Pediatric Acute Myeloid Leukemia Patients Research Articles

Deciphering molecular heterogeneity in pediatric AML using a cancer vs. normal transcriptomic approach.

Still 30-40% of pediatric acute myeloid leukemia (pedAML) patients relapse. Delineation of the transcriptomic profile of leukemic subpopulations could aid in a better understanding of molecular biology and provide novel biomarkers. Using microarray profiling and quantitative PCR validation, transcript expression was measured in leukemic stem cells (LSC, n = 24) and leukemic blasts (L-blast, n = 25) from pedAML patients in comparison to hematopoietic stem cells (HSCs, n = 19) and control myeloblasts (C-blast, n = 20) sorted from healthy subjects. Gene set enrichment analysis was performed to identify relevant gene set enrichment signatures, and functional protein associations were identified by STRING analysis. Highly significantly overexpressed genes in LSC and L-blast were identified with a vast majority not studied in AML. CDKN1A, CFP, and CFD (LSC) and HOMER3, CTSA, and GADD45B (L-blast) represent potentially interesting biomarkers and therapeutic targets. Eleven LSC downregulated targets were identified that potentially qualify as tumor suppressor genes, with MYCT1, PBX1, and PTPRD of highest interest. Inflammatory and immune dysregulation appeared to be perturbed biological networks in LSC, whereas dysregulated metabolic profiles were observed in L-blast. Our study illustrates the power of taking into account cell population heterogeneity and reveals novel targets eligible for functional evaluation and therapy in pedAML. Novel transcriptional targets were discovered showing a significant differential expression in LSCs and blasts from pedAML patients compared to their normal counterparts from healthy controls. Deregulated pathways, including immune and metabolic dysregulation, were addressed for the first time in children, offering a deeper understanding of the molecular pathogenesis. These novel targets have the potential of acting as biomarkers for risk stratification, follow-up, and targeted therapy. Multiple LSC-downregulated targets endow tumor suppressor roles in other cancer entities, and further investigation whether hypomethylating therapy could result into LSC eradication in pedAML is warranted.

Clinical significance of dysregulation of miR-381 in pediatric acute myeloid leukemia

BackgroundmicroRNA-381 is dysregulated in a variety of cancers. However, its clinical significance in pediatric acute myeloid leukemia (AML) is still unclear. The purpose of this study was to detect the expression level of miR-381 in pediatric AML patients and to explore its potential clinical significance.MethodsThe levels of miR-381 in bone marrow and serum of 102 pediatric AML patients were measured by quantitative real-time polymorperase chain reaction (qRT-PCR). The diagnostic value of serum miR-381 in pediatric AML patients was evaluated by the receiver operating characteristic (ROC) curve. A Chi square test was used to analyze the relationship between serum miR-381 and the clinical characteristics of patients. Cox regression analysis and Kaplan–Meier evaluated the prognostic value of serum miR-381 in patients. Finally, the proliferation of the cells was analyzed by the CCK-8 assay.ResultsCompared with healthy controls, the levels of miR-381 in serum and bone marrow of pediatric AML patients were significantly decreased (P < 0.001). ROC curve showed that miR-381 could distinguish pediatric AML cases from normal controls. At the same time, the downregulation of miR-381 was associated with M7 in the French–American–British (FAB) classifications and unfavorable cytogenetic risks (P < 0.05). Low serum miR-381 levels were associated with poor overall survival of pediatric AML (log-rank test, P = 0.011) and poor relapse-free survival (log-rank test, P = 0.004). Cox regression analysis confirmed that reduced serum miR-381 was an independent predictor of poor prognosis in AML (HR = 3.794, 95% CI 1.3633–10.559, P = 0.011). In addition, low expression of miR-381 significantly reduced the proliferation of cells (P < 0.05).ConclusionAll experimental results confirm that miR-381 has reduced bone marrow and serum expression in pediatric AML, and low levels of serum miR-381 have certain diagnostic and prognostic value in pediatric AML and may be a potential therapeutic target for AML.

Profiling FLT3 Mutations in Mexican Acute Myeloid Leukemia Pediatric Patients: Impact on Overall Survival.

Background: Acute myeloid leukemia (AML) is the second most frequent leukemia in childhood. The FLT3 gene participates in hematopoietic stem cell proliferation. FLT3 mutations are recurrent in AML and influence prognosis. In Mexican pediatric AML patients, FLT3 mutational profile, and their clinical impact have not been evaluated.Aim of the study: This study aimed to identify the profile of FLT3 mutations in pediatric patients with de novo AML and to assess their possible influence on overall survival (OS) and other clinical features.Methods: Massive parallel target sequencing of FLT3 was performed in 80 patients.Results: FLT3 mutations [internal tandem duplication (ITD) or tyrosine kinase domain (TKD)] were identified in 24% of them. OS was significantly lower in FLT3POS cases than in FLT3NEG (p = 0.03). The average OS for FLT3POS was 1.2 vs. 2.2 years in FLT3NEG. There were no significant differences in the children's sex, age, percentage of blasts in bone marrow aspirate, or white blood cell count in peripheral blood at diagnosis between both groups. No differences were identified stratifying by the mutational load (high > 0.4) or type of mutation. The negative effect of FLT3 mutations was also observed in patients with acute promyelocytic leukemia (APL).Conclusions: FLT3 mutational profile is described in Mexican pediatric AML patients for the first time. Mutated FLT3 negatively impacts the outcome of AML patients, even considering the APL group. The clinical benefit from treatment with tyrosine kinase inhibitors in the FLT3POS pediatric patients needs to be assessed in clinical trials. FLT3 testing may contribute to better risk stratification in our pediatric AML patients.

