SNV (n=7; DEK-NUP214, ETV6-FOXO1, FUS-ERG, NUP98-NSD1, ETV6-MNX1, FLT3-ITDx2) patients. There was otherwise no difference between presenting WBC count, FLT3-ITD, NPM1, CEBPA, remission rates or MRD status after Induction 1 therapy.Patients with any CREBBP alteration had a significantly worse 5-year event free survival (EFS) compared to patients without (25.9% vs. 45.2%; p=0.002) and this inferior EFS overlaps with contemporarily defined high-risk patients (Figure 1a). Evaluation of outcomes based on type of alteration demonstrated a similar 5-year EFS of 33.3% and 23.1% between CREBBP/fus and CREBBP/mut patients, respectively (Figure 1b; p=0.832). This poor EFS was maintained in the CREBBP/indel patients with a co-occurring t(8;21) (n=8, 5-year EFS 12.5%). When patients with co-occurring high-risk lesions were excluded from analysis, the remaining CREBBP/mut (n=27) patients maintained their poor EFS (29.6%). Despite their poor EFS, CREBBP/mut patients had an analogous overall survival (OS) to non-CREBBP patients (57.4% vs. 62.3%; p=0.499, Figure 1c), demonstrating that these patients could be successfully salvaged following relapse. In contrast, all patients with CREBBP/fus that relapsed subsequently died from their disease (OS 33.3%).Conclusions: In a large study of CREBBP alterations in pediatric patients with de novo AML, we show that these patients have a dismal EFS, regardless of alteration type. Further, despite enrichment of t(8;21), the favorable prognosis typically conferred by this alteration was abrogated by the co-occurrence of CREBBP/indel. Similarly, by excluding patients with co-occurring high-risk lesions from analysis, we show that these poor outcomes persist in a cohort of patients that would otherwise be considered low risk. Translocations between CREBBP and KAT6A in patients over 90 days of age are considered high risk on the active COG phase 3 trial. Given the inferior EFS and high salvage rates associated with other CREBBP alterations, intensification of upfront treatment, including hematopoietic stem cell transplant, should be considered in this population.The authors would like to acknowledge Astellas Pharma Global Development, Inc. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High EVI1 Expression Predicts Adverse Outcomes in Children With De Novo Acute Myeloid Leukemia

BackgroundA high ecotropic viral integration site 1 (EVI1) expression (EVI1 high) is an independent prognostic factor in adult acute myeloid leukemia (AML). However, little is known of the prognostic value of EVI1 high in pediatric AML. This study aimed to examine the biological and prognostic significance of EVI1 high in uniformly treated pediatric patients with AML from a large cohort of seven centers in China.MethodsA diagnostic assay was developed to determine the relative EVI1 expression using a single real-time quantitative polymerase chain reaction in 421 newly diagnosed pediatric AML patients younger than 14 years from seven centers in southern China. All patients were treated with a uniform protocol, but only 383 patients were evaluated for their treatment response. The survival data were included in the subsequent analysis (n = 35 for EVI1 high, n = 348 for EVI1 low).ResultsEVI1high was found in 9.0% of all 421 pediatric patients with de novo AML. EVI1 high was predominantly found in acute megakaryoblastic leukemia (FAB M7), MLL rearrangements, and unfavorable cytogenetic aberrance, whereas it was mutually exclusive with t (8; 21), inv (16)/t (16; 16), CEBPA, NPM1, or C-KIT mutations. In the univariate Cox regression analysis, EVI1 high had a significantly adverse 5-year event-free survival (EFS) and overall survival (OS) [hazard ratio (HR) = 1.821 and 2.401, p = 0.036 and 0.005, respectively]. In the multivariate Cox regression analysis, EVI1 high was an independent prognostic factor for the OS (HR = 2.447, p = 0.015) but not EFS (HR = 1.556, p = 0.174). Furthermore, EVI1 high was an independent adverse predictor of the OS and EFS of patients with MLL rearrangements (univariate analysis: HR = 9.921 and 7.253, both p < 0.001; multivariate analysis: HR = 7.186 and 7.315, p = 0.005 and 0.001, respectively). Hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) provided EVI1 high patients with a tendential survival benefit when compared with chemotherapy as a consolidation (5-year EFS: 68.4% vs. 50.8%, p = 0.26; 5-year OS: 65.9% vs. 54.8%, p = 0.45).ConclusionIt could be concluded that EVI1 high can be detected in approximately 10% of pediatric AML cases. It is predominantly present in unfavorable cytogenetic subtypes and predicts adverse outcomes. Whether pediatric patients with EVI1 high AML can benefit from HSCT in CR1 needs to be researched further.

