Outcome In Pediatric Acute Myeloid Leukemia Research Articles

Identifying a Relapse Signature in CBFB-MYH11 Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer characterized by many mutational and cytogenic subtypes with varied risk strata. The CBFB-MYH11 fusion is a subtype associated with favorable disease outcomes in pediatric patients despite a relapse rate of over 30%. While overall survival remains relatively high, above 65%, more work can be done to improve outcomes. Considering the frequent rate of relapse, identifying a genetic signature that can predict relapse at diagnosis is vital to outcome improvement. Through k-means clustering, differential expression analyses, and recursive feature elimination, we were able to identify twenty genes that were strongly associated with relapse risk in the CBFB-MYH11 fusion at the time of diagnosis. All analysis was performed in R, version 4.3.0, including all listed packages. Ribodepeleted RNA sequencing data was generated on 3023 patients and included a total of 119 diagnostic CBFB-MYH11; 64 were considered non-relapse and 55 were considered relapse. A Poly(A)-seq validation cohort was generated on 495 patients included 60 diagnostic CBFB-MYH11. Of these patients, 42 were considered non-relapse and 18 were considered relapse. Unsupervised clustering was performed using a k-means algorithm in the ComplexHeatmap package which resulted in three “clusters” of 48, 54, and 17 samples with distinct variation in z-scores when the most-variable 500 genes were plotted. These clusters were labeled 1, 2, and 3 respectively. Survival analysis was then performed using Kaplan-Meier method and curves were stratified by cluster. Clusters 1 and 2 had nearly identical event-free survival rates near 60%, yet cluster 3 had an event-free survival rate of less than 12% (p<0.001). Clusters also differentiated in overall survival, with cluster 3's survival rate near 50% compared to 80% and 95% in cluster 1 and 2 respectively (p<0.001). To identify the underpinnings of aggressive relapse in CBFB-MYH11, differential expression analysis was performed using DESeq2, contrasting cluster 3 against the other clusters. yielding a list of 6,204 differentially expressed genes (p-adjusted cutoff of 0.05, using a Benjamini-Hochberg procedure to reduce false discovery rate). The list of differentially expressed genes were input into a feature elimination machine learning model using the RandomForest package. This model identified 20 genes that were most predictive of relapse risk with diminishing returns when the number of genes was increased. The list of 20 genes in order of predictive influence includes: CBWD3, DRAP1, GNL3LP1, MAF1, GRAMD1C, MAGED2, TICAM1, MPP1, NBPF11, RAB13, RN7SL329P, HAL, TLK2P2, CD151, XRCC6, HIST1H2AM, MSANTD3-TMEFF1, CDCA4, LRRC8A, and IPCEF1. CBWD3 was the most predictive gene and is under-expressed in relapsing patients in CBFB-MYH11. When survival analysis was performed on CBWD3, above-median expressors had an event-free survival probability of near 75% and below-median expressors had an event-free survival probability of just 30% with a combined p-value of less than 0.0001. To validate our findings, we used a separate CBFB-MYH11-positive cohort that had been sequenced using Poly(A)-seq, which is sequenced at a lower depth than ribodepeleted RNA-seq data. We saw similar event-free survival estimates in both cohorts for some genes-including CBWD3 and GNL3LP1. The 20 genes of interest were then input into a Random Forest model, using 70% of the CBFB-MYH11 cohort as a training subset. When the training model was optimized, relapse was correctly predicted in 32 of 39 samples (82%) and non-relapse was correctly predicted in 40 of 45 samples (89%). Future analyses will aim to test the efficacy of the model on the testing subset and the Poly(A) cohort for validation. A LASSO-Cox regression will also be used to calculate variable coefficients for risk determination in a clinical setting. While the fusion group CBFB-MYH11 is associated with improved survival outcomes in pediatric AML, relapse rate remains high. Through k-means clustering, differential expression analysis, and recursive feature elimination, we've identified a subset of genes that have predictive power in determining relapse risk. As our machine learning model approaches improve, a robust relapse signature may be identified and used to improve patient outcomes.

Early Peripheral Blood Blast Clearance As a Prognostic Marker for Early Treatment Response in Pediatric Acute Myeloid Leukemia

Background and Significance The cure rate of pediatric acute myeloid leukemia (AML) has improved significantly; however, relapses still occur in up to 30% of the total patient population in high-income countries (HICs), and in an even higher percentage of patients in low- and middle-income countries (LMICs). Early treatment response, cytogenetics and minimal residual disease (MRD) are well-known prognostic factors for treatment outcome; however, sophisticated techniques for identifying prognostic factors are often lacking in LMICs, and are sometimes unsuccessful in HICs. Early treatment response assessed by a bone marrow (BM) aspirate on day 15 or day 22 (D22) (depending on the applicable protocol) after initiation of chemotherapy is a known prognostic factor for outcome in patients with pediatric AML, but does not allow modifications of the first induction chemotherapy course (Creutzig 2014, Abrahamsson 2011, Kern 2003). In both pediatric acute lymphoid leukemia (ALL) and adult AML, early peripheral blood blast clearance (PBBC) is a known early indicator of treatment response (Manabe 2008, Arellano 2012), whereas to the best of our knowledge, this has not yet been reported in pediatric AML. In this study, we evaluated the significance of time to complete PBBC, and rate of PBBC after initiation of chemotherapy, in relation to disease status in BM D22, as measure of early treatment response that has prognostic significance. Study Design and Methods Pediatric patients with AML, aged 0-18 years, that were treated according to the NOPHO-DBH AML-2012 protocol were included. The first 5 days of induction therapy consisted of etoposide, followed by 7 days of low-dose cytarabine and mitoxantrone or liposomal daunorubicin. Peripheral blood (PB) was assessed by morphology at the time of diagnosis, daily in the first week, and thrice weekly until D22 or until occurrence of complete clearance of blasts. BM D22 was assessed by morphology and flow cytometry. A receiver operating characteristic (ROC) analysis was performed to evaluate the predictive power of days from starting chemotherapy to complete PBBC. The correlation between time to complete PBBC and BM D22 results was analyzed using a logistic regression model. Multivariable logistic regression models were applied for the association after adjusting for effects of other covariates. Rate of PBBC was defined as the percentage of the absolute PB blast count on the day of diagnosis that cleared with each day of therapy, on average, until D22 or the day of complete PBBC. Results In total, 319 patients were included, out of whom 241 were eligible for analysis, and 78 not eligible due to missing or incomplete data. Day 9 after initiation of chemotherapy was identified as the most discriminating cut-off point for predicting BM D22 response. Patients were categorized into early PBBC (≤9 days; n=159) and delayed PBBC (>9 days; n=82) groups. Early PBBC was associated with having <5% AML cells in BM D22 as assessed by both morphology (OR 3.11; P=0.005) and flow cytometry (OR 2.73; P=0.041). Early PBBC also showed a much lower likelihood of having >15% AML cells in BM D22 by morphology (OR 0.08; P=0.002). None of the early PBBC patients had >15% AML cells in BM D22 by flow cytometry. For 217 patients (selection due to missing data [n=24]), the rate of PBBC could be calculated, and a discriminative cut-off value of 11% was identified. Patients were categorized into high rate of PBBC (>11%; n=132) and low rate of PBBC (≤11%; n=85). High rate of PBBC was associated with having <5% AML cells in BM D22 by flow cytometry (OR 4.30; P=0.007) and negative MRD status (OR 2.02; P=0.004), while also showing a strong association with a lower likelihood of >15% AML cells in BM D22 (OR 0.11; P=0.007) by morphology. None of the high rate PBBC patients had >15% AML cells in BM D22 by flow cytometry. Conclusion Both early complete PBBC and rate of PBBC after initiation of chemotherapy holds promise as prognostic marker for clinical outcome in pediatric AML, as it correlates significantly with BM D22 response. These findings may offer an early and easily accessible prognostic factor, particularly beneficial for LMICs, as well as for patients with unsuccessful MRD analyses.