A three-gene signature and clinical outcome in pediatric acute myeloid leukemia.

Although the 5-year survival rates in pediatric acute myeloid leukemia (AML) have improved over the last decades, there is a high relapse rate for Pediatric AML patients. In the present study, we mainly combine PCA with the LASSO technique to identify prognostic markers for Pediatric AML patients coming from the NCI TARGET database. Three key genes (EEF1A1, RPLP2, RPL19) associated with poor prognosis of pediatric AML has been screened by both PCA and LASSO Cox regression analysis. Simultaneously, we developed a risk score model to predict the prognosis of pediatric AML, according to risk scores, the patients were divided into high- and low-risk groups based on the median risk score. Kaplan-Meier survival analysis indicated that Pediatric AML patients with the high-risk group have a poorer survival rate than those with a low-risk group (p < 0.000). The receiver operating characteristic (ROC) analysis showed that the risk model has a good performance (AUC:0.669). Moreover, the clinicopathologic correlation showed that the expression levels of three genes were related to the central nervous system (CNS) disease and chloroma. GSEA identified that those pathways including oxidative phosphorylation, apoptosis and TGFB signaling pathway were differentially enriched. Taken together, those studies suggested that a gene panel that consists of three genes (EEF1A1, RPLP2, RPL19) may act as a potential prognostic marker.

Inhibition of Methyltransferase DOT1L Sensitizes to Sorafenib Treatment AML Cells Irrespective of MLL-Rearrangements: A Novel Therapeutic Strategy for Pediatric AML.

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

The influence of various doses of busulfan in conditioning regimes on outcome of allogeneic hematopoietic stem cell transplantation in children with acute myeloid leukemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for patients with acute myeloid leukemia (AML). The conditioning regimen administered for this patient based on busulfan (Bu) combined with cyclophosphamide (Cy), fludarabine (Flu) or some other agents. Comparisons of myeloablative conditioning (MAC) versus reduced intensity conditioning (RIC) have demonstrated a various results between relapse and toxicity in a few reports. We suppose, that dose intensity of Bu across regimens may affect treatment outcomes. Aim of this retrospective study was to evaluate the impact dose of busulfan to overall survival (OS), transplant-related mortality (TRM), relapse-free survival (RFS), toxicity, the incidence of primary graft failure and acute "graft versus host" disease (GvHD) in transplantation in children and adolescents with AML. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University. We analyzed 110 AML pediatric patients with the median age 9 (range 1–19) y.o., who underwent first allo-HSCT with Bu based conditioning in R.M. Gorbacheva Memorial Institute from 2002 to 2018. Patients were divided into 3 groups: Bu1 – patients, who received Bu at the dose 8–10 mg/kg, n = 34 (31%), in Bu2 – dose of Bu was 12 mg/kg, n = 35 (32%), in Bu3 – dose of Bu was &gt; 12 mg/kg, n = 41 (37%). In Bu1 Bu was combined with Flu in 31 (91%) pts and Cy in 3 (9%); in Bu2 – with Flu in 12 (34%), Cy in 7 (20%) and other agents in 16 (46%); in Bu3 – with Cy in 32 (78%), with Flu in 7 (17%) and other agents in 2 pts (5%) (p &lt; 0.001). Patients in Bu2 received more Cy based GvHD prophylaxis regimens (69% vs 44% in Bu1, vs 29% in Bu3, p = 0.003) and more haplo grafts (51% vs 29% in Bu1, vs 15% in Bu3, p = 0.003). The complete remission at the HSCT was observed in 79 % in Bu1, 49% in Bu2, 61% in Bu3 (p = 0.02). Probabilities of OS, RFS, TRM were estimated by using the Kaplan–Meier method. Incidence of toxicity, acute GvHD and primary graft failure – by using Mann–Whitney U-test. Transplant engraftment was achieved in 95 (86%) of patients. Graft failure occurs in the 5 patients of Bu1 group (15%), in the 6 pts of Bu2 (17%) and in the 4 pts of Bu3 (10%) (p = 0.7). Median follow-up was 2 years for Bu1 and Bu3, 1 year for Bu2. Two-year OS was similar (Bu1 = 59% vs Bu2 = 60% vs Bu3 51%, p = 0.7). Two-year OS of pts with CR before HSCT was 70% in Bu1, 82% in Bu2, 60% in Bu3, p = 0,3 and 14%, 39%, 38% for pts with progression disease (PD), respectively (p = 0.5). Two-year RFS was 74% in Bu1, 82% in Bu2, 64% in Bu3 at CR (p = 0.4); 43%, 39% and 38% in pts with progression, respectively (p = 0.9). Median of RFS were also similar for the pts in PD (4 months in Bu1, 5 months in Bu2 and Bu3, p = 0.9) and not achieved for pts at CR. Drug related toxicity grade III–IV 4 experienced in 35% pts in Bu1, 29% in Bu2, in 54% in Bu3 (p = 0.04). Mucositis and toxic hepatitis were the most common adverse events. Sinusoidal obstruction syndrome (SOS) experienced in 8 pts from different group: 4 from Bu2 (11%), 3 from Bu3 (7%) and only pts from Bu1 (3%) with previously treated of inotuzumab (p = 0.4). The most pts with VOD (3/5) had PD at the HSCT. Cumulative incidence of acute GvHD grade 2 (15% vs 14% vs 10%, p = 0.8) were not different. Acute GvHD grade III–IV was observed a bit more often in Bu3 (34%), than in Bu1 (18%) and Bu2 (17%) (p = 0.09). TRM up to D+100 was also higher in Bu3 (15%), than in Bu2 (6%) and Bu1 (0%) (p = 0.05). The transplant results of children with similar disease status of AML, received MAC or RIC conditioning with various dose of Bu, were not associated with significant differences in overall outcomes. The higher dose Bu may increase incidence of toxicity grade III–IV (p = 0.04) and acute GvHD grade III–IV (p = 0.09) with increasing of early TRM (p = 0.05).