The LSC17 Leukemic Stem Cell Signature Predicts Outcome in Pediatric Acute Myeloid Leukemia

IntroductionThe model of the leukemic stem cell was proposed nearly 60 years ago as a mechanism of leukemic pathogenesis. Recently, a 17-gene leukemic stem cell (LSC) prognostic signature, termed the LSC17, was developed (Ng et al., 2016). The LSC17 score was shown to reliably differentiate adult acute myeloid leukemia (AML) patients into high and low risk groups. However, its applicability to pediatric AML remains unknown.Prognostic indicators are sensitive to differences in age. For example, NPM1 mutations, which are highly prognostic of favorable outcome, have no significance in patients older than 65 years (Ostronoff et al., 2015). Similarly, core-binding factor AML, also associated with favorable outcomes, loses prognostic value in older patients (Prébet et al., 2009).Given that age remained a significant prognostic factor in multivariate analyses with the LSC17, we evaluated the impact of the signature in older versus younger adults in the TCGA AML dataset and further, applied it to transcriptome sequence data from pediatric AML to assess the prognostic power in younger patients.Patients and MethodsPediatric AML biological samples were collected from patients enrolled in the Children's Oncology Group protocol AAML0531 and RNA sequencing was performed by the British Columbia Genome Sciences Center (Vancouver, BC). The Cancer Genome Atlas AML RNA sequencing data was downloaded from the Broad Institute Firehose (TCGA Network et al., 2013; Broad Institute TCGA Genome Data Analysis Center, 2016). LSC17 scores were calculated from log2 RPKM normalized gene expression as described by Ng et al .ResultsWe initially verified that the LSC17 score is effective for risk stratification in adult AML by applying the method to the TCGA AML cohort, excluding PML-RARA, BCR-ABL1 fusions, and those who received no treatment (n = 155, range: 18 - 88 yrs). Patients with low LSC17 scores had significantly better overall survival (OS) compared to the high scoring group after 5 years (low-LSC17 OS: 38.0%, high-LSC17 OS: 15.2%, hazard ratio (HR) = 2.08, p < 0.001). To investigate age effects on LSC17, we divided TCGA into two groups based on the median age of 58 yrs. The low (n = 81) and high age groups (n = 78) were reanalyzed. Younger patients with low LSC17 scores fared much better (low-LSC17 OS at 5yrs: 57.9%, high-LSC17 OS at 5yrs: 16.0% p < 0.001, Fig. 1A). Strikingly, for TCGA patients above 58 years, the LSC17 signature showed no prognostic significance (p = 0.714, Fig. 1B).We then evaluated the LSC17 signature to identify risk groups using RNA sequencing from our pediatric AML cohort (n = 446, range: 0.02 - 28 yrs). Univariate survival analysis revealed strong prognostic value for both overall survival (low-LSC17 OS at 5yrs: 72.8%, high-LSC17 OS at 5yrs: 51.4%, HR = 2.06, p < 0.001, Fig. 1C) and event-free survival (low-LSC17 EFS at 5yrs: 60.3%, high-LSC17 EFS at 5yrs: 36.4%, HR = 2.04, p < 0.001). Although dividing the cohort by LSC17 scores above and below the median (as proposed by Ng. et al.) demonstrated prognostic value in pediatric AML, the choice of median as a threshold may not be ideal. Dividing pediatric AML patients into four quartiles, we found no statistical significance between the survival curves for the middle two groups. In contrast, the top and bottom quartiles show very strong trends (LSC17-q1: 77.0% at 5 years, LCS17-q4: 41.1% OS at 5yrs, HR = 3.34, p < 0.001, Fig.1D). The high-LSC17 cohort carried a higher incidence of adverse cytogenetics and recurrent mutations, such as FLT3-ITD, and conversely, the low-scoring group was found to have a higher percentage of favorable cytogenetics (Fig.1E). To determine if the prognostic significance would remain, we performed a multivariate analysis; though WBC, age, and NMP1 mutations continued to maintain prognostic significance, we found the LSC17 score to be a strong independent prognostic indicator (HR = 2.031, p < 0.001, Fig. 1F).ConclusionIn this study, we validated that the LSC17 score can be used as an effective risk stratification tool in children and young adults. The LSC17 remains a strong independent prognostic factor after controlling for age, WBC, adverse FLT3-ITD mutations and favorable NPM1 mutations. While the LSC17 can be used as an informative indicator of long term survival, and event-free survival, we showed that this signature loses prognostic value in older AML patients. [Display omitted] 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.