Genome-wide CRISPR/Cas9 screen identifies etoposide response modulators associated with clinical outcomes in pediatric AML.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.

Impact of SAMHD1 Pharmacogenetics on Clinical Outcome in Pediatric AML

Acute myeloid leukemia (AML) is a heterogeneous disease characterized in part by genetic and epigenetic alterations. Cytarabine arabinoside (Ara-C) has been the cornerstone of chemotherapy treatment for patients diagnosed with AML for decades. Following cellular uptake, ara-C is phosphorylated into its active metabolite, ara-CTP, which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in ara-C activation/inactivation pathway (Fig 1A), can impact intracellular abundance of ara-CTP and thus its therapeutic benefit. Recently, deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) was shown to limit the efficacy of ara-C by intracellularly hydrolyzing its active metabolite (PMID: 27991919). Other nucleoside analogs, such as clofarabine, fludarabine, and gemcitabine have also been shown to be substrates of SAMHD1 (PMID:30305425). Among adult AML patients, higher SAMHD1 expression in the leukemic cells has been found to correlate with poor outcome (PMID: 30341277). However, whether genetic variation in SAMHD1 has any impact on the clinical outcomes in pediatric AML patients has not been evaluated in depth. In this study, we looked into 25 single nucleotide polymorphisms (SNPs) within SAMHD1 gene for their association with clinical outcome of newly diagnosed pediatric AML patients in 2 cohorts (multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=231] clinical trials). Briefly, genotypes for the 25 SNPs within SAMHD1 genes were obtained from Illumina 2.5 Omni data of AML patients. Association analysis was conducted using logistic regression models comparing minimal residual disease (MRD) positivity after treatment with chemotherapy using an unadjusted model and an adjusted model stratified by initial risk assignment of the patient determined at diagnosis (i.e. low, standard, and high). MRD positivity was defined as one or more leukemic cell per 1000 mononuclear bone-marrow cells. Cox proportional hazard models were used to examine the association of the SNPs with EFS and OS and considered three modes of inheritance. Cox regression models were performed with or without adjusting for provisional risk group assignment at time of diagnosis. Significance levels for association of SNP with clinical outcome were set at p < 0.05.Three top SNPs significantly associated with clinical outcomes were all localized in the 3'UTR region of SAMHD1(Fig 1B). Presence of the variant allele of rs7265241 was associated with a lower EFS and OS in both AML02 and AML08 cohorts. Figure 1C shows the results for OS in AML02 (HR = 2.24, 95% CI (1.05-3.55), p=0.02) and AML08 (HR = 1.52, 95% CI (1.04-1.99), p=0.01). Another 3'UTR SNP, rs1291128 was associated with poor OS in AML02 and in the clofarabine plus ara-C treatment arm in AML08 trial. For rs1291141, T allele was associated with higher MRD positivity (OR: 2.12, p=0.01) in AML02 and poor OS (HR = 1.55, 95% CI (1.12-2.14), p=0.008) in AML08(Fig 1D). Consistent with the clinical outcome results, rs1291141 T allele was also associated with higher SAMHD1 expression in the whole blood (p=8.1e-14) and several other tissues in the GTEx database. https://gtexportal.org/home/Our results suggest that genetic variation in SAMHD1 is associated with clinical outcomes in pediatric AML patients. Future studies are aimed at looking at haplotypes and SNP-SNP combinations to establish the impact SAMHD1 pharmacogenomics on its expression within leukemic cells and subsequent response to nucleoside analogs in AML patients. SAMHD1 has also been implicated as a tumor suppressor; thus, future studies will evaluate its role both as a proliferation regulator and in drug resistance.Acknowledgements: NIH-R01-CA132946 (Lamba and Pounds), University of Florida Opportunity Seed Grant (Lamba), American Lebanese Syrian Associated Charities (ALSAC), and American Society of Hematology Bridge funding (Lamba) funded the study. We thank Drs. Campana, Coustan-Smith, and Raimondi for MRD and cytogenetic data. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Calcitonin receptor‐like (CALCRL) is a marker of stemness and an independent predictor of outcome in pediatric AML

AbstractWe have recently identified the G protein-coupled neuropeptide receptor calcitonin receptor‐like (CALCRL) as an independent prognostic biomarker and a therapeutic target in more than 1500 adult patients with acute myeloid leukemia (AML). Here, we confirmed CALCRL expression as a prognostic factor in a cohort of 284 pediatric patients with AML. High CALCRL expression was independently associated with event-free survival (hazard ratio [HR], 1.87; 95% confidence interval [CI], 1.36-2.57; P = .0001), overall survival (HR, 1.55; 95% CI, 1.06-2.27; P = .025), and cumulative incidence of relapse (HR, 2.10; 95% CI, 1.49-1.96; P < .0001) when adjusting for age, white blood cell count, and genetic risk. Despite its association with leukemia stem cell signatures, CALCRL expression remained associated with all end points when compared with the 17-gene leukemic stem cell score. The strong association of CALCRL expression with the risk of relapse also in the pediatric population supports its role as novel age-independent master regulator of relapse-initiating, drug-tolerant AML cells in humans.

Treatment of acute myeloid leukemia in children: A practical perspective.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease that requires a multifaceted treatment approach. Although outcomes for low-risk AML have improved significantly over recent decades, high-risk AML continues to be associated with an adverse prognosis. Recent advances in molecular diagnostics, risk stratification, and supportive care have contributed to improvements in outcomes in pediatric AML. Targeted approaches, for example, the use of tyrosine kinase inhibitors to treat FLT3-ITD AML, offer promise and are currently undergoing clinical investigation in pediatric patients. New approaches to hematopoietic stem cell transplantation, including the use of haploidentical donors, are significantly expanding donor options for patients with high-risk AML. This review provides an overview of recent advances in the treatment of pediatric AML that are likely to have clinical impact and reshape the standard of care.