Deep Transcriptome Sequencing of Pediatric Acute Myeloid Leukemia Patients at Diagnosis, Remission and Relapse: Experience in 3 Malaysian Children in a Single Center Study.

Deep Transcriptome Sequencing of Pediatric Acute Myeloid Leukemia Patients at Diagnosis, Remission and Relapse: Experience in 3 Malaysian Children in a Single Center Study.

Positive expression of PAX7 indicates poor prognosis of pediatric and adolescent AML patients

ABSTRACTObjective: Therapeutic advances based on risk stratification and implementation of excellent supportive care measures have significantly improved outcomes for childhood acute myeloid leukemia (AML) over the past 30 years. However, approximately half of all childhood AML cases relapse. Therefore, precise risk stratification is needed for predicting relapse potential.Methods: RNA-seq data of TARGET-AML and corresponding clinical information of pediatric and adolescent AML cases were downloaded from The Cancer Genome Atlas. Clinical information of 156 patients with gene expression data was extracted. The effects of PAX7 expression profiles on overall survival (OS) and event-free survival (EFS) were analyzed.Results: Positive expression of PAX7 indicated shorter OS and EFS, especially in patients older than 14 years. Furthermore, positive PAX7 expression also predicted shorter OS and EFS in intermediate- and low-risk group patients, compared to patients with negative PAX7 expression. In addition, patients who have received allogenic hematopoietic stem cell transplantation (allo-HSCT) in the first complete remission had better outcome than those who did not receive HSCT.Conclusions: Positive PAX7 expression in pediatric and adolescent AML patients indicates a poor outcome. Hence, the detection of PAX7 expression profiles is helpful for further stratification of intermediate- and low-risk groups.

Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation for Pediatric Acute Myeloid Leukemia in the Contemporary Era

High risk or relapsed pediatric acute myeloid leukemia (AML) is potentially curable with allogeneic hematopoietic stem cell transplantation (HSCT). Significant improvements have been made with supportive care practices, but there are limited HSCT outcome data for pediatric AML patients in the contemporary era. We report retrospective data in 87 pediatric patients with high risk or relapsed AML, who underwent HSCT at a single institution between 2008 and 2018. The median age at time of HSCT was 8 years (range: 0.6-20 years). Forty three patients (49%) were transplanted in CR1, 18 (21%) in CR2, and 26 (30%) with active disease (defined as any evidence of disease at the time of HSCT). Patients were transplanted using matched related (MRD, n=21), matched unrelated (MUD, n=25), mismatched unrelated (MMUD, n=15), haplo-identical (haplo, n=12) or umbilical cord blood (UCB, n=14) grafts. The majority of patients (78/87, 90%) received myeloablative conditioning. MRD and UCB graft recipients did not receive serotherapy. All other graft recipients (n=52) received serotherapy, the majority using an alemtuzumab-based conditioning regimen, 42/52 (81%). Haplo-identical transplants were performed using CD34+ selected grafts. Three-year overall survival (OS) and disease free survival (DFS) for the entire cohort were 52% (95% CI: 41-62%) and 49% (95% CI: 38-59%) respectively. OS differed significantly according to disease status at time of transplant (p=0.018), with 3 year OS: 60%, (95% CI: 43-74%) for those in CR1, 56%, (95% CI: 31-75%) for those in CR2 and 34% (95% CI: 17-52%) for those with active disease. OS differed by donor type (p=0.058). OS was comparable for patients with MRD (71%, 95% CI: 46-86%) and 10/10 MUD (59%, 95% CI: 37-75%) (p=0.67). Patients with alternate donor sources had worse outcomes (MMUD 33%, 95% CI: 10-59%; UCB 43%, 95% CI: 18-66%; Haplo 33%, 95% CI: 10-59%) as compared to MRD (MMUD vs MRD, p=0.042, UCB vs MRD, p=0.046, haplo vs MRD p=0.017). There was a low incidence of Grade III-IV acute GVHD (8%, n=7) and extensive chronic GVHD (9%, n=7) likely secondary to the use of alemtuzumab-based conditioning regimens. The 3-year non-relapse mortality was low at 11% (95% CI: 5-19%). The primary cause of death was relapse, with a 3-year cumulative incidence of relapse of 40%, (95% CI: 30-50%). Patients with active disease at HSCT had significantly higher cumulative incidence of relapse (sHR: 2.42, 95% CI: 1.18-4.95, p=0.016) compared to patients in remission. In the contemporary era, outcomes after allogeneic HSCT are comparable for MRD and 10/10 MUD for pediatric AML. Myeloablative and alemtuzumab based conditioning regimens were well tolerated with low rates of NRM and GVHD. Relapse remains the major cause of treatment failure. Future trials evaluating use of cellular therapies and targeted agents post-HSCT will be needed.

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR-204 in pediatric AML.

The long noncoding RNA urothelial carcinoma-associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27kip1 . Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR-204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase-3 in AML cells. Moreover, UCA1 sponged miR-204 and suppressed its expression. UCA1 overexpression inversed the miR-204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR-204 target, via the sponging interaction. Furthermore, miR-204 inhibited inducible nitric oxide synthase and cyclooxygenase-2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR-204 in pediatric AML.