Wilms\u2019 tumor gene 1 is an independent prognostic factor for pediatric acute myeloid leukemia following allogeneic hematopoietic stem cell transplantation

BackgroundSequential monitoring of Wilms’ tumor gene 1 (WT1) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) could predict relapse in adult acute myeloid leukemia (AML). However, the prognostic role of WT1 in pediatric AML after allo-HSCT is unclear. Thus, we determined to see whether sequential monitoring of WT1 after allo-HSCT could predict relapse in AML children.MethodsPediatric AML patients receiving allo-HSCT from January 21, 2012 to December 20, 2018 at the Peking University Institute of Hematology were included in this study. WT1 expression level was determined by TaqMan-based reverse transcription-polymerase chain reaction. WT1 sequential monitoring was performed 1, 2, 3, 4.5, 6, 9, and 12 months post-transplantation and at 6-month intervals thereafter. The primary end point was relapse. The secondary end points included disease-free survival (DFS), overall survival (OS), and non-relapse mortality (NRM). Kaplan–Meier analysis was used for DFS and OS estimates, while competing risk analysis was used for estimating relapse and NRM.ResultsOf the 151 consecutive patients included, the median age was 10 years (range, 1–17). The optimal cutoff value of WT1 within 1 year after allo-HSCT to predict relapse was 0.8% (80 WT1 copies/104 ABL copies), with a sensitivity of 60% and specificity of 79%. Compared with WT1 expression < 0.8%, WT1 expression ≥0.8% indicated significantly higher 5-year cumulative incidence of relapse (CIR, 35.1% vs. 11.3%; P = 0.001), lower 5-year disease-free survival (DFS, 60.4% vs. 80.8%; P = 0.009), and lower 5-year overall survival (OS, 64.9% vs. 81.6%; P = 0.038) rates. Multivariate analyses showed that WT1 was an independent risk factor for relapse (HR 2.89; 95% confidence interval (CI), 1.25–6.71; P = 0.014). Both the CIR (5-year CIR: 8.3% vs. 11.3%; P = 0.513) and DFS (5-year DFS: 91.7% vs. 80.8%; P = 0.208) were comparable between patients achieving minimal residual disease (MRD) negativity after preemptive interferon-α (IFN-α) treatment and those without MRD after allo-HSCT, which were better than those of MRD-positive patients without preemptive therapies.ConclusionsSequential monitoring of WT1 could predict relapse in pediatric AML after allo-HSCT. WT1-directed immunotherapy may have the potential to prevent relapse and improve survival.

FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy

AbstractPoint mutations of D835/I836 of the FLT3 gene have been reported in adult acute myeloid leukemia (AML), but not in pediatric AML or acute lymphoblastic leukemia (ALL). FLT3-D835/I836 mutations were found in 6 (5.4%) of 112 children with ALL older than 1 year and in 8 (16.0%) of 50 infants with ALL. Missense mutations were found in 11 patients, 3-base pair deletions in 2 patients, and a deletion/insertion in 1 patient. Remarkably, FLT3-D835/I836 mutations were found in 8 (18.2%) of 44 infants with ALL with MLL rearrangements and in 4 (21.5%) of 19 patients with hyperdiploid ALL, but they were not found in any patients older than 1 year who had TEL-AML1 (n = 11), E2APBX1 (n = 4), or BCR-ABL (n = 6) fusion genes. Although infant ALL patients with mutations had poorer prognoses than did those without mutations, pediatric ALL patients with mutations who were older than 1 year had good prognoses. We also found FLT3-D835 mutations in 2 of 11 leukemic cell lines with MLL rearrangements. FLT3 was highly phosphorylated in these cell lines with FLT3-D835 mutations, leading to constitutive activation of downstream targets such as signal transducer and activator of transcription 5 (STAT5) without FLT3 ligand stimulation. These results suggested that FLT3-D835/I836 mutations are one of the second genetic events in infant ALL with MLL rearrangements or pediatric ALL with hyperdiploidy. (Blood. 2004;103:1085-1088)

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.

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Type 1 regulatory (Tr1) T cells induced by enforced expression of interleukin-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft-versus-host disease while mediating graft-versus-leukemia effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1- RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. In order to examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.

Sensitive detection of EBV microRNAs across cancer spectrum reveals association with decreased survival in adult acute myelocytic leukemia

Epstein Barr virus (EBV) is the etiologic agent involved in numerous human cancers. After infecting the host, EBV establishes a latent infection, with low levels of messenger RNA (mRNA) and protein expression, evolved to evade immune recognition. Conversely, EBV microRNAs (miRNA) are expressed ubiquitously and abundantly within infected cells. Their role in tumor biology and clinical outcomes across the spectrum of cancer is not fully explained. Here, we applied our bioinformatics pipeline for quantitative EBV miRNA detection to examine sequencing data of 8,955 individual tumor samples across 27 tumor types representing the breadth of cancer. We uncover an association of intermediate levels of viral miRNA with decreased survival in adult acute myeloid leukemia (AML) patients (P = 0.00013). Prognostic modeling of this association suggests that increased EBV miRNA levels represent an independent risk factor for poor patient outcomes. Furthermore, we explore differences in expression between elevated and absent viral miRNA loads in adult AML tumors finding that EBV positivity was associated with proinflammatory signals. Together, given no associations were found for pediatric AML, our analyses suggests EBV positivity has the potential for being a prognostic biomarker and might represent a surrogate measure related to immune impairment in adult patients.

A Prognostic Human Splicing Signature That Precurses Leukemia

Alternative splicing is a primary mechanism used to achieve mRNA transcript and proteomic diversity in higher-order eukaryotes. While alternative splicing is a recognized oncogenic driver in a small percentage of adult acute myeloid leukemia (AML) cases (~10-15%), splicing factor mutations are rarely found in pediatric AML. To discover splicing heterogeneity within diverse cancers in the absence of known causal mutations we developed a new computational workflow called OncoSplice. This workflow incorporates unsupervised splicing pattern analysis to iteratively identify novel patient splicing-defined subtypes (splice-ICGS algorithm), in conjunction with new integrative methods for cis-regulatory motif, CLIP-Seq binding and splicing factor expression data analysis (Fig. 1a).Analysis of RNA-Seq data from over 800 AML diagnosis samples identified subtypes associated with nearly all known splicing factor mutations in addition to over a dozen novel splicing-defined subtypes. Novel splicing subtypes were confirmed in independent patient cohorts and were associated with the expression or mutation of oncogenes (MYC, TP53, NPM1), oncofusions (CBFB-MYH11, MLL, PML-RAR, RUNX1), or the regulation of diverse splicing factors (U2AF1, SRSF2, HNRNPK). Both adult and pediatric AML are most frequently characterized by a single predominant splicing signature which divides the majority of AML patients into subtypes with splicing events overlapping those found in patients with mutations in U2AF1 or SRSF2 (Figure 1b). These “U2AF1-covarying” or “SRSF2-covarying” (CV) occur independently of splicing-factor mutations and were principally linked to mis-splicing rather than differential gene expression. Unlike patients with U2AF1-S3F mutations, U2AF1-CV splicing events are associated with canonical rather than altered U2AF1 binding specificity (Fig. 1c). In both adult and pediatric AML, U2AF1-CV splice events result in a shift towards longer protein isoforms associated with stem and progenitor programs, have significantly worse outcomes (poor survival and increased relapse) and are persistent during relapse in adults (Fig. 1d). Survival correlated U2AF1-CV splicing events were statistically enriched in genes required for leukemic growth based on a published CRISPR dropout screen. U2AF1-CV splicing was not associated with prior described epigenetic AML subtypes, but appears to be dependent on the expression of MYC and downstream splicing regulators (WDR77 and PRMT5). Surprisingly, RNA-Seq analysis of CD34+ bone marrow progenitors in healthy donors finds the same overall skewing in a subset of U2AF1-CV or SRSF2-CV splicing events as those found in AML. Distinct sorted progenitor populations (HSC, Multi-Lin, CMP, GMP, MEP) further show consistent U2AF1-CV or SRSF2-CV skewing within progenitors from the same donors, indicating that this is not a cell-type associated splicing difference. Hence, our data suggest that healthy bone marrow is skewed in the healthy population towards either U2AF1 or SRSF2 splicing pathways and that these splicing profiles impact future oncogenic transformation and patient survival in AML. These data further suggest a paradigm shifting model, in which widespread coordinated pathogenic splicing occurs across cancers, likely via imbalances in splicing factor expression, regulation or mutation.Figure 1. a) Steps implemented in the OncoSplice splicing subtype discovery workflow for splicing event quantification (MultiPath-PSI), iterative unsupervised splicing subtype discovery (splice-ICGS), supervised splicing subtype discovery (Bridger) and RNA-regulatory splicing subtype prediction (RBP-Finder). b) Comparison of splicing events enriched in adult AML with splicing factor mutations identifies their coincidence with U2AF1-CV and SRSF2-CV splicing events. c) Analysis of U2AF1 binding-site preferences at the e-3 splice-site position for cassette-exon splicing events (U2AF1-S34-specific but not U2AF1-CV; occurring in U2AF1-S34; occurring in U2AF1-CV; U2AF1-CV-specific but not U2AF1-S34). d) Kaplan-Meier curves for overall survival in patients from TCGA (top) and TARGET (bottom) with associated coxph p-values (all splice-ICGS stringently classified U2AF1-CV versus all other considered AMLs). Analysis of TCGA was restricted to cytogenetically normal AMLs with no splicing factor mutations and under 60 years of age. DisclosuresMyers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Transposable Element Expression in Acute Myeloid Leukemia Transcriptome and Prognosis