Integrated Stem Cell Signature and Cytomolecular Risk Determination in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a therapeutic challenge with high mortality rates despite intensive and myeloablative therapies. Structural and sequence alterations have been linked to outcomes in pediatric AML and have been used for risk-based therapy allocation with modest success. Given the vast heterogeneity of AML, conventional cytogenetic and mutational (cytomolecular) biomarkers have not yielded a robust prognostic model: nearly one-third of pediatric patients deemed "low risk" relapse and, inversely, approximately one-third of those in "high risk" categories have favorable outcomes. AML studies in adults previously identified a leukemia stem cell score (LSC17) that was highly prognostic across five independent cohorts comprised of adult patients with diverse AML subtypes (n = 908). We reasoned that incorporating a similar scoring system in pediatric AML would lead to improved prognostic risk models. To assess for the effects of LSC17 on pediatric AML, we leveraged transcriptome sequencing data from bone marrow aspirates and peripheral blood collected from 1,503 children, adolescents, and young adults with AML at the time of diagnosis. Patients were enrolled on one of three upfront phase III Children's Oncology Group trials spanning the past three decades: CCG-2961, AAML0531, and AAML1031. In aggregate, patients with a high LSC17 score had an event free survival (EFS) of 36.9 ± 3.5% at 5 years from diagnosis compared to 55.3 ± 3.7% for those with low LSC17 scores (p &amp;lt; 0.0001). (Figure 1A) LSC17 scores were also associated with adverse overall survival (OS): 51.9 ± 3.9% versus 73.8 ± 3.5% (p &amp;lt; 0.0001) (data not shown). Intriguingly, we found that LSC17 scores significantly cluster within fusion groups and that median LSC17 scores closely correlate with survival based on fusion status (Figure 1B). Thus, when the impact of LSC17 scores was evaluated in the context of established cytomolecular risk groups, LSC17 scores were no longer predictive of outcome (Figure 1C). We then asked whether LSC gene expression data could be utilized to generate a more robust risk classification schema in the context of disease defining structural variants. Importantly, AMLs diagnosed in children, adolescents, and young adults are associated with frequent driver gene fusion alterations that also play an important role in risk stratification and transcriptional landscape (Figure 1D). We went on to confirm that AML fusion groups occupy distinct transcriptional stages of hematopoietic stem cell and myeloid progenitor maturation based on gene set enrichment analysis (GSEA) using normal hematopoiesis transcriptome experiments as their reference (data not shown). To develop more predictive biomarkers related to stemness, we used the 54 original LSC genes identified by Ng S, et al. and performed linear regression based on a least absolute shrinkage and selection operator (LASSO) algorithm to fit a Cox regression model for patients within each fusion group. The study population was divided into discovery (n = 752) and validation (n = 752) cohorts using stratified randomization based on fusion status (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A, NUP98, CBFA2T3-GLIS2, and Other/None). In the discovery cohort, we identified distinct LSC signatures that best distinguished outcome cohorts in patients with conventional high/standard risk disease (KMT2A, NUP98, and Other/None fusions) (Figure 1E). For patients deemed favorable risk (RUNX1-RUNX1T1 and CBFB-MYH11 or core binding factor/CBF), LSC signatures were not reliably predictive based on "leave one out" cross validation. Therefore, we performed multivariable analysis incorporating clinical, mutational, and transcriptional signatures to determine the factors that best discriminated outcomes with CBF AML, and found GLIS2-like transcriptional signatures were most predictive. These cytomolecular and LSC (CM-LSC) biomarkers were then combined to build a robust risk determination model that was then validated in an independent cohort (Figure 1F). This study demonstrates that a 54 LSC gene expression panel can enhance the predictive power of conventional cytomolecular markers and can more effectively partition patients into risk groups. Figure 1 Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019).

Hyperleukocytosis predicts inferior clinical outcome in pediatric acute myeloid leukemia

ABSTRACT Objectives Hyperleukocytosis (HL) is a laboratory abnormality commonly presented in patients with acute myeloid leukemia (AML). However, large cohort studies on the clinical significance of HL in pediatric AML are paucity. Moreover, the effect of stem cell transplantation in HL patients remains unknown. Methods The clinical profiles of 885 pediatric patients with AML were downloaded from the TARGET dataset. HL was defined as an initial peripheral WBC count of ≥ 100 ×109/L. We analyzed the prevalence, clinical profile and prognosis of HL in these patients. Results The frequency of HL among all the pediatric AML was 22.6%. FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD) mutation and gene fusion of NUP98/NSD1 occurred with higher incidence in HL patients. Overall, HL was associated with a low induction complete remission rate, and high risk of induction death. Moreover, HL predicted a significantly inferior 5-year event-free survival (EFS) (P < 0.001) and a trend of inferior 5-year overall survival (OS) (P = 0.059). However, compared with chemotherapy, stem cell transplantation had no significant effect on the survival of HL patients in terms of 5-year leukemia-free survival (P = 0.449) or OS (P = 0.447). Multivariate analysis revealed that HL was an independent prognosis factor for EFS (Hazard ratio:1.352, P = 0.013) but not for OS (Hazard ratio:1.225, P = 0.170) in pediatric AML. Conclusion HL might predict inferior clinical outcome in pediatric AML. SCT is an effective therapy for AML, but it may have no better effect on the survival of patients with HL, compared to chemotherapy.

Effect of Ara-C on T-Cell Function and Flotetuzumab Activity in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) in children still has a poor prognosis despite the use of maximally intensive chemotherapy associated with severe short-term and long-term side effects. Therefore, development of targeted therapeutics is necessary to improve outcomes in pediatric AML. CD123 (IL3RA) is overexpressed in most of pediatric AML patients (Bras et al., Cytometry B Clin Cytom, 963:134, 2019) and has been pursued as a target for immunotherapy. The efficacy of a dual affinity retargeting agent (CD123xCD3; MGD006 or flotetuzumab), was evaluated in two patient-derived xenograft models of pediatric AML. In addition, concurrent administration of cytarabine with MGD006 was performed to determine the effect of cytarabine on T-cell function and flotetuzumab efficacy. NSG-SGM3 mice were transplanted with 2.5 x 106 cells AML PDX cells. After 18 days post transplant, when human cells were detectable in mouse blood, mice were randomly assigned to one of 8 treatment groups - 1) untreated, 2) T-cells, 3) T-cells with MGD006 (0.5 mg/Kg, Q5d), 4) T-cells with Ara-C (50 mg/Kg, Q5d), 5) T-cells with concurrent administration of Ara-C and MGD006, 6) MDG006 and 7) Ara-C. Mice belonging to groups 2-5 were intravenously injected with 2.0 x 106 human pan T-cells (StemCell Technologies, Cat No. 70024.1), prior to i.p. administration of MGD006 and/or Ara-C. Mice were monitored daily and peripheral blood was collected periodically to evaluate leukemia progression (CD45+CD3-) and T-cell expansion (CD3+CD45+) by flow cytometry. Mice were euthanized when they showed systemic signs of leukemia based on weight and body condition score. The growth of human cell percentage in mouse blood over time was plotted and Kaplan-Meier survival plots were generated. On the day after treatment was terminated, AML cell percentage was greatly reduced, in mice treated with T-cells + MGD006 (Fig. 1, group 3) or T-cells + MGD006 + Ara-C (group 5), compared to the other groups. In addition, exposure to MGD006 (groups 3 and 5) enhanced expansion of adoptively transferred T-cells compared to AML PDX mice receiving T-cells alone (group 2). The ability of MDG006 to enhance the expansion of T-cells in vivo was not attenuated by treatment with Ara-C. Similar results were obtained in a second PDX model (Fig. 2). Taken together, MGD006 enhanced T-cell engraftment with or without Ara-C accompanied by marked reduction in the burden of AML blats in the peripheral blood. As expected, MGD006 in the absence of the effector T-cells (group 6) had minimal effect on reducing leukemic burden or survival (Fig. 3A, B). Mice injected with T-cells alone (group 2) showed 40-day improvement in survival, likely due to the allogeneic effect of T-cells. Regardless, the addition of MGD006 with T-cells (group 3) amplified the effect as mice did not reach experimental endpoints upon study termination at 210 days (Fig. 3B, brown line). Ara-C treatment (group 7) delayed leukemia progression and prolonged median survival by 22.5 days compared to untreated mice (Fig. 4A, B). Consistent with the T-cell expansion induced by Ara-C (Fig. 1), mice treated with T-cells + Ara-C (group 4) survived longer (median survival 180 days) than those treated with Ara-C (group 7) or T-cells alone (group 2) (median survival 116 and 135 days respectively). Mice administered with MGD006 concurrently with Ara-C following T-cell injection (group 5) also did not reach experimental endpoints upon study termination (Fig. 3B, purple line). These mice had 0.1% residual AML cells when the study was terminated (Fig. 3A, solid purple line), which was significantly lower than mice receiving T-cells + MGD006 (group 3, 2% AML cells, P=0.0047). These data demonstrate the activity of MGD006 in the presence of T-cells in prolonging survival in pediatric AML PDX models. Inclusion of Ara-C to this regimen was more efficient in reducing AML burden. Disclosures Bonvini: MacroGenics, Inc.: Employment, Equity Ownership. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership.

Prognostic impact of mitochondrial DNA D-loop variations in pediatric acute myeloid leukemia.

The role of mitochondrial DNA (mt-DNA) changes, especially those in the regulatory D-loop region in Acute Myeloid Leukemia (AML) remains investigational. Consecutive 151 de novo pediatric AML patients, (≤18 yr) were prospectively enrolled from June 2013-August 2016, to assess the prognostic impact of mt-DNA D-loop variations (somatic/germline) on survival. For each patient, D-loop region was sequenced on baseline bone marrow and buccal swab, and mother’s blood sample. In 151 AML subjects, 1490 variations were found at 237 positions; 80.9% were germline and 19.1% somatic. The mean number of variations per position was 6.3. Variations with frequency ≥6 were analyzed for their impact on survival and 4 categories were created, namely “somatic-protective”, “somatic-hazardous”, “germline-protective” and “germline- hazardous”. Although, somatic-protective could not predict event free survival (EFS) or overall survival (OS), somatic-hazardous [(OS) HR = 2.33, p = 0.06] and germline-hazardous [(OS) HR = 2.85, p < 0.01] significantly predicted OS and EFS. Notably, the germline-protective, could significantly predict EFS (HR = 0.31, p = 0.03) and OS (HR = 0.19, p < 0.01), only when variations at ≥2 positions were present. On multivariate analysis, three positions namely 16111, 16126, 16362 and karyotype were found to be predictive of EFS. A prognostic index (PI) was developed using nomogram PI = (0.8*karyotype) + (1.0*c16111) + (0.7*t16362) + (1.2*t16126). Hazard ratio for EFS increased significantly with increasing PI reaching to a maximum of 3.3 (p < 0.01). In conclusion, the impact of mt-DNA D-loop variations on outcomes in pediatric AML depends on their nature (germline/somatic), position and mutational burden, highlighting their potential role as evolving prognostic biomarkers.

Metabolomics Profiling Reveals Markers for Chemosensitivity and Clinical Outcomes in Pediatric AML Patients

Acute myeloid leukemia (AML) is a clinically challenging disease with high interpatient variability in response to chemotherapy. Despite continuing advances in treatment options, current 5-year survival rates for pediatric AML are suboptimal at ~60%. The heterogeneous nature of AML contributes significantly to the variability in treatment response and survival outcomes. Several known genetic lesions and cytogenetic features contribute to disease progression. However, our understanding of how molecular mechanisms contribute to variation in treatment outcomes is still limited. Previous metabolomics studies have successfully identified significant metabolic alterations in hematological malignancies, but very few metabolomics studies have been conducted for the pediatric AML patient population. In this study, we used global and targeted metabolomics to identify differential metabolite abundance associated with chemosensitivity and treatment outcomes in pediatric AML patients.Serum metabolomics profiles were generated with serum samples obtained at diagnosis from patients treated in the multicenter AML02 study (n=94, NCT00136084). Clinical outcomes tested for association included half-maximal inhibitory concentration (IC50) of cytarabine, minimal residual disease (MRD), relapse free survival (RFS), and overall survival (OS). Global metabolomics profiling was performed using liquid chromatography/mass spectrometry (LC/MS). Targeted metabolomics profiling was generated for a select group of organic acids and acylcarnitines. The organic acid panel included eight metabolites related to the tricarboxylic acid cycle and glycolysis. The acylcarnitine panel featured 20 varieties of acylcarnitines detectable in human serum. Statistical analyses were performed using MetaboAnalyst and various R packages.A total of 3205 features were detected in the global metabolome, with 124 known metabolites and 3081 unknown features. All metabolites were used for association analysis, while annotated metabolites were used in pathway analyses. Association analysis of clinical endpoints vs. metabolome identified 10 known metabolites significantly associated with IC50 values, 17 associated with MRD, 7 associated with RFS, and 7 associated with OS (p<0.05). Targeted metabolomics generated the absolute abundance profile of 8 organic acid metabolites and 20 acylcarnitine metabolites in patient samples. Spearman correlation analysis identified five acylcarnitines significantly correlated with IC50 values. Among the significant metabolites, the most interesting is pantothenic acid, showing higher serum abundance associated with poorer IC50, MRD, and RFS outcomes. Pantothenic acid is an essential component for Coenzyme A synthesis, leading into energy production through the tricarboxylic acid cycle. A previous study has shown a reduced capacity for pantothenic acid uptake in leukemia cells resistant to daunorubicin. Our results suggest a similar relationship for pantothenic acid uptake and cytarabine resistance.Pathway enrichment analysis identified 11 metabolic pathways showing significant association with IC50 values and 12 pathways associated with MRD (FDR<0.05). Some of the most significantly associated pathways included alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and pantothenic acid based CoA biosynthesis. Overall, differences in chemosensitivity and clinical outcomes appear to be most closely related to amino acid synthesis and energy production.This study identifies several metabolites and metabolic pathways significantly associated with chemosensitivity and clinical endpoints in pediatric AML patients. These results help expand on previously conducted AML pilot studies, and metabolomics studies on other cancer types, to further clarify the metabolic differences associated with interpatient variability in chemotherapy response for AML patients. Continued metabolic profiling of AML patient populations can help establish targetable pathways that can be used to improve treatment efficiency for AML. In addition, in vitro functional modeling to validate results of the metabolomics study are currently underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Integrated Genome Wide Association Study (GWAS) Identifies SNPs Associated with Outcome in Pediatric AML

Acute myeloid leukemia (AML) treatment response remains poorly understood. Although multiple studies have focused on understanding the transcriptomic and epigenetic landscape of AML, a genome-wide analysis of SNPs in pediatric AML has not yet been investigated in depth. Thus, we sought to identify genetic variants predictive of AML response, relapse, and survival in pediatric AML patients. For this study, we generated genome-wide SNP data patients (n=160) treated on the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) using Infinium Omni 2.5M Exome Beadchip. Standard GWAS QC procedure was followed in order to remove SNPs with call rate < 95%, monomorphic SNPs, SNPs with MAF<5% and samples with call rate<95%. Following QC, a risk-adjusted multi-outcome integrative GWAS was performed to identify SNPs associated with minimal residual disease (MRD) following induction I, relapse-free survival (RFS) and overall survival (OS). We performed a risk-adjusted analysis to identify 21 SNPs mapping to 14 genes at an endpoint-integrative p value <2x10-5. Table 1 provides list of genes with SNPs significantly associated with MRD, RFS, OS as well as in the integrated analysis at <2x10-5. Of interest multiple SNPs in DICER1, which is a key enzyme required for the biogenesis of microRNAs and small interfering RNAs were significantly associated with clinical outcome with promise integrated analysis at p = 0.000011, supported by associations with MRD, RFS and OS at p <0.002 (Figure 1A). DICER1 is over-expressed in AML with its expression under the influence of hematopoietic transcript factor, GATA1. RAI14, a retinoic acid induced 14 is a prognostic marker of poor response in solid tumors and has been associated with development of drug resistance. Multiple SNPs in RAI14 were significantly associated with clinical endpoints. Figure 1B shows RAI14 SNP rs336474 with C allele significantly associated with better RFS (p= 0.027) and OS (p=0.007), with an integrated p= 0.000004. SNP in upstream of RBFOX1, a RNA binding fox-1 homolog 1 and within intron of GRIN2A, glutamate ionotropic receptor NMDA type subunit 2A were significantly associated with MRD, RFS and OS (all p<0.005) and integrated p =0.00001 (Figure 1C). SNPs within genes involved in pyrimidine metabolism such as UPP2, a uridine phosphorylase; tumor suppressor genes such as JPH3, which codes for junctophilin; LILRB4 which encodes for a Leukocyte Immunoglobulin Like Receptor B4, that regulates inflammatory responses and cytotoxicity; HACE1 a potential tumor suppressor involved in the solid tumors pathophysiology; ANK2, an ankyrin family protein with role in cell proliferation and motility; BIRC8, which is implicated in CML disease progression etc. In conclusion, our results demonstrate significance of genome-wide investigation of SNPs to identify novel and clinically relevant SNPs of prognostic significance in childhood AML. We will present the in depth results of our integrated GWAS analysis as well as validation in independent patient cohorts. In summary, our results constitute one of the first integrated GWAS analyses to identify SNPs of prognostic significance in pediatric AML.Acknowledgments: We are thankful for funding from NIH R01-CA139246 and ALSAC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Apoptosis: A biomarker of high-risk phenotype in pediatric acute myeloid leukemia?

Dysregulation of apoptosis has been explored in acute myeloid leukemia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown. This study was aimed to analyze percentage of apoptosis and apoptosis mediated through the intrinsic pathway with clinical outcomes in patients with pediatric AML. This prospective study included pediatric AML patients enrolled from July 2013 to August 2016. Annexin-V (marker of total apoptosis) and caspase-9 expression (marker of intrinsic pathway) was determined in baseline bone marrow (BM) samples by flow cytometery and compared with controls (unaffected BM of solid tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and event-free survival (EFS) were compared using log-rank test. A total of 151 AML patients were enrolled, median age 10 (range: 0.7-18years). Annexin-V expression in blast cells was significantly high in AML patients as compared to BM of subjects with solid tumors (P=0.01) and PB of healthy subjects (P=0.04). Caspase-9 expression in blast cells was not significantly different. Median annexin-V expression was significantly higher in patients with WBC count ≥11000/mm3 (P=0.02), poor-risk cytogenetics (P=0.02), the absence of RUNX1-RUNX1T1 translocation (P=0.004), and the absence of NPM1 mutation (P=0.05). Patients with high annexin-V expression had significantly inferior OS (P=0.05) in univariate analysis but not in multivariate analysis (P=0.32). Apoptosis as a whole was found to be activated in baseline BM samples of AML patients. High apoptosis may be associated with high-risk phenotype in this disease.

Myeloid Sarcoma Predicts Superior Outcome in Pediatric AML; Can Cytogenetics Solve the Puzzle?

BackgroundThe purpose of our study was to evaluate the clinical, cytogenetic, and molecular features, and survival outcomes in patients with acute myeloid leukemia (AML) with myeloid sarcoma (MS) and compare them with patients with AML without MS. Patients and MethodsThis was a retrospective analysis of de novo pediatric AML patients with or without MS diagnosed at our cancer center between June 2003 and June 2016. ResultsMS was present in 121 of 570 (21.2%), the most frequent site being the orbit. Patients with MS had a younger median age (6 years vs. 10 years) and presented with higher hemoglobin and platelet but lower white blood cell count compared with patients without MS. Further, t (8; 21) (P < .01), loss of Y chromosome (P < .01), and deletion 9q (P = .03) were significantly higher in patients with AML with MS. Event-free survival (EFS; P = .003) and overall survival (OS; P = .001) were better among patients with AML with MS (median EFS 21.0 months and median OS 37.1 months) compared with those with AML without MS (median EFS 11.2 months and median OS 16.2 months). The t (8; 21) was significantly associated with MS (odds ratio, 3.92). In a comparison of the 4 groups divided according to the presence or absence of MS and t (8; 21), the subgroup of patients having MS without concomitant t (8; 21) was the only group to have a significantly better OS (hazard ratio, 0.53; 95% confidence interval, 0.34-0.82; P = .005). ConclusionAlthough t (8; 21) was more frequently associated with MS, it did not appear to be the reason for better outcome.

Successes and challenges in the treatment of pediatric acute myeloid leukemia: a retrospective analysis of the AML-BFM trials from 1987 to 2012

Overall survival (OS) of pediatric patients with acute myeloid leukemia (AML) increased in recent decades. However, it remained unknown whether advances in first-line treatment, supportive care, or second-line therapy mainly contributed to this improvement. Here, we retrospectively analyzed outcome and clinical data of 1940 pediatric AML patients (younger than 18 years of age), enrolled in the population-based AML-BFM trials between 1987 and 2012. While 5-year probability of OS (pOS) increased from 49 ± 3% (1987–1992) to 76 ± 4% (2010–2012; p < 0.0001), probability of event-free survival only improved from 41 ± 3% (1987–1992) to 50 ± 2% (1993–1998; p = 0.02) after introduction of high-dose cytarabine/mitoxantrone, but remained stable since then. Non-response and relapse rates stayed constant despite intensified first-line therapy (p = 0.08 and p = 0.17). Reduced fatal bleedings and leukostasis translated into fewer early deaths (8.1%vs. 2.2%; p = 0.001). Strikingly, pOS after non-response (13 ± 5% (1987–1992) vs. 43 ± 7% (2005–2010); p < 0.0001) or relapse (19 ± 4% vs. 45 ± 4%; p < 0.0001) improved. After 1999, more relapsed or refractory patients underwent hematopoietic stem cell transplantation (HSCT) with increased pOS after HSCT (29 ± 5% (1993–1998) vs. 50 ± 4% (2005–2010); p < 0.0001). Since efficacy of salvage therapy mainly contributed to better outcome in pediatric AML, our analysis indicates that a better allocation of patients, who cannot be cured with conventional chemotherapy, to an early “salvage-like” therapy is necessary.

Successes and Challenges of Pediatric AML: A Report on Survival, Salvage Therapy and Causes of Deaths in the AML-BFM Study Group from 1987 -2012

Background:Over the past 30 years, prospective randomized trials led to a markedly increased overall survival (OS) for pediatric patients with acute myeloid leukemia (AML). While hematopoietic stem cell transplantation (HSCT) was previously recommended to all patients with available matched sibling donor in first remission, recent protocols mainly restricted allogeneic HSCT to patients with very high relapse risk, refractory disease or after relapse. At the same time, the first-line treatment was heavily intensified, while supportive care was greatly improved. Also recent studies for relapsed patients demonstrated an improved, but still poor outcome (5yr-pOS: 24-38%). Thus, the factors that mainly contributed to the markedly increased OS in pediatric AML remain largely unknown. Here we present a retrospective analysis, which we performed to reveal those factors, but also to identify the obstacles that may hamper further improvement.Patients and Methods:From 1/1987 to 12/2012, a total of 1940 patients with de novo AML were enrolled in four consecutive trials of the AML-Berlin/Frankfurt/Muenster study group (AML-BFM-87, -93, -98, -04 and the following registry). Median follow-up was 10 years. Five year estimates of EFS and OS (pEFS, pOS), cumulative incidences (CI) of early death (ED), death in nonresponse (NR), death in complete remission (CR) or deaths of patients with one or more relapses were analyzed retrospectively over time in 3-year and 9-year periods and also according to study periods.Results:In this large population of 1940 patients with, we sought to systematically decipher the causes of death, incidences and survival after relapse or NR and the impact of HSCT in pediatric AML. As previously shown, the 5yr-pOS markedly increased from 52±4% (1987-1989) to 72±5% (2011-2012) (p<0.0001), but improvement of 5yr-pEFS was unsteady from 43±3% to 55±5%. This implies that factors after the first event are responsible for the better survival. Accordingly, the OS of patients with NR (n=48, OS 13±5% [AML-BFM 87], vs. n=65, OS 41±6% [AML-BFM 04]; p<0.001) and relapse (n=97, OS 19±4% [AML-BFM 87] vs. n=204, OS 42±4%, [AML-BFM 04]; p<0.001) improved remarkably (Figure 1). Treatment intensification resulted generally in less disease-related deaths in most subgroups, but in turn was associated with higher treatment-and procedure-related mortality over the last 25 years. Incidences of ED significantly decreased (p=0.002). More specifically, disease-related causes within this group decreased, whereas treatment-related deaths did not change (Figure 2). Patients with NR also showed less disease-related deaths in recent compared to the earlier cohorts (p=0.001), and no change in treatment-related lethality (p=0.43). In the recent cohort, we observed a trend of fewer deaths in CR. Treatment-related deaths decreased significantly (p=0.02). CI of death after relapse showed a remarkable decrease over time. This is mostly affecting disease-related deaths (p<0.011), while deaths due to HSCT increased (p=0.008). Deaths in patients with more than one relapse did not change, and the only significant change within this subgroup is a higher incidence of HSCT-related deaths. The increase of HSCT in first CR and after NR/relapse from 25% 1987 - 1995 to about 40% after 1995 translates into a higher incidence of procedure-treatment related deaths. Nevertheless OS of patients with HSCT after NR/relapse increased significantly (OS 35±5% [1987-1995], 37±4 [1996-2004] and 51±4% [2005-2012] (p=0.001)). The comparison within study periods showed no trend for a better pEFS and pOS in the second half of a study compared to the first so an improvement due to general measures like better supportive care cannot be proven. In a Cox regression analysis of OS there was a significant trend for better OS over the study periods (HR 0.88, 95% confidence interval 0.72-0.82) but not for better OS in the second half (HR 0.95, 0.82-1.09).Conclusion:In conclusion, efficacy of salvage therapy and better therapy strategy is one of the main reasons for improved outcome in pediatric AML within the last 3 decades. However, stable and in some subgroups decreased treatment-related mortality reflects better supportive care with respect to intensified therapy. Of note, procedure-related mortality increased, but nevertheless survival after HSCT improved. [Display omitted] [Display omitted] DisclosuresRasche:Jazz Pharma: Other: Travel accomodation. Reinhardt:Celgene: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

MiR-155 expression and correlation with clinical outcome in pediatric AML: A report from Children's Oncology Group.

Aberrant expression of microRNA-155 (miR-155) has been implicated in acute myeloid leukemia (AML) and associated with clinical outcome. We evaluated miR-155 expression in 198 children with normal karyotype AML (NK-AML) enrolled in Children's Oncology Group (COG) AML trial AAML0531 and correlated miR-155 expression levels with disease characteristics and clinical outcome. Patients were divided into quartiles (Q1-Q4) based on miR-155 expression level, and disease characteristics were then evaluated and correlated with miR-155 expression. MiR-155 expression varied over 4-log10-fold range relative to its expression in normal marrow with a median expression level of 0.825 (range 0.043-25.630) for the entire study cohort. Increasing miR-155 expression was highly associated with the presence of FLT3/ITD mutations (P < 0.001) and high-risk disease (P < 0.001) and inversely associated with standard-risk (P = 0.008) and low-risk disease (P = 0.041). Patients with highest miR-155 expression had a complete remission (CR) rate of 46% compared with 82% in low expressers (P < 0.001) with a correspondingly lower event-free (EFS) and overall survival (OS) (P < 0.001 and P = 0.002, respectively). In a multivariate model that included molecular risk factors, high miR-155 expression remained a significant independent predictor of OS (P = 0.022) and EFS (0.019). High miR-155 expression is an adverse prognostic factor in pediatric NK-AML patients. Specifically, high miR-155 expression not only correlates with FLT3/ITD mutation status and high-risk disease but it is also an independent predictor of worse EFS and OS.

A Recurrent Immunophenotype at Diagnosis Independently Identifies High Risk Pediatric AML: A Report from the Children's Oncology Group Trial AAML0531

Background: Risk stratification of pediatric patients with acute myeloid leukemia (AML) combines cytogenetics and molecular markers at diagnosis with detection of measurable residual disease (MRD) by multidimensional flow cytometry (MDF) after initial induction chemotherapy (EOI1). However, using MDF to distinguish between favorable and poor risk groups by diagnostic immunophenotype has not been sufficiently reproducible for clinical implementation.Objective: We sought to characterize a recurrent, unique diagnostic immunophenotype identified among patients treated on Children's Oncology Group trial AAML0531 and determine the clinical characteristics, frequency, and outcome of patients with this immunophenotype in a large cohort.Patients and Methods: Of the 1022 newly diagnosed pediatric patients with de novo AML who enrolled in protocol AAML0531 (exclusive of patients with promyelocytic leukemia and Down syndrome), 812 who submitted a specimen for MDF at diagnosis and gave consent to MRD testing were eligible for the study.Results: A recurrent diagnostic phenotype was identified by a combination of high intensity CD56 expression, a lack of HLA-DR, dim to negative CD38 expression and dim to negative CD45 expression (designated as RAM phenotype). At diagnosis 19 patients (2.3%) expressed this phenotype and MRD was subsequently detected by MDF in 16/19 patients at EOI1 at a level of 0.02%-41% (median 0.3). Eleven patients with this phenotype submitted bone marrow specimens for MDF analysis at all 3 additional monitoring time points during therapy and in 8 patients MDF analysis revealed disease persistence throughout treatment. The experimental cohort (RAM cohort, N=19) and 2 control cohorts, one consisting of all other patients with CD56 positive AML (CD56+ control, N=166) and the other of patients with CD56 negative AML (CD56- control, N=627), were analyzed for clinical outcomes.Patients in the RAM cohort were significantly younger with a median age at diagnosis of 1.26 years [range 0.75-16.89] than those in the CD56+ control (median 7.57 years [range 0.01-29.8], p=0.002) and the CD56- control (median 10.64 [range 0.02-23.94], p<0.001). All patients in the RAM cohort were classified as standard risk per study protocol compared to 55% (p<0.001) for the CD56+ control and 46% (p<0.001) for the CD56- control. Patients in the RAM cohort were FLT3-ITD- and wild type for CEBPA, NPM1, and WT1. Of them, 7 had normal cytogenetics, 1 had trisomy 8, and 11 had other cytogenetic abnormalities not associated with prognostic subgroups, including the 11q23/MLL rearrangement. According to morphology, patients in the RAM cohort were more often classified as having megakaryoblastic AML (38%) compared to the CD56+ control (2.0%, p<0.001) and the CD56- control (4.0%, p<0.001).MDF analysis detected MRD in 84% of RAM cohort compared to 29% (p<0.001) for the CD56+ control and 33% (p<0.001) for the CD56- control cohorts. MRD detected at EOI1 was compared to morphologic complete remission (CR). Morphologic CR at EOI1 was achieved in 11 (58%) patients in the RAM cohort (58%) versus 71% of patients in the CD56+ (p=0.250) and 74% of patients in the CD56- (p=0.116) control cohorts. Patients in the RAM cohort had a 3-year event free survival (EFS) of 16% versus 52% for those in the CD56+ (p=0.003) and 51% for those in the CD56- (p<0.001) control cohorts. The 3-year overall survival (OS) for patients in the RAM cohort was 26% versus 66% for those in the CD56+ (p<0.001) and 70% for those in the CD56- (p<0.001) control cohorts. Multivariable Cox regression analysis revealed that the diagnostic RAM immunophenotype was an independent predictor of adverse outcome (hazard ratios [and 95% confidence intervals]: OS= 3.4 (1.84 - 6.18), relapse risk from CR=4.97 (2.67 - 9.24), DFS from CR=4.27 (2.04 - 8.95)).Conclusions: A definitive diagnostic phenotype is identified that is highly predictive of poor outcome in pediatric AML. The survival of patients with the RAM phenotype is similar to or worse than those with high-risk molecular features. It occurs in 10% of infants (<1 year old) with AML who lack known risk factors, do not respond to standard therapy, and have extremely poor outcomes. This phenotype can be identified rapidly at diagnosis, allowing clinicians to predict failure before initiating therapy, and thereby allowing administration of more effective alternative therapies and minimizing exposure to non-efficacious treatment. DisclosuresBrodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Pardo:Hematologics Inc.: Employment. Voigt:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

A Combination of EVI1 and PRDM16 Expression Clarified the Clinical Features of Intermediate/High Risk Patients in Pediatric Acute Myeloid Leukemia

BackgroundSeveral molecular markers, such as FLT3-internal tandem duplication (ITD), NPM1, CEBPA are well known to correlate with mortality in patients with acute myeloid leukemia (AML). Recently, a number of gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3. However, DNMT3A, IDH1/2, and TET2 are rare in pediatric patients with AML, thus accurate risk evaluation remained challenging even after incorporating these molecular markers.On the other hand, overexpression of the EVI1 gene is reported to be associated with adverse outcome in pediatric AML. Moreover, we have previously reported that measuring of PRDM16 gene expression was a powerful tool to predict the prognosis of pediatric AML. PRDM16 gene is highly homologous to the MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 gene. In this study, we investigated EVI1 gene expression to verify the prognosis of EVI1 gene expression and the relationship between EVI1 and PRDM16 gene expression.MethodsBetween 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 116 patients were excluded from the study because of misdiagnosis and unavailability of their RNA samples. Therefore, 369 patients were analyzed. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of EVI1 and association between EVI1 and PRDM16 genes.ResultsA total of 58 of 369 patients (15.7%) showed high expression of EVI1 gene. Overexpression of EVI1 gene was strongly associated with dismal prognosis; low risk (LR; 1 of 123 patients, or 0.8%); intermediate risk (IR; 38 of 147 patients, or 25.9%); high risk (HR; 6 of 50 patients, or 12%); and non-complete remission (Non-CR; 13 of 49 patients, or 26.5%), (P < 0.001). Overexpression of EVI1 correlated with the following characteristics: younger age at diagnosis; M4, M5, and M7 subtype; higher coincidence of MLL-rearrangement; and lower coincidence of t(8;21), and inv(16). EVI1 overexpression was very frequent among patients with de novo pediatric AML and IR/non-CR groups. Furthermore, more than half of patients in M6 (5 of 8 patients, or 62.5%) were EVI1 high expression. Interestingly, no patients with EVI1 high expression in M7 had a fusion of CBFA2T3-GLIS2. Patients with EVI1 overexpression also more frequently harbored a complex karyotype and monosomy 7. The frequencies of patients with high or low EVI1 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 1% vs 99%, P < 0.001; inv(16), 0% vs 100%, P < 0.001; NUP98-JARID1A, 83% vs 17%, P < 0.001; OTT-MAL, 100% vs 0%, P = 0.02; and KIT, 5% vs 95%, P = 0.003. The overall survival (OS) and event-free survival (EFS) among patients with EVI1 high expressions were significantly lower than that among patients without such gene aberrant expression (3-year OS 54% vs. 77%, P=0.008G3-year EFS: 34% vs 58%, P<0.001), respectively.On the other hand, a total of 84 of 369 patients (22.8%) showed high expression of PRDM16 gene. The OS and EFS among PRDM16 overexpressing patients were significantly worse than those among low expression group (3-year OS: 51% vs 81%, P<0.001; 3-year EFS: 32% vs 64%, P<0.001), respectively. Remarkably, concerning 125 patients with high EVI1 and/or PRDM16 expression, their prognosis was much worse than that of patients without these high expression (3-year OS: 54% vs 84%, P<0.001; 3-year EFS: 32% vs 68%, P<0.001) , respectively.ConclusionsWe investigated EVI1 and PRDM16 gene expression in de novo pediatric AML patients, and their high expression was associated with inferior survival, respectively. We suggest that high EVI1 and/or PRDM16 expression is useful marker for adverse outcome. On the other hand, low EVI1 and PRDM16 expressions are useful to elucidate low risk patients. DisclosuresNo relevant conflicts of interest to declare.

Excellent Outcome in Pediatric AML with Response Guided Chemotherapy without Allogeneic HSCT in First Complete Remission: Results from Protocol DB-AML01

Background: Contemporary treatment schedules for pediatric acute myeloid leukemia (AML) have improved 5 year-overall survival rates up to 70% . These schedules usually consist of 4 to 5 intensive courses mainly based on cytarabine (AraC) and anthracyclines, with allo-HSCT upfront in a subset of patients . Long-term side effects such as late cardiotoxicity caused by high cumulative anthracycline dosages are a major concern. Therefore, the DCOG/BSPHO collaboration adapted the NOPHO 2004 protocol to DB-AML01 to reduce cumulative anthracyclines from 420-450 mg/m² to 300-330 mg/m² and to omit HSCT in CR1.Material: The treatment consisted of 5 chemotherapy courses: first induction course AIET (AraC conventional dose and Idarubicin (Ida) 36 mg/m2); second induction course AM (AraC conventional dose and Mitoxantrone (Mitox) 30 mg/m2) when blasts at day 15 < 5% or FLA-DNX (high dose AraC and Daunoxome (DNX) 180 mg/m2) when blasts > 5% at day 15 or t(8;21) AML; 3 consolidation courses (high dose AraC with Etoposide as course 3 and 5 and in between high dose AraC alone). Cumulative dose of AraC is 43,4 gr/m2 and of anthracyclines 300 mg/m² for patients receiving AIET+AM or 330 mg/m² for patients receiving AIET/FLA-DNX.Results: From 2010 to 2014 a total of 113 children with AML, median age 6.0 yrs (range 0-16), gender 56 males, were included. Second induction course was AM in n=75 and FLA-DNX in n= 33. Death in induction occurred in 4 patients, CR rate was 102/113 (90.3%) and 7 patients were refractory after induction. The later events were death in CR (n=2), relapse in CR (n=40), and secondary malignancies (n=0). HSCT was given to 3 patients in CR1 (physician's decision). Median follow up time is 2.6 years (range (range 1-5). Probability of EFS at 2 years was 60% (95% CI 49-69%) and 2-yr OS was 76 (95% CI 66-83%)77.9%. Especially inv 16 (n=10) and t(8;21) AML (n=16) had an excellent outcome (inv 16:2-yr EFS: 70% (95% CI 32-89%) and 2-yr OS 91% (95% CI 51-99%) resp t(8;21):2-yr EFS: 87% (95% CI 57-97%) and OS: 94% (95% CI 63-99%)), EFS nor OS in other genetic subgroups such as FLT3-ITD and MLL rearrangements were significantly different from the total group. Children with an older age (> 10 years) (n=34) or high WBC count (>50.109/l) (n=41) had the poorest 2-yr EFS: 51 (95% CI 33-67%) resp EFS: 40% (95% CI 24-56%), whereas OS was not significantly worse. For the patients with a relapse, 32 achieved a second CR and 4 a partial remission (90%) . In second CR 34 patients received an allo-HSCT. Until now 26 of the 40 patients are still in second CR.Conclusion: The DB-AML01 outcome data suggest that the choice of high cumulative doses of AraC with moderate cumulative doses of anthracyclines up to 300-330 mgr/m² results in a favorable overall survival for particularly those children with t(8;21), inv (16) AML, young age or lower WBC counts. Children with relapsed AML could be rescued with second line treatment and allo-HSCT. These results underscore the relevance of identification of subgroups of pediatric AML patients, based on age, WBC count and t(8;21) or inv 16, curable with "lower risk" treatment arms without allo-HSCT in CR1. DisclosuresKaspers:Janssen-Cilag: Research Funding.