Myeloid sarcoma is associated with poor clinical outcome in pediatric patients with acute myeloid leukemia.

The impact of myeloid sarcoma (MS) on clinical outcome of pediatric acute myeloid leukemia (AML) patients remains controversial. Moreover, little is known about the role of stem cell transplantation (SCT) in such patients. Clinical data of patients with AML under 18years of age were retrieved from the TARGET dataset. We analyzed the prevalence, clinical profile, molecular characteristics, and prognosis of MS in these patients. Among 884 pediatric patients with AML, the frequency of MS was 12.3%. Pediatric AML with MS was associated with age under 1-year, abnormal cytogenetics, and KMT2A rearrangement. Moreover, MS was associated with a low complete remission rate, high induction death, poor 5-year EFS, and OS. KMT2A rearrangement had a negative impact on clinical outcome in AML patients with MS. In addition, SCT had no significant effect on the survival of AML patients with MS. Multivariate analysis revealed that MS was an unfavorable prognostic factor in pediatric AML in terms of EFS (Hazard ratio 1.670, P < 0.001) and OS (Hazard ratio 1.623, P = 0.004). The presence of MS at diagnosis of pediatric AML is associated with poor clinical outcomes, particularly when associated with KMT2A rearrangements. Moreover, pediatric patients with AML and MS may not benefit from SCT.

Clinical features and prognosis of normal karyotype acute myeloid leukemia pediatric patients with WT1 mutations: an analysis based on TCGA database

ABSTRACT Objectives: To explore the clinical features and prognosis of normal karyotype acute myeloid leukemia (NK-AML) pediatric patients with WT1 mutations. Methods: The clinical data and prognostic information of 220 NK-AML pediatric patients were selected from target-AML project of The Cancer Genome Atlas (TCGA) database. Survival analyses were performed for NK-AML pediatric patients with different combinations of mutations. Results: We found that 28(12.7%) NK-AML patients harbored WT1 mutations. The positive rate of FLT3-ITD in the WT1-mutated group was higher than that in the WT1 wild-type group (P = 0.002). In contrast, WT1 mutation and NPM1 mutation were mutually exclusive (P = 0.013). Furthermore, the WT1-mutated group suffered lower rates of complete remission (CR) (P < 0.001 and P < 0.001, respectively) but higher rates of minimal residual disease (MRD) (P = 0.003 and P = 0.021, respectively) after both one and two courses of induction chemotherapy. Patients with WT1 mutations had significantly worse overall survival (OS) and event-free survival (EFS) in both univariate (P < 0.001 and P = 0.007, respectively) and multivariate survival analyses (P < 0.001 and P < 0.001, respectively). The stratification analysis showed that for FLT3-ITD positive patients, WT1 mutations predicted shorter OS (P = 0.003) and EFS (P < 0.001). Conclusion: WT1 mutations conferred an independent poor prognosis for NK-AML pediatric patients.

Novel therapies for pediatric acute myeloid leukemia: building future strategies through incorporation of treatment currently used in adults

In Japan, acute myeloid leukemia (AML) accounts for approximately 25% of all pediatric leukemias, with approximately 150 cases of newly diagnosed AML occurring annually. Approximately 10% of patients have primary induction failure and 30% of patients, who initially achieve remission in primary treatments, subsequently relapse. Novel treatment modalities need to be developed to further improve the prognosis of pediatric AML patients. AML is a heterogeneous genetic disease characterized by changes in the genome of hematopoietic progenitor cells. Recent studies that have made progress in research related to the pathogenesis of AML have suggested that genotype-specific treatment strategies are associated with increased efficacy. Potential new therapeutic alternatives for pediatric AML include: tyrosine kinase inhibitors, monoclonal or bispecific T-cell engager antibodies, chimeric antigen receptor T-cell therapy, and metabolic agents. This review highlights the current landscape of novel therapeutic approaches for childhood AML, including the results of both preclinical and clinical trials, as well as introducing the results of several preceding adult clinical studies, which could potentially be translated into pediatric AML patients.

Machine Learning to Predict 5-Year Survival among Pediatric Acute Myeloid Leukemia Patients and Development of OSPAM-C Online Survival Prediction Tool

Machine Learning to Predict 5-Year Survival among Pediatric Acute Myeloid Leukemia Patients and Development of OSPAM-C Online Survival Prediction Tool

MiR-100 regulates cell viability and apoptosis by targeting ATM in pediatric acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common pediatric malignancy and a major cause of morbidity and mortality in children. miR-100 is associated with progression of various diseases including AML. The aim of this study was to explore the underlying molecule mechanisms of miR-100 involved in AML. The expressions of miR-100 and ataxia telangiectasia mutated (ATM) in pediatric AML patients and cell lines were monitored using qRT-PCR and western blot assays. MTT assay was carried to evaluate cell viability. Cell apoptosis was measured by flow cytometry. The binding sites between miR-100 and ATM were predicted by mirtarbase database. Luciferase reporter assay was used to confirm the relationship between miR-100 and ATM. miR-100 expression was highly expressed in bone marrow of AML patients and cell lines. Moreover, Knockdown of miR-100 led to the inhibition of viability and promotion of apoptosis in Kasumi-1 and MV-4-11 cells. miR-100 harbored the 3′UTR of ATM. Meanwhile, the expression of ATM was downregulated in bone marrow of AML patients and AML cell lines. Subsequently, a negative correlation between miR-100 and ATM in bone marrow of AML patients was also observed. Furthermore, ectopic expression of ATM repressed cell viability while enhanced apoptosis. Notably, loss of ATM attenuated the effect of miR-100 depletion on cell viability and apoptosis in AML cells. miR-100 participates in cell viability and apoptosis by targeting ATM in pediatric AML.