Over half of the human genome is comprised of transposable elements (TE). Despite large-scale studies of the transcriptome in cancer, a comprehensive look at TE expression and its relationship to various mutations or prognosis has not been performed. We characterized the expression of TE in 178 adult acute myeloid leukemia (AML) patients using transcriptome data from The Cancer Genome Atlas. We characterized mutation specific dysregulation of TE expression using a multivariate linear model. We identified distinct patterns of TE expression associated with specific mutations and transcriptional networks. Genes regulating methylation was not associated with significant change in TE expression. Using an unpenalized cox regression analysis we identified a TE expression signature that predicted prognosis in AML. We identified 14 candidate prognostic TE transcripts (TEP) that classified AML as high/low-risk and this was independent of mutation-based and coding-gene expression based risk-stratification. TEP was able to predict prognosis in independent cohorts of 284 pediatric AML patients and 19 relapsed adult AML patients. This first comprehensive study of TE expression in AML demonstrates that TE expression can serve as a biomarker for prognosis in AML, and provides novel insights into the biology of AML. Studies characterizing its role in other cancers are warranted.

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.

CXCR4 Overexpression is a Poor Prognostic Factor in Pediatric Acute Myeloid Leukemia With Low Risk: A Report From the Japanese Pediatric Leukemia/Lymphoma Study Group.

Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear. This retrospective study examined the prognostic significance of CXCR4+ in pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. In the total cohort (n = 248), no significant differences were observed between CXCR4+ patients (n = 81) and CXCR4- patients (n = 167) in terms of 3-year overall survival (OS) (69.4% vs. 75.2%, P = 0.44). However, there was a significant difference in 3-year OS between CXCR4+ and CXCR4- patients in the low-risk (LR) group (n = 93; 79.2% vs. 98.3%, P = 0.007). CXCR4+ patients in the t(8;21) AML without KIT mutation group had a significantly worse 3-year OS than CXCR4- patients (n = 44; 76.1% vs. 100.0%, P = 0.01). Multivariate Cox regression analysis identified CXCR4+ as a poor prognostic factor for OS in LR AML patients (hazard ratio, 11.47; P = 0.01). Consistent with the data for survival analysis, CXCR4+ patients in the t(8;21) AML group had a higher incidence of splenomegaly than CXCR4- patients (25.9% vs. 5.9%, P = 0.03). These results suggest that CXCR4+ is a poor prognostic factor for LR patients, particularly t(8;21) patients without KIT mutation. The poor outcome was only applicable to OS, not relapse-free survival (RFS); thus, CXCR4+ may be associated with a poor prognosis after recurrence. Intensive therapy, including administration of CXCR4 antagonists, may be promising for pediatric AML patients with LR.

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.