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Abstract 593 Cytarabine (ara-C) is one of the most widely used chemotherapeutic agents in the treatment of acute myeloid leukemia (AML). Inter-patient variability in clinical response following ara-C treatment has been correlated with leukemic cell concentration of ara-C triphosphate (ara-CTP), the active metabolite of the drug. NT5C2 is an inactivating enzyme in the activation pathway of ara-C, and catalyzes conversion of ara-C monophosphate to ara-C. We identified genetic variations in NT5C2 and investigated the association of NT5C2 genetic variations with its mRNA expression and with intracellular ara-CTP accumulation. The coding exons of NT5C2 were resequenced using genomic DNA from EBV-transformed B-lymphoblastoid HapMap cell lines derived from 90 CEPH (representing 30 trios with European ancestry) and 90 YRI (30 trios with African ancestry) sample. Thirty-nine genetic variants (one insertion-deletion and 38 SNPs), including three nonsynonymous SNPs (Thr3Ala, Lys47Arg, Gln136Arg), were identified. The strongly linked SNPs (R2 > 0.8) in CEPH and YRI were grouped into ten distinct groups. NT5C2 mRNA expression levels were determined in all samples and was significantly associated with ethnicity (p = 8.5 × 10-6), with subjects of African ancestry having significantly higher NT5C2 mRNA expression as compared to the subjects with European ancestry (Figure A). Several NT5C2 SNPs were associated with its mRNA expression in both the ethnic groups. To determine the clinical implication of NT5C2 SNPs, we investigated selected germline NT5C2 SNPs in pediatric AML patients treated on the St Jude AML 97 protocol (n=55). Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hrs after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher when given as short daily infusion, as compared to continuous infusion (0.56 ±0.5 vs. 0.33± 0.31 nmol ara-CTP/2×107leukemic cells; p = 0.014). The inter-patient variability for blast ara-CTP concentration was 40-fold in the short infusion arm and 101-fold in continuous infusion arm. Karyotypes, gender, age, and ethnicity had no significant influence on the leukemic ara-CTP levels. NT5C2 Group 1 SNPs were found to be significantly associated with intracellular ara-CTP levels in leukemic blasts from ara-C-treated Pediatric AML Patients (Figure B). These results suggest that genetic variation in NT5C2 could influence its activity and/or expression and may predict the variability observed in intracellular levels of the ara-C active metabolite, ara-CTP, which in turn can influence treatment response. Disclosures: No relevant conflicts of interest to declare.

Abstract 114 To identify genes that influence responses to cytarabine (ara-C) treatment, we explored the association of gene expression in leukemic cells at diagnosis with multiple pharmacological and clinical end-points in children with acute myeloid leukemia (AML) treated with ara-C on the St. Jude AML97 clinical trial. We applied a novel statistical procedure, PRojection Onto the Most Interesting Statistical Evidence (PROMISE; PR), to identify genes with expression levels associated with clinical and pharmacological endpoints. To do this, we first defined the following values of the clinical and pharmacological variables as “therapeutically beneficial” :higher leukemic cell ara-C triphosphate levels, lower DNA synthesis values on days 1 and 2 of treatment relative to baseline, decreases in leukocyte counts on day 2 of treatment, improved response and decreased risk of relapse, death, or second malignancy. We considered a gene to show a therapeutically beneficial pattern of association if its expression was positively correlated with ara-CTP levels, negatively correlated with DNA synthesis levels, negatively correlated with decrease in leukocyte counts on day 2, positively correlated with better treatment response, and negativelycorrelated with the risk of relapse or death. A gene showed a therapeutically detrimental pattern of association if its expression had the opposite correlations with the clinical and pharmacological variables. We performed five variable (PR5 using early pharmacologically interesting phenotype measures) or seven variable (PR7 all the above indicated phenotypes) PROMISE analyses. PR5 identified 275 beneficial probe sets and 69 detrimental probe sets (p ≤ 0.005). PR7 analysis identified 112 beneficial probe sets and 115 detrimental probe sets (p ≤ 0.005). To confirm these results, we performed a PROMISE for a cohort of patients treated with ara-C and other agents on the AML02 protocol. Gene expression in leukemic cells at diagnosis was analyzed for a beneficial or detrimental pattern of association with three phenotypes (PR-3); diagnostic blast ara-C cytotoxicity, minimal residual disease (MRD) and event-free survival (EFS). Eighty-one probe sets identified by PR5 or PR7 analyses in the initial cohort were confirmed in the PR-3 analysis of AML02 data. Genes identified in the present study may serve as predictive markers of response and candidates for future drug development. Disclosures: No relevant conflicts of interest to declare.

10034 Background: In the setting of hematopoietic stem cell transplantation (HSCT), donor natural killer (NK) cells exhibit potent anti-leukemic effects without causing graft-versus-host disease. We hypothesized that the transplantation of purified haploidentical NK cells may be a safe and effective form of consolidation therapy that will reduce the risk of relapse among children with acute myeloid leukemia (AML) who are not treated with HSCT. In this pilot study, we assessed the safety, feasibility, and engraftment of NK cell infusions in 10 patients with AML in first remission.Methods: Patients received cyclophosphamide, 60 mg/kg on day -7; fludarabine, 25 mg/m2/day on days -6 through -2; and IL-2, 1 million units/m2, every other day for 6 doses starting on day -1. On day -1, the donor underwent apheresis and the product was purified for CD56+ cells by a two-step procedure. The entire purified product was infused on day 0.Results: The 10 patients had a median age of 2.5 years (range, 8 months to 21 years) and a median leukocyte count of 62 x 109/L (range, 4 to 487) at diagnosis. Leukemic cell genetic abnormalities included CBFβ-MYH11in 4 cases, RBM15-MKL1in 2 cases, MLL-ENL and MLL-AF9 in 1 case each; 2 cases had no detectable abnormalities. Patients received a median of 29 × 106/kg NK cells (range, 5 to 81 × 106/kg). All patients had detectable donor NK cells at one or more time points: donor NK cell chimerism ranged from 0% to 30% during the first 4 weeks after the infusions and was greater than 1% in 9 cases at week 1, 4 cases at week 2, 5 cases at week 3, and 3 cases at week 4. One patient had prolonged NK engraftment (189 days), but no non-hematological toxicity. Grade 3–4 non-hematological toxicity was limited to one respiratory viral infection and one episode of febrile neutropenia. Median length of hospitalization was 2 days (range, 0–3) and median time to neutrophil recovery was 12 days (range, 9–56). With a median follow-up time of 637 days, all patients remain in remission. Conclusions: Haploidentical NK cells can be safely administered to AML patients who are in remission. All patients demonstrated temporary engraftment, which may have antileukemic effects. We have recently opened a new trial to evaluate the efficacy of NK cell therapy in children in first remission of AML. No significant financial relationships to disclose.

Cytarabine (ara-C) is one of the most effective agents for the treatment of AML; however development of drug resistance remains a major limitation. To identify genes that impact ara-C therapy, we explored the association of leukemic blast gene expression (measured using the Affymetrix U133A microarray) with intracellular ara-CTP pharmacokinetic and pharmacodynamic variables (DNA synthesis relative to baseline measured in leukemia cells, peripheral blast clearance) measured in leukemia cells from 42 patients treated under St. Jude AML97 protocol. In this study, patients received either a daily short infusion of ara-C or a continuous infusion of ara-C in combination with cladribine (CdA). Probe sets with mRNA expression levels (at diagnosis) that positively correlate with leukemic blast ara-CTP levels at day 1 (after ara-C alone) and day 2 (after ara-C and CdA), negatively correlate with extent of inhibition in days 1 and day 2 DNA synthesis relative to baseline, and negatively correlate with absolute blast count at 48 hours relative to baseline were considered to exhibit a therapeutically beneficial pattern of association. Probe sets with correlations opposite to those mentioned above were considered to exhibit a therapeutically detrimental pattern of association. An association pattern test found statistically significant evidence (p ≤ 0.005, FDR ≈ 0.30) that the expression of 304 probe sets associates with a beneficial pattern of association and of 61 probe sets associates with a detrimental pattern of association. These results include 34 cell cycle genes with beneficial association patterns and 2 cell cycle genes with detrimental association patterns (PPP1R13B and CUL4B). Also, 2 tumor necrosis factor genes (TNFRSF25 and FASLG) showed significant association with beneficial association patterns. The significant probe sets included RAS oncogene pathway genes, RAP2A and RAB9A with a detrimental pattern of association and the RAS-like gene RIT2 with a beneficial pattern of association. It has been recently shown that AML patients with mutations in RAS genebenefit from high ara-C doses1. The erythroblastic leukemia viral oncogene homologs EGFR and THRB demonstrated a statistically significant beneficial pattern of association. The T-cell leukemia homeobox gene also showed a statistically significant beneficial pattern of association. We further analyzed these 365 genes using Ingenuity Pathway Analysis tools and identified 16 networks with scores greater than 11 (p value < 10−11). In addition to the apoptotic genes, genes in the metabolic pathway of ara-C as well as few previously identified genes were also found to be associated with the pharmacokinetic and/or pharmacodynamic measures of ara-C in circulating blasts. In conclusion, our study has identified molecular signatures (in leukemic blasts at diagnosis) associated with the ara-CTP pharmacokinetics and pharmacodynamics. Validation of these signatures will help in better understanding of ara-C response as well as the mechanism underlying drug resistance. We are concentrating on these candidate genes and pathways for functional validation studies.

Background Differential gene expression analysis typically applies a statistical hypothesis testing procedure to the (log-transformed) data for each gene and then adjusts for multiple testing. However, it is not always clear whether a parametric or non-parametric test of hypothesis is preferred for a specific application. In a less complex application, a statistical test or graphical assessment of the fit of the assumed parametric model (e.g. the normal distribution) could be used to make this determination. However, in the analysis of microarray data it is unclear how to assess assumption adequacy for each gene and use it to guide subsequent analysis. Therefore, we introduce the principle of empirical Bayes assumption adequacy averaging and use it to develop a robust method to perform differential expression analysis. The proposed method combines results from the t-test and nonparametric rank-sum test according to the results of assessing the assumption of normality using the Shapiro-Wilk procedure. Through this averaging process, the proposed method is able to rely more heavily on the statistical test that the data suggests is superior for each individual gene. Subsequently, we observed that the proposed method has desirable power and robustness properties in a series of traditional and bootstrap-based simulation studies. Additionally, the proposed method showed greater concordance in gene selection across two studies of gene expression in acute myeloid leukemia than did the t-test or rank-sum test. Finally, we note that the principle of empirical Bayes assumption adequacy averaging can be used to develop robust procedures for a wide variety of experiments. from UT-ORNL-KBRIN Bioinformatics Summit 2008 Cadiz, KY, USA. 28–30 March 2008

To define the complement of genetic lesions in pediatric AML, we performed high resolution genome-wide analysis on bone marrow blasts from 111 cases using Affymetrix 100K and 500K SNP microarrays and genomic resequencing. Cases included t(8;21) (N=20), inv(16) (N=16), t(15;17) (N=7), MLL rearranged (N=16), FAB-M7 (N=9), miscellaneous cytogenetic abnormalities (N=24) and normal karyotype (N=19). Germline DNA was available for 67 of the cases. The mean number of somatic copy number abnormalities (CNA) was 2.38 (range 0–45) with 1.32 gains (range 0–41) and 1.06 losses (range 0–12). Focal CNAs were detected at the breakpoints of known chromosomal translocations, most commonly in inv(16) cases (7/16) and t(8;21) cases (4/20). Based on this observation, we examined focal CNAs that affected the 5’ or 3’ regions of genes for their possible involvement in cryptic translocations. CNAs involving NUP98 and MLL led to the identification of two cases with t(5;11)[ NUP98-NSD1 ], and two with t(6;11)[ MLL-MLLT4 ] not evident on cytogenetic analysis. RT-PCR for these fusions identified two additional NUP98-NSD1 cases. Identification of focal CNAs are thus useful in identifying clinically significant translocations in AML. Other recurring somatic CNAs were uncommon. Twelve regions containing a single gene were identified, including amplification of EBF , PRDM5 , CCDC26* , GDF10 , ABCC4*, and deletion of FAM20C* , TUSC1 , GPC5 , A2BP1 , INSR, BCOR* ( * involved in N>2 cases). The most frequent abnormality was amplification of CCDC26 at 8q24.21 ( N =15; focal in 4 cases, broad in 11) which encodes a putative mediator of retinoic acid receptor signaling. The focal amplifications are predicted to abolish expression of normal transcripts. Seventeen additional recurring CNAs involving 2 to 25 genes were identified, many of which contain known or putative tumor suppressor genes or oncogenes, including deletions of regions at 7q36, 9p21 ( CDKN2A/B ), 11p14-p12 ( WT1 , WIT1 ), and amplification at 19p13 ( JUNB, LYL1 ). These regions showed significant correlation between copy number and local gene expression in an integrated analysis of CNA and Affymetrix U133A gene expression data. Copy-neutral LOH was uncommon and recurrent only with LOH of chromosome 13 in 3 cases. Sequence analysis has been completed for several genes involved in recurring CNAs ( CCDC26, TUSC1, FBXW7 ) and for genes known to be mutated in AML ( N/KRAS, PTPN11 , BRAF, SOS1 , FLT3 , CEPBA , NPM1, AML1, CKIT , GATA1 ). No mutations were identified in CCDC26 , TUSC1 , FBXW7 , BRAF , or SOS1 . Marked differences in the combination of CNA and sequence mutation were identified across the different genetic subtypes of AML. FAB M7 cases had the highest frequency of CNA (mean 9.3 lesions/case) but had sequence mutations limited to GATA1 . Mutations of FLT3 , CEPBA and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, where as RAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 30% of the cases with recurring translocations had no other sequence or numerical abnormalities. These data demonstrated that, in contrast to pediatric ALL, AML is characterized by relatively few recurring CNAs, and that spectrum of CNA and sequence mutation is significantly associated with disease subtype.

Cytarabine (Ara-C) is a prodrug requiring conversion to its 5′-triphosphate (ara-CTP) form for antileukemic effect. Alteration of deoxycytidine kinase (DCK), a rate-limiting enzyme in this conversion, is associated with ara-C resistance. We sought to identify and determine the functional and clinical significance of DCK genetic variants. Sixty-four genetic polymorphisms were identified, including 3 coding changes (Ile24Val, Ala119Gly, and Pro122Ser amino acid substitutions), in subjects of European or African ancestry. The activity of recombinant DCK-24Val, DCK-119Gly, and DCK-122Ser was 85±5%, 66±3%, and 43±4%, respectively, that of wild-type (WT) DCK. These 3 DCK mutants also demonstrated altered substrate kinetics. Lymphoblast cell lines derived from subjects heterozygous for these coding changes had DCK activity significantly lower than that of homozygous-WT lymphoblasts (176.7±15 vs. 340.8±19 pmol CdA-MP/min/mg protein; p=0.0004). Moreover, for a 3′UTR 35708T>C SNP, the C allele was significantly associated with lower DCK mRNA expression in lymphoblast cell lines (p=0.02). To determine the clinical implications of DCK SNPs, we screened pediatric AML patients treated on the St Jude AML 97 protocol (n=55) for the 3 coding variants and the 3UTR SNP. Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hours after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher in patients given a short daily infusion than in those given a continuous infusion (0.56±0.5 vs. 0.33±0.31 nmol ara-CTP/2x107 leukemia cells; p = 0.014). The inter-patient variability of blast ara-CTP concentration was 40-fold (0.06–2.43 nmol/2x107 cells) when ara-C was administered as short infusion and 101-fold (0.012–1.22 nmol/2x107 cells) when given as a continuous infusion. Karyotype, sex, age, and ethnicity were not significantly associated with leukemia-cell ara-CTP level. An Ala119Gly or Pro122Ser non-synonymous polymorphism was present in 1 patient each; the patient heterozygous for Ala119Gly (continuous infusion arm) had the lowest intracellular concentration of ara-CTP (0.012 nmol/2x107 cells; Figure). Both of these patients had relapses, despite the presence of favorable cytogenetic abnormalities [inv16 and t(8;21)]. The 3′UTR 35708 T>C SNP was significantly associated with lower intracellular ara-CTP concentration in patients receiving ara-C as a continuous infusion (35708T allele 0.5±0.3 vs. 35708C allele 0.22±0.17 nmol/2x107 cells; p= 0.04; Figure). These results suggest that genetic polymorphism of DCK influences its activity and expression and may underlie interpatient variability in intracellular ara-CTP concentration, which in turn can influence treatment response. Influence of DCK SNPs on leukemic blast ara-CTP levels in AML patients receiving ara C-as continuous infusion Influence of DCK SNPs on leukemic blast ara-CTP levels in AML patients receiving ara C-as continuous infusion

Purpose Mice lacking the Arf tumor-suppressor gene develop eye disease reminiscent of persistent hyperplastic primary vitreous (PHPV). The current work explores mechanisms by which Arf promotes eye development, and its absence causes a PHPV-like disease. Methods Chimeric mice were made by fusing wild-type and Arf(-/-) morulae. In these experiments, wild-type cells are identified by transgenic expression of GFP from a constitutive promoter. PCR-based genotyping and quantitative analyses after immunofluorescence staining of tissue and cultured cells documented the relative contribution of wild-type and Arf(-/-) cells to different tissues in the eye and different types of cells in the vitreous. Results The contributions of the Arf(-/-) lineage to the tail DNA, cornea, retina, and retina pigment epithelium (RPE) correlated with each other in wild-type Arf(-/-) chimeric mice. Newborn chimeras had primary vitreous hyperplasia, evident as a retrolental mass. The mass was usually present when the proportion of Arf(-/-) cells was relatively high and absent when the Arf(-/-) proportion was low. The Pdgfrbeta- and Sma-expressing cells within the mass arose predominantly from the Arf(-/-) population. Ectopic Arf expression induced smooth muscle proteins in cultured pericyte-like cells, and Arf and Sma expression overlapped in hyaloid vessels. Conclusions In the mouse model, loss of Arf in only a subset of cells causes a PHPV-like disease. The data indicate that both cell autonomous and non-cell autonomous effects of Arf may contribute to its role in vitreous development.

To obtain a comprehensive registry of oncogenic lesions in pediatric acute lymphoblastic leukemia (ALL), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of heterozygosity (LOH) in leukemic blasts and matched remission samples from 250 ALLs. We studied B-progenitor ALLs with high hyperdiploidy, n=39; ETV6-RUNX1, n=47; MLL rearranged, n=11; TCF3-PBX1, n=17; BCR-ABL1, n=9; low hyperdiploidy, n=23; hypodiploidy, n=10; unclassified cases, n=42; and 50 T-lineage ALLs. Four arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 loci at a mean inter-marker distance of 4.8 kb. Data was analyzed using dChipSNP and a modified array normalization algorithm using only SNPs from regions known to be diploid by routine karyotyping. Copy number abnormalities were confirmed by FISH and genomic quantitative PCR. Complementary methylation analysis and sequencing of candidate genes was performed. 84% of B-ALLs and 96% of T-ALLs had at least one region of somatic deletion, and excluding cases with high hyperdiploidy, 68% of B-ALLs and 50% of T-ALLs had at least one region of somatic amplification. These included previously identified abnormalities including chromosomal duplications in hyperdiploid B-ALL; 1q duplication in TCF3-PBX1 ALL; and deletions of 9p21 (harboring CDKN2A/B, 70% of T-ALLs, 34.5% of B-ALL), 12p13 (ETV6; 25.5% of B-ALLs, 10% of T-ALLs), 6q16 (22 cases) and 11q (15 cases). The resolution of the arrays enabled precise mapping of the minimal regions of deletion at 9p21 to CDKN2A, and at 12p13 to ETV6. Combined LOH and copy number analysis identified several patterns of 9p21 abnormality: focal hemizygous deletion with corresponding LOH; focal homozygous and flanking hemizygous loss with corresponding LOH, indicating two focal deletional events; and focal homozygous loss with LOH of all of 9p or chromosome 9, indicating loss of the normal 9 or 9p and duplication of the chromosome or chromosomal arm containing the focal deletion. Copy-neutral LOH without any focal deletion in the affected region was uncommon. Deletions involving other genes with potential roles in leukemogenesis were identified including BTG1 (17 cases), ERG (10), FHIT (14), mir-16/-15a (19), MYB (5), NF1 (9), the glucocorticoid receptor NR3C1 (11), PTEN (4), and RB1 (20). Furthermore, deletions, translocations, amplifications, and point mutations of genes that regulate B-cell development and differentiation, including EBF, PAX5, Ikaros and Aiolos, were identified in 40% of B-ALL. For each of the listed genes, cases were identified that contained focal deletions limited to the specific gene. Overall, 73.6% B-ALL and 88% T-ALLs harbored deletions of one of the common lesions listed above, with 48% of B-ALLs and 48.5% of T-ALLs having multiple common lesions. The average number of deletions per case was 3.8 and 5.7 for B and T-lineage ALLs respectively. By contrast, when hyperdiploid cases were excluded, it was rare to find more than 2 regions of amplification in a single case, and the majority of cases contained no amplifications. These findings show the power of high-resolution copy number analysis for the identification of new genetic lesions in cancer, and demonstrate that multiple genetic abnormalities contribute to leukemogenesis in pediatric ALL.

Patients with acute myeloid leukemia (AML) who receive intensive chemotherapy regimens containing high-dose cytarabine are at high risk for bacterial sepsis due to prolonged neutropenia and severe mucositis. To determine if the use of prophylactic antibiotics during periods of neutropenia reduced bacterial infections and viridans streptococcal infections (VSI) in particular, we reviewed the charts of 66 AML patients who were treated on our front line protocol (AML02) from October 2002 to June 2006. During this time, several consecutive prophylactic antibiotic strategies were implemented for use after intensive myelosuppressive therapy. All patients also received prophylactic antifungal therapy. Overall, patients who received any prophylactic antibiotic regimen had an 86% (95% CI: 66–95%) reduction in VSI (P

To identify a comprehensive registry of oncogenic lesions in pediatric acute myeloblastic leukemia (AML), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of-heterozygosity (LOH) in leukemic blasts from 112 cases of pediatric AML and corresponding remission samples from 63 of these cases. The analyzed cases included t(8;21)[AML1-ETO] (n=20), inv(16)[CBFβ-MYH11](n=16), t(15;17)[PML-RARα] (n=7), MLL rearranged (n=17), FAB M7 (n=9) and normal cytogenetics or miscellaneous cytogenetic abnormalities (n=43). Four SNP arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 markers at a mean inter-marker distance of 4.8 kb. Combined data were analyzed using dChipSNP and a modified array normalization algorithm using only those SNPs from regions known to be diploid by routine karyotyping. These analyses not only detected known whole or partial chromosomal losses or gains, but also detected numerous copy number abnormalities that were not evident by conventional cytogenetics. Somatic DNA copy number abnormalities were identified in 102 (91.1%) cases. The mean number of lesions per patient was 3.2 (range 1–12), with a mean of 2.13 deletions/whole chromosome losses and 1.02 amplifications/whole chromosome gains per patient. Deletions were detected in the leukemic blasts from over 90% of patients, whereas amplifications were only seen in the leukemic blasts from 54% of patients. The vast majority of deletions were focal (

Chromosomal aberrations are a hallmark of acute lymphoblastic leukemia (ALL) but alone fail to induce leukemia. To identify cooperating oncogenic lesions, we performed genome-wide analysis of leukemic blasts from 242 pediatric ALL patients using high-resolution 100K and 250K Affymetrix single nucleotide polymorphism arrays, and genomic DNA sequencing. Remarkably, our analyses identified deletion, amplification, point mutation and structural rearrangement in genes encoding regulators of B lymphocyte development in over 40% of B-progenitor ALL. PAX5, which encodes a transcription factor critical for B cell commitment and differentiation, was the most frequent target of somatic mutation, being altered in 31.7% of cases. The most frequent PAX5 mutations were copy number alterations with mono-allelic loss in 53 cases, biallelic loss in 3 cases, and an internal amplification in 1 case. PAX5 deletions and the amplification were confirmed by FISH and/or RT-PCR, and were present in over 90% of blasts. Twenty-five of the mono-allelic deletions were confined to PAX5, with the majority deleting only a subset of PAX5 exons. Thus, of the 57 cases with PAX5 copy number changes, the majority had haploinsufficiency of PAX5 (n=30), or generated hypomorphic alleles that produce proteins that lack the DNA-binding domain (n=20) or the transcriptional activation domain (n=7). Four cases contained cryptic PAX5 translocations: PAX5-ETV6 (n=2), PAX5-FOXP1 and PAX5-ZNF521 (1 case each). In addition to the structural alterations, sequencing identified 14 B-ALLs with PAX5 point mutations. Mutations were identified in the DNA-binding paired domain, homeodomain and transactivation domains. The mutations were somatically acquired, and present in a dominant clone. Gel-shift and transcriptional reporter assays demonstrated reduced DNA binding and/or transcriptional activity for each of the identified PAX5 fusion proteins and point mutants. In addition to the PAX5 mutations, deletions were also detected in the B cell regulatory genes Ikaros (20 cases), Aiolos (3), EBF (8), E2A (1), LEF1 (3), and RAG1/2 (11). Importantly, genomic sequencing of Ikaros and EBF revealed no point mutations. Although the high frequency of mutations in genes regulating B-cell development was unexpected, even more surprising was the marked difference in the frequency and type of mutations among the various genetic subtypes of ALL. Hypodiploid ALLs had alterations in B-cell development genes in 100% of cases, with broad deletions of one PAX5 allele, mutation of the other PAX5 allele in 50% of the cases, and mono-allelic deletion of other regulators of B-cell development, frequently with multiple genes affected within a single case. In contrast ETV6-RUNX1 cases exhibited a much more restricted pattern of mutations, with 27% showing focal PAX5 deletion without alterations of the retained allele. At the other end of the spectrum were hyperdiploid ALLs, with only rare cases harboring a deletion of one of these genes. These data demonstrate that disruption of pathways controlling B cell development and differentiation contributes to the pathogenesis of ALL. Moreover, the approach used provides a rational roadmap for the application of genome-wide approaches to the identification of new molecular lesions in cancer.

Patients with acute myeloid leukemia (AML) and hyperleukocytosis are at increased risk of early death from neurological and pulmonary complications due to leukostasis. Although leukapheresis is often used for rapid cytoreduction in these patients, a recent adult study showed no difference in the rate of early mortality between patients who underwent leukapheresis and historical controls. The scarcity of information about leukapheresis in childhood AML led us review our experience of 106 children with AML and hyperleukocytosis (i.e., initial leukocyte count ≥ 100 × 109/L) treated between 1968 and 2002. The presenting clinical features, early complications, and clinical outcomes during the first 2 weeks of remission induction therapy were analyzed according to two treatment eras: early (1968–1982) vs. recent (1983–2002), when leukapheresis was available. The entire cohort had a median age of 9.7 years (range, 0–19.9 years); initial leukocyte count of 161 × 109/L (100 to 1,600); hemoglobin concentration of 8.2 g/dL (2.9–15.4); and platelet count of 38.5 × 109/L (0 to 300). The presenting features were comparable between patients treated in the two eras with the exception of higher hemoglobin concentrations among those treated in the recent era (p=0.03). Platelet transfusion prior to chemotherapy was used more often in the recent era (p=0.001). Half of the cases (53 of 106) had FAB M4 or M5 subtypes. Twenty-one patients (19.8%) had grade 3 or 4 neurological, respiratory, and/or renal complications (according to NCI common criteria) at initial presentation, and 35 patients (33%) had one or more of these complications during the first 2 weeks after diagnosis (17 neurological; 20 respiratory; 16 renal). The frequency of the complications did not differ significantly between patients treated in the two eras. Patients with FAB M4/M5 AML were significantly more prone than others to respiratory (p=0.005) and renal (p=0.0002) complications during the first two weeks of therapy. Seventeen patients (16%) died during the first 2 weeks after diagnosis. The rate of early death was significantly higher in the early era than in the recent era (16/70 vs. 1/36, p=0.01). The time between admission and initiation of chemotherapy was significantly shorter (20.2 vs. 33.6 hours, p

Therapy-related leukemia (t-ML) is a rare but devastating complication of anticancer treatment, including treatment of acute lymphoblastic leukemia (ALL). Although a number of treatment factors have been identified that contribute to the development of t-ML, little is known about the biology of the disease or the genetic risk factors. To identify possible genetic risk factors for and characteristics of t-ML among ALL patients, we used the Affymetrix GeneChip® Human Mapping 100K set to genotype over 100,000 single nucleotide polymorphisms (SNPs) in germline DNA from 13 cases with t-ML and from 13 matched controls, as well as DNA of the t-ML blast samples of the 13 cases. Controls were matched for treatment protocol, ALL risk classification, irradiation, prior use of G-CSF, and race. Germline allele frequencies differed (at the p < 0.01 level) for 309 SNPs (203 genes) between t-ML cases and controls. Cytogenetically, most of the cases had translocations or inversions of 11q23; however, three cytogenetically-detected regions of chromosomal loss were also detected (on chromosome 5, 7, and 9, each found only once among the cases). These chromosomal deletions were all detected as regions of loss of heterozygosity (LOH) and decreased copy number by paired intra-patient germline vs t-ML blast analyses of SNP calls. However, the paired SNP data indicated 216 regions of LOH (FDR = 2.5%) corresponding to a total of 81 non-contiguous regions (Figure), most of which were not detected as losses cytogenetically, and 38 shared (among pairs) regions of LOH. As many as 45 noncontiguous and 12 contiguous LOH regions were found among those cases with no cytogenetically defined regions of loss, including patients with simple balanced translocations. In addition to cytogenetically-defined cases, using SNP analyses we detected additional t-ML cases with regions of LOH on 5q (n=1), on chromosome 7 (n=4), and on chromosome 9 (n=2). In fact, only one patient, whose cytogenetics showed a balanced translocation involving 11q23, had no extended regions of LOH detected by SNP analysis. Altogether, 5 of the 13 cases demonstrated LOH on chromosome 7. Using a change-point model, we detected 18 regions of copy number increase and 21 regions of copy number decrease in the blasts compared to paired germline DNA (p Figure: 81 regions of inferred LOH in case t-ML blast DNA compared to germline DNA indicated losses involving multiple autosomes. Figure:. 81 regions of inferred LOH in case t-ML blast DNA compared to germline DNA indicated losses involving multiple autosomes.

We reviewed the records of 408 patients who were less than 21 years old at the time of diagnosis of acute myeloid leukemia (excluding patients with Down syndrome or acute promyelocytic leukemia) treated on five consecutive institution protocols from 1980–2002 to investigate prognostic factors for attainment of second complete remission (CR2) and overall survival (OS) after first relapse. Of the 320 (78%) patients who achieved CR, 158 patients suffered hematologic relapses at a median of 11.9 months (range, 2.9–119.9 months) from the time of diagnosis. Forty-one patients relapsed on therapy and 117 relapsed after the completion of all planned therapy. For patients who relapsed off therapy, 38 were diagnosed with relapse because of symptoms suggestive of recurrent leukemia and 78 were diagnosed at the time of a routine follow up (information not available for one patient). After relapse, 20 patients received palliative care, 82 received chemotherapy alone, 36 received chemotherapy followed by hematopoietic stem cell transplant (SCT), and 20 proceeded directly to SCT. Eighty-five (54%) patients attained CR2. In univariate analyses, factors associated with the achievement of CR2 include initial therapy (chemotherapy alone, 56%; autologous SCT, 71%; allogeneic SCT, 20%; p=0.008) and time from the diagnosis of AML to relapse (≤ 1 year, 44%; > 1 year, 65%; p=0.010). Logistic regression analysis demonstrated that patients with male gender (odds ratio [OR], 2.46; 95% confidence interval [CI], 1.14–5.28; p=0.021) and greater time from diagnosis to relapse (OR, 1.05; CI, 1.01–1.09; p=0.016) were more likely to achieve CR2, whereas patients with M7 morphology (OR, 0.11; CI, 0.01–1.04; p=0.054) and allogeneic SCT in first remission (OR, 0.17; CI, 0.03–0.85; p=0.031) were less likely to achieve CR2. At the time of last follow up, 19 patients were alive, 115 died of progressive disease, and 24 died of regimen-related toxicity. The 2-year OS estimate ± SE for the entire cohort of 158 patients was 15.8% ± 2.8%. For patients who relapsed off therapy, there was no significant difference in outcome between those whose relapse was diagnosed at the time of a routine follow up and those diagnosed because of symptoms. Cox proportional-hazards regression modeling indicated that M7 morphology (hazard ratio [HR], 3.06; CI, 1.44–6.51; p=0.004) and allogeneic SCT in first remission (HR, 2.17; CI, 1.22–3.87; p=0.008) were associated with significantly worse OS after relapse. In fact, there were no survivors in these two groups of patients. In contrast, age 1–10 years (HR, 0.53; CI, 0.36–0.77; p=0.001), M2 morphology (HR, 0.57; CI, 0.39–0.85; p=0.006), allogeneic SCT after relapse (HR, 0.34; CI, 0.22–0.53; p

The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine (A), daunorubicin (D), and etoposide (E) as Induction I; all subsequent therapy is risk-adapted. Pharmacokinetic, pharmacodynamic, and gene expression studies are performed after exposure to high-dose or low-dose cytarabine. Patients with no response (NR, ≥ 25% bone marrow blasts) to Induction I receive low-dose ADE + gemtuzumab ozogamicin (GO) as Induction II, whereas all others receive ADE. Minimal residual disease (MRD) is monitored by flow cytometry and is defined as ≥ 0.1% cells with leukemia-associated immunophenotypes among bone marrow mononuclear cells. Patients who are MRD+ after induction therapy receive GO as a single agent before consolidation chemotherapy or stem cell transplantation. Among the 112 patients enrolled since October 2002, 37 had -7, FAB M6 or M7, FLT3 internal tandem duplication (ITD), or MDS or treatment-related AML and were classified as high-risk (HR); 32 had AML1-ETO, CBFβ-MYH11, or MLL-AF9 (low-risk; LR); the remaining 43 were considered as standard-risk (SR). The rate of complete response (CR

Abstract 68 Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown. Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort. We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1). We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations. Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR. Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures: Evans: St. Jude Children's research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity's Board of Directors or advisory committees.