Recurrent Gene Mutations in Pediatric Patients with AML By Targeted Sequencing ―the Jccg Study, JPLSG AML-05―

Introduction Treatment outcomes of pediatric acute myeloid leukemia (AML) have improved via stratification therapy. However, relapse and mortality are still observed in 40% and 30% patients, respectively. Recently, comprehensive analyses using next-generation sequencing have revealed novel genetic alterations in adult AML patients, but insufficient analyses have been performed in pediatric AML patients. Further studies are warranted to evaluate the relationship between a more detailed genetic background and prognosis in pediatric AML patients. Methods We performed targeted sequencing using a 343-gene custom panel and next-generation sequencer in 302 pediatric patients with de novo AML who participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group from 2006 to 2010. One hundred and twenty patients with core binding factor (CBF)-AML, 52 with KMT2A rearrangements, 56 with normal karyotype (NK), 18 with complex karyotype (CK), and 57 with other karyotypes except for patients with acute megakaryocytic leukemia were enrolled. Correlations among gene mutations, other cytogenetic alterations, and clinical characteristics were investigated. Results and Discussion We found the following 6 recurrent gene mutations, which have not been frequently reported in pediatric AML: KMT2C (n = 13, 4.3%), PHF6 (n = 6, 2.0%), MGA (n = 13, 4.3%), TET2 (n = 11, 3.6%), JAK3 (n = 12, 4.0%), and GATA2 (n = 11, 3.6%). KMT2C mutations have been reported in adult and pediatric patients with CBF-AML and in a few patients with other types of AML; however, their clinical significance has not been revealed. Six of 13 patients with KMT2C mutations were found in CBF-AML, whereas the other 7 patients were detected in non CBF-AML (1 patient in KMT2A-rearrangement, 1 in CK, and 5 in other karyotypes). Loss of function mutations in KMT2C have also been reported to be associated with chemotherapy resistance in pediatric AML patients. Although no significant differences were found, 4 of 13 patients with KMT2C mutations did not achieve complete remission (p = .072), and 3 patients relapsed in &lt;12 months. Significant differences were observed in event-free survival (EFS) between patients with and without KMT2C mutations (3-year EFS 36.9% vs. 56.8%; p = .019). In adult AML, PHF6 mutations have been detected in 3% of patients and have been reported as poor prognostic factors. In 6 patients with PHF6 mutations, 3 patients had a normal karyotype, and 2 patients had a complex karyotype. No PHF6 mutations were detected in AML patients with CBF or KMT2A rearrangements. PHF6 mutations were significantly frequently detected in AML patients with myelodysplasia-related changes (4/6, p = .008). Although 5 of 6 patients with PHF6 mutations archived complete remission, finally, 5 patients relapsed or died. There were significant differences in EFS between patients with or without PHF6 mutations (3-year EFS 16.7% vs. 58.2%; p = .041). MGA mutations have been reported thus far in a few AML patients. MGA is a negative regulator of MYC signaling; thus, loss of functions of MGA is considered to contribute to tumorigenesis. However, the clinical significance of MGA mutations has not been reported. MGA mutations were significantly frequently detected in AML patients with FAB M2 (7/13, p = .020). In our cohort, 2 of 3 AML patients with DEK-NUP214 had MGA mutations (p = .004). We did not find correlations between MGA mutations and prognosis (3-year OS 83.3% vs. 75.5%; p= .488, 3-year EFS 66.7% vs. 56.7%; p = .507) TET2 mutations have been found in 8%-19% of adult AML cases and are considered poor prognostic factors. In previous reports, TET2 mutations were also detected in 3.8% of pediatric AML patients, but because of the small number of patients, the association between TET2 mutations and prognosis has not been revealed. TET2 mutations were significantly frequently detected in older AML patients (median age 13.95 vs. 9.0 years; p = .022) and in AML patients with NK (5/11, p = .035). Contrary to previous reports on adult AML, TET2 mutations were not associated with prognosis (3-year OS 77.9% vs. 75.9%; p= .688, 3-year EFS 54.5% vs. 57.4%; p = .907). This suggested that TET2 mutations have a different clinical significance between adult and pediatric AML patients. In conclusion, mutations in the KMT2C and PHF6 genes were associated with poor prognosis, but TET2 mutations were not. Further analysis should be needed about TET2 mutations. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents &amp; Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

Prognostic Significance of FOXO3 in Pediatric Acute Myeloid Leukemia (AML) Patients Treated with Bortezomib Addition to Standard Therapy: Results from a Children's Oncology Group Phase 3 Clinical Trial

