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Cytarabine is the primary chemotherapeutic agent used for treatment of acute myeloid leukemia (AML). Disease relapse after initial remission remains one of the most pressing therapeutic challenges in the treatment of AML. Relapsed disease is often resistant to cytarabine and subsequent salvage therapy is ineffective. Recent studies have shown that some microRNAs (miRNAs) are associated with prognosis, but have not yet explored the role of miRNAs in cellular response to cytarabine. We identified 20 miRNAs that associate with the in vitro cytarabine chemo-sensitivity or apoptotic response of eight AML cell lines. Out of the 20 miRNAs, data on 18 miRNAs was available in AML patients from The Cancer Genome Atlas database. Our stepwise-integrated analyses (step 1 – miRNA–target mRNA that were significantly correlated in AML patients; step 2 – mRNAs from step 1 with significant association with overall survival (OS)) identified 23 unique miRNA–mRNA pairs predictive of OS in AML patients. As expected HOX genes (HOXA9, HOXB7, and HOXA10) were identified to be regulated by miRs as well as predictive of worse OS. Additionally, miR107-Myb, miR-378-granzyme B involved in granzyme signaling and miR10a-MAP4K4 were identified to be predictive of outcome through integrated analysis. Although additional functional validations to establish clinical/pharmacologic importance of miRNA–mRNA pairs are needed, our results from RNA electrophoretic mobility shift assay confirmed binding of miR-10a, miR-378, and miR-107 with their target genes GALNT1, GZMB, and MYB, respectively. Integration of pathogenic and pharmacologically significant miRNAs and miRNA–mRNA relationships identified in our study opens up opportunities for development of targeted/miRNA-directed therapies.

Motivation: Normalization is critical in DNA copy number analysis. We propose a new method to correctly identify two-copy probes from the genome to obtain representative references for normalization in single nucleotide polymorphism arrays. The method is based on a two-state Hidden Markov Model. Unlike most currently available methods in the literature, the proposed method does not need to assume that the percentage of two-copy state probes is dominant in the genome, as long as there do exist two-copy probes. Results: The real data analysis and simulation study show that the proposed algorithm is successful in that (i) it performs as well as the current methods (e.g. CGHnormaliter and popLowess) for samples with dominant two-copy states and outperforms these methods for samples with less dominant two-copy states; (ii) it can identify the copy-neutral loss of heterozygosity; and (iii) it is efficient in terms of the computational time used. Availability: R scripts are available at http://publichealth.lsuhsc.edu/PAIR.html. Contact: zfang@lsuhsc.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: There is a substantial body of works in the biology literature that seeks to characterize the cyclic behavior of genes during cell division. Gene expression microarrays made it possible to measure the expression profiles of thousands of genes simultaneously in time-course experiments to assess changes in the expression levels of genes over time. In this context, the commonly used procedures for testing include the permutation test by de Lichtenberg et al. and the Fisher’s G-test, both of which are designed to evaluate periodicity against noise. However, it is possible that a gene of interest may have expression that is neither cyclic nor just noise. Thus, there is a need for a new test for periodicity that can identify cyclic patterns against not only noise but also other non-cyclic patterns such as linear, quadratic or higher order polynomial patterns. Results: To address this weakness, we have introduced an empirical Bayes approach to test for periodicity and compare its performance in terms of sensitivity and specificity with that of the permutation test and Fisher’s G-test through extensive simulations and by application to a set of time-course experiments on the Schizosaccharomyces pombe cell-cycle gene expression. We use ‘conserved’ and ‘cycling’ genes by Lu et al. to assess the sensitivity and CESR genes by Chenet al. to assess the specificity of our new empirical Bayes method. Availability and implementation: The SAS Macro for our empirical Bayes test for periodicity is included in the supplementary materials along with a sample run of the MACRO program. Contact: mkocak1@uthsc.edu Supplementary information: Supplementary data are available at Bioinformatics online.

To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Prognostic features in acute megakaryoblastic leukemia in children without Down syndrome: a report from the AML02 multicenter trial and the Children’s Oncology Group Study POG 9421

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Background Older age has historically been an adverse prognostic factor in pediatric acute myeloid leukemia (AML). The impact of age relative to that of other prognostic factors on the outcome of patients treated in recent trials is unknown.