The proteasome degrades unneeded and damaged proteins. Tumor cells highly depend on increased protein production and their degradation suggesting that malignant cells with a high proliferation index will be more sensitive to proteasome inhibition. The addition of the proteasome inhibitor Bortezomib (Velcade, 'BTZ') to standard pediatric AML chemotherapy (cytarabine, daunorubicin and etoposide, 'ADE') depleted leukemia-initiating cells in a phase 2 clinical trial in pediatric AML (pedi-AML) patients. A randomized phase 3 clinical trial was then conducted by the Children's Oncology Group (COG) comparing ADE and ADE+BTZ treatment regimes in pedi-AML (AAML1031). To determine if there were specific protein expression profiles that correlated with response to BTZ-containing chemotherapy, we analyzed key components of the proteome of pedi-AML that participated in the trial using reverse phase protein arrays (RPPA). RPPA-based expression of 293 validated antibodies was tested in 500 leukemia samples and compared to expression in CD34+ samples from healthy individuals (n=30). Among all proteins, FOXO3 expression was identified as the protein with the highest influence on outcome in the ADE group. The expression of FOXO3 was prognostic for event-free survival (EFS) in both univariate (HR = 0.56, 95% CI = 0.34-0.90, P = 0.017) and multivariate (HR = 0.55, 95% CI = 0.34-0.88, P = 0.013) analysis. All patients were divided into two clusters (low and high) based on their FOXO3 expression level using median FOXO3 expression in normal CD34+. Kaplan-Meier survival analysis showed poor OS (3 year OS 65.3% vs. 73.9%, P = 0.03) and EFS (3 year EFS, 42.8% vs. 55%, P = 0.01) in low FOXO3 expressors (n=119) compared to patients with high FOXO3 expression (n=291) (fig. 1A). Notably, the poor prognostic effect of low FOXO3 for OS was seen in ADE (3 year OS 60% vs. 72.3%, P = 0.03), but not in ADE-BTZ (3 year OS 70.3% vs. 75.3%, P = 0.23) (fig. 1B). This suggests that in particular patients with low FOXO3 may be eligible candidates for BTZ-addition. To validate our findings, we performed knockdown (KD) of FOXO3 using a short hairpin approach in OCI-AML3 (p53WT) and THP-1 (p53null) cell lines. KD FOXO3 in OCI-AML3 had a short-term growth advantage compared to controls (Day 4, P = 0.004), but not KD FOXO3 THP-1 cells suggesting a role for p53 in the FOXO3 functional pathway. KD FOXO3 cells were more resistant to doxorubicin and etoposide combination therapy than controls (P = 0.04), confirming our clinical observations. Since therapeutic regimes in AML are currently shifting towards Bcl-2 inhibition by Venetoclax (ABT-199, 'ABT'), we were eager to test whether BTZ and ABT could act in synergy, and if this is related to FOXO3 expression. Single low dose BTZ and ABT did not reduce cell numbers after 3 days, but were effective when used in combination (&lt;12% survival, P &lt; 0.001) in OCI-AML3 and THP-1. Although cell counts were lower regardless FOXO3 status in both cell lines, flow cytometry analysis using Annexin V staining revealed significant more apoptosis in KD FOXO3 THP-1 compared to controls. Using RPPA-based profiling, we identified a cluster of pedi-AMLs that benefitted from BTZ-addition to standard therapy. We hypothesize that AML patients with low FOXO3 levels have less activation of their apoptotic transcriptional programs and that this subgroup may benefit from proteasome blockage to prevent FOXO3 proteasomal degradation. The use of FOXO3 as potential biomarker may identify a prognostically adverse cluster in pedi-AML that could stratify patients for ADE+BTZ therapy. Patients with low FOXO3 expression may potentially benefit from ADE+BTZ in combination with ABT, a novel and promising combination approach in AML that warrants further investigation. Figure 1. A. Kaplan meier analysis comparing overall survival (OS) and event-free survival (EFS) between low (n=119, blue) and high (n=291, red) FOXO3 expression in pedi-AML patients. Children with low FOXO3 had significantly poorer OS (P = 0.033, Wilcoxon) and EFS (P = 0.011, Wilcoxon). B. Overall survival between ADE (solid) and ADE+BTZ (dashed) treated patients based on low (blue) or high (red) FOXO3 protein expression. The poor prognostic effect of FOXO3 is present in ADE, but not in ADE+BTZ treated patients (P = 0.028, Wilcoxon). Figure 1 Disclosures No relevant conflicts of interest to declare.

Adoptively Transferred Donor-Derived Cytokine Induced Memory-like NK Cells Persist and Induce Remission in Pediatric Patient with Relapsed Acute Myeloid Leukemia after Hematopoietic Cell Transplantation

Acute myeloid leukemia (AML) accounts for 18% of pediatric leukemias. For high-risk AML, standard treatment includes multi-agent chemotherapy followed allogeneic hematopoietic cell transplantation (HCT). Despite a high remission rate, 50% of patients relapse and have a poor prognosis with &lt; 20% of patients surviving more than 3 years. Salvage chemotherapy alone or combined with donor lymphocyte infusions (DLI) have little curative potential, and new treatment strategies are needed for relapsed-refractory AML. Previous studies have shown that natural killer (NK) cells can be stimulated ex vivo with IL-12/15/18 to generate a memory-like phenotype with enhanced anti-leukemia effect. In adults with relapsed-refractory AML, adoptive transfer of MHC-haploidentical cytokine-induced memory-like (CIML or ML) NK cells induced remission in 54% of patients (PMID27655849). The infused donor ML NK cells expand in vivo but are rapidly eliminated following recovery of recipient T cells, providing a window of therapeutic activity of 2-3 weeks. We sought to test the safety and efficacy of ML NK cells for treatment of pediatric/young adult patients with post-HCT relapsed AML. We hypothesized that ML NK cells derived from the HCT donor would be well-tolerated, exhibit anti-leukemia activity, and expand with prolonged persistence following transfer into pediatric AML patients. Here, we report the results of the first pediatric patient treated on a phase I clinical trial using ML NK cell therapy for relapsed AML after allogeneic HCT (NCT03068819). Briefly, patients are treated with FLAG (fludarabine, cytarabine and granulocyte colony stimulating factor) salvage chemotherapy to reduce the bulk of AML and provide lymphodepletion for ML NK cell expansion. Two weeks after chemotherapy, a non-mobilized leukapheresis product is collected from the original HCT donor and processed into a T cell-based DLI and ML NK cells. The T cell DLI (1 x 106 T cells/kg) is immediately infused, and the ML NK cells are generated by stimulation with IL-12/15/18 ex vivo for 12-16 hours and then infused (10x106/kg). An 18-month-old male with infant AML had relapse of his leukemia 3 months after MHC-haploidentical HCT. Treatment with chemotherapy, including mitoxantrone and daunorubicin-cytarabine liposome, failed to induce remission. At the time of enrollment on the phase I trial, he had AML blasts in his bone marrow (Table 1). He was treated with FLAG chemotherapy followed by infusion of DLI and ML NK cells from the original haploidentical HCT donor. Assessment at 30 days, 3 months and 6 months post NK cell infusion showed complete remission with no evidence of leukemia and full donor engraftment. Remarkably, donor-derived ML NK cells expanded to 77% of donor lymphocytes on day 28 and still comprised 24% percent of lymphocytes at 6 months post infusion (Figure 1A-C). The expanded donor NK cell phenotype was consistent with ML NK cells (e.g., NKG2A+KIR+) utilizing CyTOF multidimensional analysis previously confirmed to identify ML NK cells (Figure 1D). The ML NK cells were functional as demonstrated by leukemia-triggered IFN-γ production immediately ex vivo from day 7-28 samples (Figure 1E-F). The patient's clinical course was complicated by mild gastrointestinal graft-versus-host disease that resolved with low-dose steroids and tociluzimab. These early results demonstrate proof-of-principle that adoptive transfer of donor-derived ML NK cells in combination with DLI is feasible and offers a novel immunotherapy option for patients with relapsed AML after HCT. Moreover, in this T and NK cell compatible immune environment post-HCT, donor ML NK cells expand and persist robustly in vivo for &gt; 6 months without exogenous cytokine support and have potent anti-leukemic activity. Thus, ML NK cells are a cellular therapy platform to treat AML that has relapsed after allogeneic HCT. Disclosures Cashen: Celgene: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Novartis: Other: Speaker's Bureau. Fehniger:Horizon Pharma PLC: Other: Consultancy (Spouse); Cyto-Sen Therapeutics: Consultancy.

Area-Based Socioeconomic Disparities in Survival of Children with Newly Diagnosed Acute Myeloid Leukemia: A Report from the Children's Oncology Group

Introduction: Income, education, and health insurance coverage have been shown to influence access to appropriate oncology care, impacting detection and treatment. We sought to evaluate the role of area-based measures of socioeconomic status in contributing to outcome disparities on the two most recent Children's Oncology Group (COG) Phase 3 clinical trials for acute myeloid leukemia (AML), AAML0531 and AAML1031. We hypothesized that pediatric AML patients from low income and low education zip codes have inferior five-year overall survival (OS) and event-free survival (EFS) relative to patients from middle income and more educated zip codes. Methods: Patients enrolled on AAML0531 and AAML1031 were included. Patients with Down syndrome (n=5), FLT3/ITD high allelic ratio (n=264), in AAML1031 Arm D (n=332), and patients whose zip code was not able to be mapped to a US Census area were excluded (n=60). Patients were observed from enrollment on study through last available follow up. Zip code level median annual household income was the primary exposure and was categorized as follows: Poverty: &amp;lt;$24,250 (federal poverty line); Low: $24,250-56,516; Middle/High &amp;gt;$56,516. Secondary exposures of interest included zip code level educational attainment and insurance type (Medicaid Only vs other insurance) at AML diagnosis. Standard descriptive statistics were used to compare patient characteristics by levels of exposure; the Kaplan Meier method was used to estimate OS (defined as time from study entry to death) and EFS (time from study entry until failure to achieve CR during induction, relapse, or death). Cox proportional hazards models were used to estimate hazard ratios (HR) for OS and EFS. Measures of association were adjusted for known risk factors for mortality including cytogenetic/mutation risk group, gemtuzumab (GO) receipt, race, and age. Logistic regression analyses were used to estimate odds ratios (OR) for early mortality (defined as death during induction). Results: Of 2387 patients enrolled on AAML0531 and AAML1031, 1726 met inclusion criteria for the overall analysis. Due to missing covariate data, 1467 patients were included in the final model. Race/ethnicity differed significantly by area-based income, area-based education, and insurance type with a higher proportion of Black and Hispanic patients living in poverty, low income, and low education areas, and having Medicaid only insurance. Lower area-based income was associated with lower OS (43% in poverty vs. 61% in low income vs. 68% in middle/high income; p = 0.004) and EFS (34% in poverty vs. 46% in low income vs. 54% in middle/high income; p = 0.005), shown in Figure 1. Lower area-based educational attainment was also associated with lower OS (58% in Quartile 4 (lower education) vs. 70% in Quartile 1 (higher education); p = 0.005 across quartiles) and EFS (44% in Q4 vs. 54% in Q1; p = 0.03 across quartiles). Patients with Medicaid Only insurance had lower OS (59 ± 5% vs. 66 ± 3%: p = 0.01) but similar EFS (48 ± 5% vs. 50 ± 3%: p = 0.33). In a full multivariable model, differences in survival by area-based educational attainment and insurance type resolved suggesting that observed crude associations were explained by confounding by area-based income combined with established risk factors. Patients from middle/high income areas experienced 25% lower risk of mortality compared to patients from low income areas (OS: crude HR 0.74 95% CI 0.62, 0.89; adjusted HR 0.79 95% CI 0.63, 0.99) with similar differences in EFS (crude HR 0.79 95% CI 0.69, 0.92; adjusted HR 0.77 95% CI 0.65, 0.89). There was no meaningful confounding of the income-survival association detected as evidenced by unchanged magnitudes of association following adjustment for area-based education, insurance, and established risk factors. Area-based low income was associated with both higher risk of early death (crude OR: 2.43 95% CI 1.04, 5.69) and treatment-related mortality on therapy (11.1 ± 10.5% vs. 3.7 ± 2.5%, p = 0.03) compared to area-based middle/high income. Conclusions: Lower area-based income and education were associated with significantly inferior EFS and OS among patients with AML on the last two Phase 3 COG trials. Moreover, zip-code based low SES is an independent risk factor for mortality in pediatric AML. Additional studies to understand mechanisms of observed socioeconomic disparities in treatment outcomes will inform interventions that may mitigate these inequities. Disclosures Fisher: Pfizer: Research Funding; Astellas: Other: Data Safety Monitoring Board Chair for an antifungal study; Merck: Research Funding.