Medulloblastomas that display a large cell/ anaplastic morphology and overexpress the cellular c-MYC gene are highly aggressive and carry a very poor prognosis. This so-called MYC-subgroup differs in its histopathology, gene expression profile, and clinical behavior from other forms of medulloblastoma. We generated a mouse model of MYC-subgroup medulloblastoma by transducing Trp53-null cerebellar progenitor cells with Myc. The cardinal features of these mouse medulloblastomas closely mimic those of human MYC-subgroup tumors and significantly differ from mouse models of the Sonic Hedgehog (SHH)- and WNT-disease subgroups. This mouse model should significantly accelerate understanding and treatment of the most aggressive form of medulloblastoma and infers distinct roles for MYC and MYCN in tumorigenesis.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Cytosolic 5'-nucleotidase II (NT5C2) is involved in the development of 1-β-d-arabinofuranosylcytosine (ara-C) resistance and has been associated with clinical outcome in patients receiving ara-C-based chemotherapy. NT5C2 inactivates ara-C by dephosphorylating ara-C monophosphate to ara-C. In this study, we sequenced NT5C2 in genomic DNA samples from International HapMap project panels with European [Centre d'Etude du Polymorphisme Humain (CEU); n = 90] or African [Yoruba people in Ibadan, Nigeria (YRI); n = 90] ancestry. We identified 41 genetic variants [one insertion-deletion and 40 single nucleotide polymorphisms (SNPs)], including three nonsynonymous SNPs (Y3A, K47R, and Q136R). Twenty-five SNPs were novel and 16 overlapped with the HapMap data. Subjects with African ancestry had NT5C2 mRNA expression levels that was significantly higher than those with European ancestry (p = 0.005). Furthermore, there was a correlation between NT5C2 mRNA expression and ara-C sensitivity in CEU but not in YRI cell lines. None of the nonsynonymous SNPs demonstrated any effect on NT5C2 activity. The genotypes of several SNPs were significantly associated with NT5C2 mRNA expression and/or ara-C sensitivity in CEU cell lines, but very few were significant in YRI cell lines. Of most interest, SNPs (linkage disequilibrium group CEU.12) in the 5'-untranslated region were associated with NT5C2 expression and ara-C sensitivity in HapMap cell lines and with NT5C2 mRNA expression and ara-C sensitivity in diagnostic leukemic blasts from pediatric patients with acute myeloid leukemia. Functional genomics analysis demonstrated that the promoter SNP rs11191612 was associated with altered luciferase activation in reporter assays and altered DNA-protein binding in gel shift assays. These results suggest that genetic variations in NT5C2 influence its expression and, potentially, cellular responses to nucleoside analogs.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(−)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3 activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

Acute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy.Using 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγ(null) mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration-time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγ(null) mice bearing AML xenografts.Sorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P.001 for combination twice daily vs all other treatments listed).Sorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML.

Introduction: Resistance and relapse remain major obstacles in the treatment of acute myeloid leukemia (AML). Pre-existence and persistence of drug resistant leukemia stem cells (LSCs) is considered one of the major causes of relapse. A previous study (Ng et al., 2016) reported a prognostic signature of 17 genes (LSC17 score) differentially expressed in LSC+ compared to LSC- cell fractions that predicted outcome in patients with AML thereby classifying patients into high and low risk groups. The goal of this study is to determine the validity of LSC17 score in pediatric AML patients and to enhance its clinical utility by exploring a new score with limited number of stem cell genes. Methods: 150 pediatric patients with AML enrolled in the multicenter AML02 clinical trial (ClinicalTrials.gov Identifier: NCT00136084) with Affymetrix U133A microarray gene expression data and clinical data were included in the study. Since only 14 of the 17 genes were represented on the Affymetrix U133A gene chip we tested the validity of the LSC14 score using the previously defined equation (Ng et al, 2016) with multiple clinical endpoints such as minimum residual disease (MRD), event free survival (EFS) and overall survival (OS). To reduce the model complexity, we applied a penalized regression algorithm called the least absolute shrinkage and selection operator (LASSO) implemented in the glmnet R-package using event free survival (EFS) as an outcome variable. Score of the new equation, which included three genes, was designated as pediatric-LSC3 (pLSC3). pLSC3 was tested in the AML02 cohort for association of high or low pLSC3 (based on the median value) with clinical endpoints mentioned above. pLSC3 score equation was validated using publically available gene-expression data from 117 pediatric relapse enriched AML patient cohort enrolled in Children's Oncology Group (COG) protocol (TARGET database). COX-proportional hazard models and Log rank test were used for survival data analysis. Results: AML02 cohort: Patients with high LSC14 scores (greater than median), had significantly worse MRD (p In a multivariate COX regression model, pLSC3 score groups was the only significant covariate (table 1). It explained 13.1% of variability in EFS and 11.6% of variability in OS, while other prognostic factors such as risk groups, FLT3 status, treatment arm and age collectively explained 15.1 and 12.1 % of variability. Discussion: In summary, our results show validity of a previously defined LSC14 score in a pediatric AML population from the multicenter AML02 clinical trial. To enhance the clinical utility, score equation was further simplified and the final score (pLSC3) was derived from three genes: DNMT3B, which encodes for DNA methyltransferase; CD34, an important cell surface marker for early-undifferentiated LSCs; and GPR56, a G protein coupled receptor of significance in AML. Given that there is need to refine classification of a highly heterogeneous group of patients with standard risk AML, we show that differentiating standard risk patients based on pLSC3 score should be considered in the future. We show the relevance of pLSC3 in two independent cohorts, opening up opportunities to improve treatment outcomes of pediatric patients with AML. Disclosures No relevant conflicts of interest to declare.

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 Pathway enrichment analysis identified 11 metabolic pathways showing significant association with IC50 values and 12 pathways associated with MRD (FDR 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. Disclosures No relevant conflicts of interest to declare.

Introduction B progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor. Methods To identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing. Results Using integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in "double/triple hit" lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion. We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL. Conclusions These results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. Disclosures McKay: ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

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 Acknowledgments: We are thankful for funding from NIH R01-CA139246 and ALSAC. Disclosures No relevant conflicts of interest to declare.

Pediatric de novo acute myeloid leukemia (AML) is an aggressive malignancy with current therapy resulting in cure rates of only 60%. To better understand the cause of the marked heterogeneity in therapeutic response and to identify new prognostic markers and therapeutic targets a comprehensive list of the genetic mutations that underlie the pathogenesis of AML is needed. To approach this goal, we examined diagnostic leukemic samples from a cohort of 111 children with de novo AML using single-nucleotide-polymorphism microarrays and candidate gene resequencing. Our data demonstrate that, in contrast to pediatric acute lymphoblastic leukemia (ALL), de novo AML is characterized by a very low burden of genomic alterations, with a mean of only 2.38 somatic copy-number alterations per leukemia, and less than 1 nonsynonymous point mutation per leukemia in the 25 genes analyzed. Even more surprising was the observation that 34% of the leukemias lacked any identifiable copy-number alterations, and 28% of the leukemias with recurrent translocations lacked any identifiable sequence or numerical abnormalities. The only exception to the presence of few mutations was acute megakaryocytic leukemias, with the majority of these leukemias being characterized by a high number of copy-number alterations but rare point mutations. Despite the low overall number of lesions across the patient cohort, novel recurring regions of genetic alteration were identified that harbor known, and potential new cancer genes. These data reflect a remarkably low burden of genomic alterations within pediatric de novo AML, which is in stark contrast to most other human malignancies.

To characterize the biology and optimal therapy of acute mixed-lineage leukemia in children, we reviewed the pathologic and clinical features, including response to therapy, of 35 patients with mixed-lineage leukemia. The majority of cases (91%) had blasts cells that simultaneously expressed either T-lineage plus myeloid markers (T/myeloid, n = 20) or B-lineage plus myeloid markers (B/myeloid, n = 12). Overall survival rates for the B/myeloid and T/myeloid subgroups were not significantly different from each other or from the rate for acute myeloid leukemia (AML) but were inferior to the outcome in children with acute lymphoblastic leukemia (ALL). Patients who failed to achieve complete remission with AML-directed therapy could often be induced with a regimen of prednisone, vincristine, and L-asparaginase. Analysis of gene-expression patterns identified a subset of biphenotypic leukemias that did not cluster with T-cell ALL, B-progenitor ALL, or AML. We propose that treatment for biphenotypic leukemia begin with one course of AML-type induction therapy, with a provision for a switch to lymphoid-type induction therapy with a glucocorticoid, vincristine, and L-asparaginase if the patient responds poorly. We also suggest that hematopoietic stem cell transplantation is often not required for cure of these patients.

BACKGROUND: Diagnosing invasive aspergillosis is difficult but might be improved by detection of circulating galactomannan. Although galactomannan antigenemia has been well studied in the detection of invasive aspergillosis in adult patients, little is known about the expression of circulating galactomannan in immunocompromised children with invasive aspergillosis. METHODS: We studied the expression of galactomannan antigen by enzyme immunoassay (EIA) in 990 serum samples from 56 pediatric oncology patients (ages 3 months to 18 years) of whom 17 had proven or probable invasive aspergillosis defined by the European Organization for Research and Treatment of Cancer-Mycoses Study Group criteria. Any sample with a galactomannan EIA Galactomannan index value of ≥0.5 was considered positive. RESULTS: At least 1 serum sample was positive for 11 of 17 pediatric oncology patients (65.7% sensitivity, 95% confidence interval: 38.3–85.7) with invasive aspergillosis. Galactomannan EIA was positive in 99 of 304 samples from patients with proven or probable invasive aspergillosis, and 7 of 686 (1.0%) samples from 39 control subjects resulted in a positive galactomannan EIA result. At least 1 sample tested positive in 5 of the 39 controls (12.8%, 95% confidence interval: 4.3–27.4). No significant association between accuracy and patient age was observed. Among the 7 evaluable galactomannan-positive patients with IA, the galactomannan EIA produced a positive result before clinical or radiographic evidence of infection in 6 cases, with a lead-time to diagnosis ranging from 1 day to 34 days (median: 10 days). In the remaining case, a positive galactomannan was observed on the same day as diagnosis by non-EIA methods. CONCLUSIONS: The presence of circulating galactomannan is predictive of invasive aspergillosis in most pediatric oncology patients. Galactomannan antigenemia may precede clinical, microbiologic, or radiographic evidence of invasive aspergillosis.

Background The percentage of myeloperoxidase (MPO)-positive blast cells is associated with prognosis in adult acute myeloid leukemia (AML), but this association is unsubstantiated in pediatric AML. Procedure We retrospectively compared cytochemical MPO results with outcome in 154 patients younger than 21 years treated on three consecutive institutional protocols for newly diagnosed AML (1987–2001). Patients with FAB M0 and M7 AML (no MPO expression) or M3 AML (100% MPO expression) and Down's syndrome were excluded. Results Median MPO expression was higher in FAB M2 subtype than in other subtypes (P

Background Detection and quantification of adenovirus (ADV) in peripheral blood specimens has become an increasingly important tool in the management of immunosuppressed patients. Investigators have described the use of whole blood (WB), peripheral blood mononuclear cells (PBMC), serum and plasma but no studies have compared the utility of these different sample types for use in a clinical diagnostic assay. Objectives To determine the optimal blood compartment for quantitative real-time measurement of adenovirus in peripheral blood specimens. Study design WB, PBMC, and plasma representing 338 samples from 148 patients were tested for ADV by quantitative real-time PCR (qrt-PCR) and the results compared for concordance of both qualitative sensitivity and viral load among positive specimens. Results There was no significant difference in qualitative sensitivity among the three tested specimen types. Quantitative values of WB and plasma were similar and tended to be greater than those found in PBMC samples. Comparison of consecutive positive samples within individual patients showed that viral loads tracked similarly over time, irrespective of the sample type tested. Conclusion While WB and plasma do not offer a significant increase in sensitivity over PBMC, they may offer benefits in terms of reduced processing costs and laboratory turn around time.

Deoxycytidine kinase (DCK) is a rate-limiting enzyme in the activation of nucleoside analogs such as cytarabine (ara-C), gemcitabine, clofarabine, and others. The present study was undertaken to identify and to determine the functional consequences of genetic variants in DCK. We sequenced 1.5 kilobases of the DCK proximal promoter and all seven coding exons in International HapMap Project panels (n = 90 each) with European (Centre d' Etude du Polymorphisme Humain; CEPH) or African (Yoruba people in Ibadan, Nigeria; YRI) ancestry. Sixty-four genetic polymorphisms, including three nonsynonymous coding changes (I24V, A119G, and P122S) were identified. Compared with DCK-wild-type (WT) protein, the activity of the recombinant DCK24Val, DCK119Gly, and DCK122Ser proteins was 85 +/- 5, 66 +/- 3, and 43 +/- 4%, respectively. DCK119Gly and DCK122Ser mutants had lower Km (p0.01) and Vmax (p0.001) compared with DCK-WT protein. Lymphoblast cell lines from subjects heterozygous for the coding changes had significantly lower DCK activity compared with homozygous WT subjects. Ethnic differences were observed, with African ancestry subjects demonstrating significantly higher DCK mRNA expression compared with subjects with European ancestry. In both CEPH and YRI subjects, the C allele of a 3'-untranslated region single-nucleotide polymorphism (SNP) (35708 CT) was significantly associated with lower DCK mRNA expression. This SNP was strongly linked with other intronic SNPs, forming a major haplotype block in both ethnic groups. In an exploratory analysis, the 35708C allele was also associated with lower blast ara-C-5'-triphosphate (ara-CTP) levels in acute myeloid leukemia patients receiving ara-C as continuous infusion. These results suggest that genetic variation in DCK influences its activity and expression and may predict the variability observed in intracellular levels of the ara-C active metabolite ara-CTP.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116,204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the 13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.

We have recently identified nine distinct molecular subgroups of ependymoma across all age groups, three in each major anatomical compartment of the CNS: spinal (SP), posterior fossa (PF), and supratentorial (ST). These nine molecular subgroups are genetically, epigenetically, transcriptionally, demographically, and clinically distinct. Pediatric intracranial ependymomas were either affiliated with one of two supratentorial subgroups characterized by RELA (ST-EPN-RELA) or YAP1 (ST-EPN-YAP1) fusion genes, or with the PF-EPN-A subgroup in the hindbrain. Observing distinct outcomes among children with PF-EPN-A tumors, the largest molecular subgroup, we tested the hypothesis that further molecular diversity exists among these tumors. The genome-wide DNA methylation profiles of 575 pediatric PF ependymomas were analyzed by unsupervised consensus hierarchical clustering and principal component analysis, which identified three distinct molecular subtypes of PF-EPN-A tumors: PFA-1, PFA-2, and PFA-3. PFA-1 and PFA-2 showed different chromosomal copy number alterations and comprised tumors almost exclusively from infants, while PFA-3 ependymomas were generally from older children. The PFA-3 subtype was significantly enriched for gain of 1q. Distinct developmental gene expression profiles and radiological characteristics on pre-operative MR imaging suggested separate anatomic origins for the infant subtypes, PFA-1 and PFA-2. Survival analyses identified PFA-3 as having the poorest outcome. Multivariate analysis revealed molecular subgroup, grade of resection and gain of 1q as independent prognostic markers among PF-EPN-A tumors. We conclude that further molecular refinement of pediatric PF-EPN-A tumors, with their division into three distinct molecular subtypes, has clinical utility and the potential for enhanced risk assessment or therapeutic stratification.

Peripheral blood samples were separated into aliquots of whole blood and plasma and tested using an automated PCR system. More positive results were obtained from plasma (43), compared to whole blood (34). Plasma results did not correlate well with those in whole blood and tended to produce higher viral loads.

Using broad interrogation of clinically relevant drug absorption, distribution, metabolism, and excretion (ADME) genes on the DMET platform, we identified a genetic variant in SLCO1B1 (rs2291075; c.597C>T), encoding the transporter OATP1B1, associated with event-free (P = 0.006, hazard ratio = 1.74) and overall survival (P = 0.012, hazard ratio = 1.85) in children with de novo acute myeloid leukemia (AML). Lack of SLCO1B1 expression in leukemic blasts suggested the association might be due to an inherited rather than a somatic effect. rs2291075 was in strong linkage with known functional variants rs2306283 (c.388A>G) and rs4149056 (c.521T>C). Functional studies in vitro determined that four AML-directed chemotherapeutics (cytarabine, daunorubicin, etoposide, and mitoxantrone) are substrates for OATP1B1 and the mouse ortholog Oatp1b2. In vivo pharmacokinetic studies using Oatp1b2-deficient mice further confirmed our results. Collectively, these findings demonstrate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of AML and suggest that inherited variability in host transporter function influences the effectiveness of therapy.

Quantitative detection of cytomegalovirus (CMV) DNA has become a standard part of care for many groups of immunocompromised patients; recent development of the first WHO international standard for human CMV DNA has raised hopes of reducing interlaboratory variability of results. Commutability of reference material has been shown to be necessary if such material is to reduce variability among laboratories. Here we evaluated the commutability of the WHO standard using 10 different real-time quantitative CMV PCR assays run by eight different laboratories. Test panels, including aliquots of 50 patient samples (40 positive samples and 10 negative samples) and lyophilized CMV standard, were run, with each testing center using its own quantitative calibrators, reagents, and nucleic acid extraction methods. Commutability was assessed both on a pairwise basis and over the entire group of assays, using linear regression and correspondence analyses. Commutability of the WHO material differed among the tests that were evaluated, and these differences appeared to vary depending on the method of statistical analysis used and the cohort of assays included in the analysis. Depending on the methodology used, the WHO material showed poor or absent commutability with up to 50% of assays. Determination of commutability may require a multifaceted approach; the lack of commutability seen when using the WHO standard with several of the assays here suggests that further work is needed to bring us toward true consensus.

The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from +0.6 to −1.0 log 10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log 10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard.

Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis. Pediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here the authors analyse the genomes, exomes and transcriptomes of 37 such tumours and identify genetic alterations whose nature, timing and potential interactions are key events with prognostic significance in pediatric adrenocortical tumorigenesis.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Despite substantial progress in the management of childhood acute myeloid leukemia (AML), only about 50% of patients are cured by intensive chemotherapy. The long-term results of clinical trials may reveal principles that can guide the development of future therapy. From 1980 to 2000, 251 patients

Febrile non-haemolytic or allergic reactions occur in 0.1-30% of transfusions; physicians often premedicate patients with acetaminophen or diphenhydramine to prevent these reactions. The effectiveness of this practice has not been demonstrated. In this retrospective review of all transfusions at our institution during 2002, 385 patients received 7900 evaluable leucoreduced, irradiated blood products (4280 single-donor apheresis platelets and 3620 packed red blood cells). Febrile reactions occurred in 0.95% of 4108 transfusions with, and 0.53% of 3792 transfusions without, acetaminophen premedication. Allergic reactions occurred in 0.90% of 4315 transfusions with, and 0.56% of 3585 transfusions without, diphenhydramine premedication. In a multivariate analysis that adjusted for age, patient category, transfusion location, product, transfusion history, and reaction history, premedication with acetaminophen was associated with a statistically non-significant increase in the odds of a febrile reaction (odds ratio 1.74; 95% confidence interval 0.71-4.23; P = 0.22), and diphenhydramine with a non-significant increase in allergic reactions (odds ratio 1.74; 95% confidence interval 0.99-3.06; P = 0.054). Reactions occurred in only 1.3% of the 518 transfusions to patients with a history of two or more prior reactions. Febrile and allergic transfusion reactions were rare in paediatric patients transfused with leucoreduced, irradiated blood products, whether premedication was used or not.

Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARα], t(8;21)[AML1-ETO], inv16 [CBFβ-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation.

The methodological advancement in microarray data analysis on the basis of false discovery rate (FDR) control, such as the q-value plots, allows the investigator to examine the FDR from several perspectives. However, when FDR control at the “customary" levels 0.01, 0.05, or 0.1 does not provide fruitful findings, there is little guidance for making the trade off between the significance threshold and the FDR level by sound statistical or biological considerations. Thus, meaningful statistical significance criteria that complement the existing FDR methods for large-scale multiple tests are desirable. Three statistical significance criteria, the profile information criterion, the total error proportion, and the guide-gene driven selection, are developed in this research. The first two are general significance threshold criteria for large-scale multiple tests; the profile information criterion is related to the recent theoretical studies of the connection between FDR control and minimax estimation, and the total error proportion is closely related to the asymptotic properties of FDR control in terms of the total error risk. The guide-gene driven selection is an approach to combining statistical significance and the existing biological knowledge of the study at hand. Error properties of these criteria are investigated theoretically and by simulation. The proposed methods are illustrated and compared using an example of genomic screening for novel Arf gene targets. Operating characteristics of q-value and the proposed significance threshold criteria are investigated and compared in a simulation study that employs a model mimicking a gene regulatory pathway. A guideline for using these criteria is provided. Splus/R code is available from the corresponding author upon request.

Summary. In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one-third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four-colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty-six of the 54 AML cases (85·2%) had immunophenotypes that allowed detection of 0·1–0·01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26·5%) had ≥ 0·1% AML cells by flow cytometry. We found that core binding factor-associated AML had a significantly better early treatment response. Mean (± standard error) 2-year survival estimate was 33·1 ± 19·1% for patients with ≥ 0·1% AML cells by flow cytometry after induction therapy, but 72·1 ± 11·5% for those with

Impact of genetic variation in FKBP5 on clinical response in pediatric acute myeloid leukemia patients: a pilot study

We developed Copy Number Segmentation by Regression Tree in Next Generation Sequencing (CONSERTING), an algorithm for detecting somatic copy-number alteration (CNA) using whole-genome sequencing (WGS) data. CONSERTING performs iterative analysis of segmentation on the basis of changes in read depth and the detection of localized structural variations, with high accuracy and sensitivity. Analysis of 43 cancer genomes from both pediatric and adult patients revealed novel oncogenic CNAs, complex rearrangements and subclonal CNAs missed by alternative approaches.

We propose in this paper a set-valued (SV) system model, which is a generalized form of Logistic (LG) and Probit (Probit) regression, to be considered as a method for discovering genetic variants, especially rare genetic variants in next generation sequencing studies, for a binary phenotype. We propose a new set-valued system identification method to estimate all the underlying key system parameters for the Probit model and compare it with the LG model in the setting of genetic association studies. Across an extensive series of simulation studies, the Probit method maintained Type I error control and had similar or greater power than the LG method which is robust to different distributions of noise: logistic, normal or t distributions. Additionally, the Probit association parameter estimate was 2.7–46.8 fold less variable than the LG log-odds ratio association parameter estimate. Less variability in the association parameter estimate translates to greater power and robustness across the spectrum of minor allele frequencies (MAFs), and these advantages are the most pronounced for rare variants. For instance, in a simulation that generated data from an additive logistic model with odds ratio of 7.4 for a rare single nucleotide polymorphism with a MAF of 0.005 and a sample size of 2300, the Probit method had 60% power whereas the LG method had 25% power at the α=10−6 level. Consistent with these simulation results, the set of variants identified by the LG method was a subset of those identified by the Probit method in two example analyses. Thus, we suggest the Probit method may be a competitive alternative to the LG method in genetic association studies such as candidate gene, genome-wide, or next generation sequencing studies for a binary phenotype.

Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.

Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Pediatric cancers are distinct from adult cancers in both their genomic alterations and therapeutic responses. Fms-like tyrosine kinase 3 (FLT3) mutations, especially internal tandem duplications (ITD), are among the most common mutations in acute myeloid leukemia (AML). FLT3-ITD mutations occur in approximately 15% of pediatric and 25-30% of adult AML, and are generally associated with poor prognosis. However, a number of studies have suggested that FLT3-ITD-positive(+) AML requires additional cooperative mutations. The objective of this study was to characterize the mutational landscape in a cohort of FLT3-ITD+ pediatric AML patients (median age,12.6 years; range, 2.8-19.2 years) enrolled to the AML02 and AML08 trials using samples obtained at diagnosis (n=34) and paired diagnosis/relapse samples (n=5). Children with promyelocytic leukemia were excluded. Samples were analyzed by RNASeq, a targeted 95 gene next generation sequencing (NGS) panel, and whole exome sequencing (WES). At diagnosis, 58.8% of the samples contained fusion genes; 41.2% were NUP98-NSD1, 11.8% were novel fusions (NSD1-CAPRIN1, NSD1-RALBP1, RUNX1-BCL11B, ZEB2-BCL11B), and 5.9% were previously reported fusions (CBFB-MYH11, DEK-NUP214). The NGS panel identified that WT1 and NPM1 were routinely mutated at a frequency of 32.4% and 20.6% respectively. While the NPM1 mutation was either a 4bp insertion at amino acid (a.a.) 287 or 288, WT1 mutations were heterogenenous with missense mutations, insertions and deletions all being reported. WT1 mutations and NUP98-NSD1 co-associated in 7 patients, 1 patient also harbored a TYK2 mutation; in the remaining 7 patients with NUP98-NSD1 fusions, a mutation in RAD21 or NRAS was observed in 2 patients. For samples with other fusions (n=6), we detected an average of 1 additional mutation per sample, which included mutations (variant allele frequency; VAF) in DNMT3A (0.44), IDH2 (0.49), KIT (0.37), NPM1 (0.51), PLCG2 (0.44), RAD21 (0.55), and SMC1A (0.47). No fusion genes were observed in 13 patients. In this latter subset, mutations in NPM1 (n=6) and WT1 (n=3) were observed. Other alterations that were identified in these samples included mutations in DNMT3A, IDH2, PLCG2, and PRKCB, which co-occurred with NPM1 mutations. Three patients did not harbor a fusion gene or a gene mutation by our analysis. When looking at cumulative incidence of relapse or resistant disease, our study results are concordant with previous reports where a NUP98-NSD1 fusion associated with worse prognosis (hazard ratio [HR] = 3.2, p = 0.02), but FLT3-ITD allelic ratio >0.4 was not prognostic (HR = 1.1, p=0.87). NPM1 mutations were not significantly associated with better prognosis (HR = 0.2, p = 0.11). We next sought to identify relapse specific alterations by analysis of paired diagnosis/relapse samples by RNASeq, NGS panel, and WES. Notably, the FLT3-ITD mutation was maintained at relapse in all samples. From the NGS panel, we observed the emergence of a MED12 mutation (P1751Q, VAF 0.37) and WT1 mutation (p.S152*, VAF 0.19) at relapse; a mutational switch in WT1 from diagnosis to relapse was also observed (5bp insertion at a.a. 157 to 2bp insertion at a.a. 158). By RNASeq analysis, we found a novel relapse specific fusion gene, LUZP6-OSBL1A. From exome sequencing, mutations in transcription factors were observed at relapse such as CREBBP, GLI3, and TBX20. Our analysis of relapse specific genes showed recurrent mutations in HUWE1, OGT, NACAD, and UNC13A. Intriguingly, both OGT and HUWE1 have been implicated in cancer metabolic reprogramming, and regulate MYC transcriptional programs. OGT is an O-Linked β-N-acetylglucosamine (O-GlcNAc) transferase involved post-translational modification of serine and threonine residues. HUWE1 is an E3 ubiquitin ligase that has been established as a tumor suppressor and previously reported to be mutated in AML. In conclusion, we demonstrate that additional genomic alterations are observed in the majority of pediatric FLT3-ITD+ AML samples evaluated, with a high proportion of samples containing fusion genes, WT1 and NPM1 mutations. We also identified novel fusion genes and mutations that have not been previously reported in pediatric FLT3-ITD+ AML, including relapse specific mutations. These results provide further biological insight into the genomic heterogeneity of pediatric FLT3-ITD+ AML, warranting further investigations in larger patient cohorts. Disclosures Inaba: Arog: Research Funding.

We measured baseline serum mannose binding lectin (MBL) levels in 91 patients with childhood leukemia to determine their predictive value for the development of febrile neutropenia or specific infections. Median MBL levels did not differ significantly between patients who developed febrile neutropenia, bacterial infection, or disseminated fungal infection and those who did not. In addition, low MBL levels were not associated with an increased cumulative incidence of infection or with a shorter time to first infection. This preliminary study suggests that baseline MBL levels may not be clinically useful to identify pediatric leukemia patients who are at increased risk of infection. Additional studies are required to determine whether serial MBL measurements may be valuable for this purpose. Pediatr Blood Cancer 2008;50:866–868. © 2007 Wiley-Liss, Inc.

We describe a filtration-based culture method to enhance sensitivity of routine culture for recovery of fungi from respiratory specimens. The new method resulted in an 8.3% (P = 0.0039) increase in recovery for all organisms and a 6.4% (P = 0.0391) for Candida species when compared to the conventional culture technique.

Genetically engineered mouse models (GEMMs) of human cancer are important for advancing our understanding of tumor initiation and progression as well as for testing novel therapeutics. Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. GEMMs faithfully recapitulate the histopathology, molecular, cellular, morphometric, neuroanatomical and neurochemical features of human retinoblastoma. In this study, we analyzed the genomic and epigenomic landscape of murine retinoblastoma and compared them to human retinoblastomas to gain insight into shared mechanisms of tumor progression across species. Similar to human retinoblastoma, mouse tumors have low rates of single nucleotide variations. However, mouse retinoblastomas have higher rates of aneuploidy and regional and focal copy number changes that vary depending on the genetic lesions that initiate tumorigenesis in the developing murine retina. Furthermore, the epigenetic landscape in mouse retinoblastoma was significantly different from human tumors and some pathways that are candidates for molecular targeted therapy for human retinoblastoma such as SYK or MCL1 are not deregulated in GEMMs. Taken together, these data suggest there are important differences between mouse and human retinoblastomas with respect to the mechanism of tumor progression and those differences can have significant implications for translational research to test the efficacy of novel therapies for this devastating childhood cancer.

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

Purpose: The purpose of this study was to evaluate clinical implications of CD33 single-nucleotide polymorphisms (SNP) in pediatric patients with acute myeloid leukemia (AML) treated with gemtuzumab-ozogamicin (GO)–based therapy. Experimental Design: We genotyped four CD33 SNPs: rs35112940 (G>A; Arg304Gly), rs12459419 (C>T; Ala14Val), rs2455069 (A>G; Arg69Gly), and rs1803254 (G>C; 3′UTR) in pediatric patients undergoing induction chemotherapy containing GO (COG-AAML03P1 trial; n = 242) or not containing GO (St. Jude AML02 trial; n = 172). Results: CD33 SNPs were correlated significantly with clinical characteristics and treatment outcome. The coding SNPs, rs35112940 and rs12459419, were significantly associated with clinical endpoints in COG-AAML03P1 but not in the St. Jude AML02 trial. Specifically, among white patients in COG-AAML03P1, the 3-year overall survival (OS) rate from remission was 84% ± 8% for those homozygous (GG) for rs35112940 versus 68% ± 15% for the other genotypes (P = 0.018); these patients also had a lower relapse risk and superior disease-free survival. Likewise, patients homozygous for variant allele (TT) for rs12459419 were more likely to have favorable risk disease than CC and CT genotypes (52% vs. 31%, P = 0.034) and significantly lower diagnostic blast CD33 expression than other genotypes (P < 0.001). Conclusion: Our data suggest that genetic variations in CD33 could impact clinical outcome of GO-based therapy in pediatric AMLs. Clin Cancer Res; 19(6); 1620–7. ©2013 AACR.

Differential expression analysis of sequence-count expression data involves performing a large number of hypothesis tests that compare the expression count data of each gene or transcript across two or more biological conditions. The assumptions of any specific hypothesis-testing method will probably not be valid for each of a very large number of genes. Thus, computational evaluation of assumptions should be incorporated into the analysis to select an appropriate hypothesis-testing method for each gene. Here, we generalize earlier work to introduce two novel procedures that use estimates of the empirical Bayesian probability (EBP) of overdispersion to select or combine results of a standard Poisson likelihood ratio test and a quasi-likelihood test for each gene. These EBP-based procedures simultaneously evaluate the Poisson-distribution assumption and account for multiple testing. With adequate power to detect overdispersion, the new procedures select the standard likelihood test for each gene with Poisson-distributed counts and the quasi-likelihood test for each gene with overdispersed counts. The new procedures outperformed previously published methods in many simulation studies. We also present a real-data analysis example and discuss how the framework used to develop the new procedures may be generalized to further enhance performance. An R code library that implements the methods is freely available at www.stjuderesearch.org/depts/biostats/software.

To identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphism–array analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n = 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-of-heterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.21 (5% of patients) to a segment 486 kb proximal to the MYC locus. 13q deletions exhibited proximal and distal breakpoint cluster regions. Among the most common novel lesions were deletions at 15q15.1 (4% of patients), with the smallest deletion (70.48 kb) found in the MGA locus. Sequence analysis of MGA in 59 samples revealed a truncating mutation in one CLL patient lacking a 15q deletion. MNT at 17p13.3, which in addition to MGA and MYC encodes for the network of MAX-interacting proteins, was also deleted recurrently.