A 5-Gene Ara-C, Daunorubicin and Etoposide (ADE) Drug Response Score As a Prognostic Tool to Predict AML Treatment Outcome

Introduction: Cytarabine, daunorubicin and etoposide (ADE) are commonly used for remission and intensification of pediatric acute myeloid leukemia (AML). However, development of drug resistance is a major cause of treatment failure. In this study, we performed a comprehensive evaluation of expression levels of genes of pharmacological significance (pharmacokinetic /pharmacodynamic) to ADE and derived a drug response score predictive of treatment outcomes in pediatric AML patients. Methods: This study included 163 cases (median age=8.79 year, range= (0.013-21.1)) with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression and clinical data available. We used a penalized LASSO regression algorithm (glmnet R-package) to fit a cox regression model on diagnostic leukemic cell gene expression levels of 66 genes of pharmacological significance to ADE. We performed 1000 bootstraps of LASSO regression with event free survival (EFS) as the outcome variable and the five genes represented in at least 95% of the models were included to build an ADE-Response Score (ADE-RS) equation. Patients were classified into low or high score groups using recursive portioning implemented in Rpart-R package and evaluated for association with minimal residual disease after induction I (MRD1), EFS and overall survival (OS). ADE response score equation was further validated using RNA-Seq gene-expression data obtained from diagnostic samples of 432 pediatric AML patients enrolled in Children's Oncology Group (COG) AAML0531 and AAML03P1 treatment protocols. Results: After applying LASSO regression, we defined the equation: ADE-RS = (0.128 x DCTD) - (0.0993 x TOP2A) + (0.212 x ABCC1) - (0.113 x MPO) - (0.126 x CBR1) to develop ADE-response score (ADE-RS), followed by classifying patients into low (60%; 98 patients) or high (40%; 65 patients) score groups. Patients in the high ADE-RS group had significantly worse EFS (HR=4.07(2.43-6.84), P &amp;lt; 0.0001; Figure 1A) and OS (HR= 4.54(2.42-8.49), P&amp;lt;0.0001) and higher proportion of MRD1 positive patients (P=0.014; Figure 1B) compared to patients in the low ADE-RS group. These results were validated in an independent COG cohort, where patients in the high score group demonstrated higher MRD1 positivity (P=0.0005; Figure 1D) and inferior EFS (HR=1.32(1.01-1.73), P=0.044; Figure 1C). We recently developed a six-gene leukemic stem cell score (pLSC6 score) that was associated with risk-groups (P&amp;lt;0.00001) and outcome (oral presentation ASH2018, Under-review, Leukemia). We further integrated pLSC6 and ADE-RS and observed significantly better prediction of treatment outcomes in AML02, COG and TCGA cohorts. Based on pLSC6 and ADE-response scores, patients were classified into three groups; 1) Low/Low:pLSC6/ADE-RS; for patients with low pLSC6 and low ADE-RS. 2) Low/High:pLSC6/ADE-RS: for patients in low pLSC6 and high ADE-RS or vice versa.3) High/High:pLSC6/ADE-RS: for patients in high pLSC6 and high ADE-RS. In all study cohorts, patients in low/low pLSC6-ADE-RS group demonstrated better outcomes compared to the low-high and the high/high score groups (EFS in AML02 cohort; Figure 1E and OS; Figure 1F). In a multivariable cox-regression models that included pLSC6-ADE response score groups, MRD1 status, risk groups, WBC at diagnosis and age in AML02 cohort, high pLSC6-ADE score group was found significantly associated with poor EFS (HR=6.02(2.71-13.2), P&amp;lt;0.00001; Figure 1G) and was the only significant predictor of poor OS (HR=8.3(2.9-23.99), P&amp;lt;0.00001; Figure 1H) Discussion: In this study, we defined a pharmacological response score focused on key genes of PK/PD significance to ADE. We further integrated LSC score with the ADE response score to improve our ability to predict treatment outcome in AML patients across different clinical trials. ADE-RS was composed of five genes: DCTD, which is a deaminase, involved in ara-C inactivation; CBR1, a carbonyl reductase involved in inactivation of daunorubicin (DNR); MPO, myeloperoxidase, an etoposide activator; ABCC1, an efflux transporter of DNR and etoposide; and TOP2A, DNA topoisomerase II alpha, which is a target for DNR and etoposide. Integrated pLSC6 and ADE-RS has a potential to predict treatment outcomes using diagnostic gene expression levels and accordingly develop treatment strategies to improve treatment outcome. Figure 1 Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding.