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With the introduction of genomic technologies, more hereditary cancer syndromes with hematologic malignancies are being described. Up to 10% of hematologic malignancies in children and adults may be the result of an underlying inherited genetic risk. Managing these patients with hereditary hematologic malignancies, including familial leukemia, remains a clinical challenge because there is little information about these relatively rare disorders. This article covers some of the issues related to the diagnosis and interpretation of variants associated with hereditary hematologic malignancies, including the importance of an accurate family history in interpreting genetic variants associated with disease. The challenges of screening other family members and offering the most appropriate early malignancy detection is also discussed. We now have a good opportunity to better define hereditary cancer syndromes with associated hematologic malignancies and contribute to clinically effective guidelines.

B lymphoblastic leukemia (B-ALL) in adults has a higher risk of relapse and lower long-term survival than pediatric B-ALL, but data regarding prognostic biomarkers are much more limited for adult patients. Microarray-based genome-wide profiling studies in pediatric B-ALL patients have revealed recurrent abnormalities in B-cell development and cell cycle regulation. IKZF1 alterations convey a negative prognostic impact in pediatric B-ALL, but their significance is not well characterized in adult B-ALL. CDKN2A alterations have been associated with a poorer prognosis in adult Ph+ ALL, possibly by mediating resistance to targeted therapy. The copy number landscape of adult B-ALL has not been fully assessed and is likely distinct from its pediatric counterpart. In addition, the copy number changes in relapsed adult B-ALL, including comparison with initial diagnostic samples, have not been characterized. We identified 70 adult B-ALL patients (median age 45 years, range 18-83) from 1998-2013 at three institutions. DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) diagnostic bone marrow clots, as well as relapse samples when available, and assessed with the OncoScan FFPE Express genome-wide single nucleotide polymorphism (SNP) assay (Affymetrix). Copy number alteration (CNA) and loss of heterozygosity (LOH) analysis was performed using Nexus Software V7 (Biodiscovery) and in-house coding. For cases with adequate DNA, IKZF1 sequencing was also performed. Clinical data included age, gender, hematologic laboratory values at presentation, CSF involvement, receipt of allogeneic transplant, cytogenetic profile, presence of t(9;22), event-free survival (EFS), and overall survival (OS). Recurrent deletions in the diagnostic samples were noted at several loci, including CDKN2A (48.6%), IKZF1 (40%), ICR (38.6%), PAX5 (24.2%), BTG1 (17.1%), ETV6 (15.7%), BTLA (14.3%), RB1 (5.7%), EBF1 (5.7%), LEF1 (2.9%), and TCF3 (2.9%). Recurrent gains were identified at the following loci: ERG (30%), ETS2 (21.4%), MYB (20%), UBASH3B (20%), PTEN (20%), PRKCH (18.6%), CDK6 (17.1%), and ETV6 (12.9%). In addition, LOH was observed in all loci with recurrent CNAs named above. 27 samples obtained at the time of relapse were screened for the recurrent CNAs identified in the diagnostic samples. The cumulative incidence of CNAs at these genes was lower in the relapse cohort than the initial diagnostic cohort, although there was a higher frequency of CNAs at PAX5, BTLA, ETS2, RB1, LEF1, PTEN, and TCF3 in the relapse samples. 16 patients had samples available at both the time of diagnosis and time of relapse. The average number of CNAs at diagnosis and relapse was the same, but analysis of paired samples revealed frequent changes at the previously defined loci of CNAs. There were a number of new CNAs at these loci in relapse samples, but there was also a similar incidence of reversion to the copy neutral state in loci that previously had CNAs in the diagnostic samples. Sequencing data was available on 26 diagnostic samples for the IKZF1 gene. Only two samples had mutations corresponding to amino acid changes. One of these two had a heterozygous deletion at IKZF1, indicating that the mutated isoform was the only one expressed. 11 other samples had a SNP without a corresponding amino acid change. 14 relapse samples were sequenced, two of which harbored a point mutation in IKZF1. Neither of these samples had a CNA at this locus. 10 of the remaining 12 relapse samples had a SNP without a corresponding amino acid change. When correlated with outcomes, no individual CNA heralded a significant prognostic impact in the entire cohort or in subgroup analyses stratified by presence of t(9;22) for either EFS or OS. However, the combination of both CDK2NA and IKZF1 deletions (26%) correlated with a significantly worse OS than having only one or neither of these deletions (both vs. CDKN2A only: p=0.028, both vs. IKZF1 only: p=0.027, both vs. neither deleted: p=0.048). Age was the only other covariate significant in univariate analyses for OS, yet IKZF1/CDKN2A co-deletion remained significant in multivariate analysis adjusting for age. Adult B-ALL demonstrates recurrent copy number changes, and the pattern of CNAs is often different at relapse than at diagnosis indicating clonal evolution. When identified in diagnostic samples, co-deletion of CDKN2A /IKZF1 is a negative prognostic marker in adult B-ALL. Disclosures South: Illumina: Consultancy, Honoraria; Affymetrix: Consultancy, Honoraria; ARUP Laboratories: Employment; Lineagen Corporation: Consultancy. Bixby:Seattle Genetics, Inc.: Research Funding. Gastier-Foster:Bristol-Myers Squibb: Research Funding.

Abstract 289 Children with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL), and consistently demonstrate poorer outcomes due to higher rates of both relapse and treatment-related mortality compared to other children with ALL. The biology of ALL in DS is unique, with lower frequency of the classic cytogenetic lesions generally observed in childhood ALL, and increased frequency of JAK2 mutations and CRLF2 rearrangements, which are not clearly associated with adverse prognosis in DS-ALL. In order to improve risk stratification and identify potential novel therapeutic targets in this vulnerable population, we analyzed 90 DS-ALL cases for prognostically significant copy number abnormalities (CNAs) in a collaborative cohort from the Children's Oncology Group (n=30), St. Jude Children's Research Hospital (n=22), AIEOP (n=16), Texas Children's Cancer Center (n=10), UKALL 2003 (n=6) and an archival UK sample (n=1), and Utah's Primary Children's Medical Center (n=5). Copy number profiling was performed using 500K, 6.0, CytoScan HD, and OncoScan FFPE Express arrays (Affymetrix), and Human CNV370-Duo arrays (Illumina). Gene expression profiling was performed using U133 Plus2.0 arrays (Affymetrix). Copy number was analyzed with Nexus Copy Number (BioDiscovery, Inc.) and gene expression was analyzed with Partek Genomics Suite (Partek, Inc.) and Gene Set Enrichment Analysis (Broad Institute). Deletions of a focal region on 22q11.22 (present in 28.9% of cases, similar to the incidence previously reported in a non-DS cohort [Mangum et al, ASH 2011:741]), and deletions of IKZF1 (present in 20.0% of cases), were significantly associated with poor event-free survival (EFS) (5-year EFS was 88.6 ± 6.3% in wild-type cases [n=31], 68.1 ± 13.3% in cases with deletion of 22q11.22 only [n=15], and 60.0 ± 21.9% in those with deletion of IKZF1 only [n=5]; combined deletion [n=6] was associated with an even poorer EFS (33.3 ± 19.3%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 2664 Background: We have recently shown that a subset of pediatric Burkitt lymphoma (pBL) show gain of 13q31, containing the microRNA (miRNA) MIR-17–92 locus, as well as associated increased expression of miRNA from that locus. PTEN, a tumor suppressor phosphatase that negatively regulates the PI3K/AKT/mTOR pro-survival pathway, is negatively regulated by MIR-17–92. Therefore, we hypothesized that PTEN protein and mRNA expression would be decreased in pBL cases with 13q31 locus gain and high expression of MIR-17–92. Because PTEN negatively regulates the PI3K/AKT/mTOR pathway, we also assessed AKT activation in pBL tissues. Methods: We assessed PTEN protein expression by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) pBL tissues from a cohort of patients (n=25) where MIR-17–92 expression levels and status of the 13q31 locus have been previously determined (Schiffman/Miles, Brit J Haematol 155:477, 2011). From FFPE tissues, we assayed PTEN mRNA levels by quantitative RT-PCR. The results of protein and mRNA expression were then correlated with presence or absence of a 13q31 gain and the expression level of MIR17. To evaluate AKT activation in pBL, we assayed for phospho-(p)-AKT by IHC. In addition, PTEN, AKT, and p-AKT protein levels were evaluated by western blotting in Raji and Ramos BL cell lines. Results: PTEN protein was weakly expressed in 5/25 cases (20%) of pBL. PTEN protein showed moderate nuclear and cytoplasmic staining in a subset of paracortical and germinal center lymphocytes in reactive lymph node control tissues, and vascular endothelial cells provided internal positive control in tumor tissue. pBL tissues showed decreased relative PTEN mRNA expression compared to reactive lymph node controls (1.56 vs. 2.61, p Discussion: pBL shows decreased PTEN mRNA, and most cases are negative for PTEN protein expression. Unexpectedly, higher MIR-17–92 expression level or 13q31 gain did not show an inverse relationship with PTEN expression at the protein or mRNA level. Despite decreased PTEN expression, p-AKT was detected in only 1/25 pBL cases and was not detected in BL cell lines. These findings suggest that decreased PTEN expression does not lead to AKT activation in pBL. Ongoing studies will investigate the potential of alternate PTEN targets and/or alternate targets for MIR-17–92 in pBL. Disclosures: No relevant conflicts of interest to declare.

Abstract 741 Identifying prognostic biomarkers in childhood acute lymphoblastic leukemia (ALL) is imperative for risk-stratification and intensifying therapy for children at high risk of remission induction failure or relapse. IKZF1 deletions/mutations were recently shown to correlate with poor outcome in ALL, highlighting genetic alterations as prognostic markers (NEJM 360:470, 2009). We recently described focal deletions in VPREB1 located within the lambda variable chain region which are not part of normal light chain rearrangement in precursor B-cell ALL in a local Utah cohort and the TARGET cohort of high-risk ALL patients treated on the Children's Oncology Group P9906 trial. Upon further inspection, we identified a second focal deletion in chromosome 22q11.22, nearly 80 kilobases (Kb) upstream from VPREB1 in the same lambda region. We characterized and correlated this deletion with outcome in childhood ALL using the Utah Cohort (N=60), TARGET P9906 cohort (N=221), and St. Jude Children's Research Hospital cohort (SJCRH, N=298). Microarray data was analyzed (Utah = Molecular Inversion Probe 330K [Affymetrix]; TARGET = SNP 500K & U133A [Affymetrix]; SJCRH = SNP 500K/6.0 & U133A [Affymetrix]) by Nexus Copy Number and Nexus Expression (BioDiscovery, Inc.). Each cohort contains the focal 22q11.22 loss (Hemizygous: Utah = 21%, TARGET = 16%, SJCRH = 18%; and Homozygous: Utah = 7%, TARGET = 16%, SJCRH = 10%). It spans 142 Kb, with the most common recurring region just under 10 Kb in length. The deleted segment encodes no known genes. In the SJCRH cohort, 22q11.22 loss differs by subtype: Down's syndrome 93%, BCR-ABL1 47%, ETV6-RUNX1 46%, Pseudodiploid 38%, Hyperdiploid 30%, Other 33%, MLL 12%, TCF3-PBX1 12%, T-ALL 7%, Hypodiploid 0%. In the TARGET cohort, 22q11.22 loss confers a trend for worse event-free survival (EFS; P=0.17) and overall survival (OS; P=0.06). Compared to normal 22q11.22/IKZF1 (77% EFS) or IKZF1 alternations alone (45% EFS), combined 22q11.22 loss plusIKZF1 alterations drastically reduces EFS to 15% (P Disclosures: Shams: BioDiscovery, Inc.: Employment.

Abstract 1192 T cell acute lymphoblastic leukemia (T-ALL) is a challenging clinical entity. Over half of adult, and about 20% of pediatric, T-ALL patients either relapse or fail to achieve remission, and despite salvage attempts outcomes are poor. To discover new acquired genetic changes that occur in T-ALL, as well as those that contribute to disease relapse, we studied zebrafish (Danio rerio) T-ALL samples using array comparative genomic hybridization (aCGH). Five different D. rerio T-ALL models have been described, and all 5 develop neoplasias that clinically and molecularly resemble human T-ALL. We performed aCGH on 14 de novo T-ALLs representing 4 of these models. To evaluate possible similarities between human and zebrafish copy number aberrations (CNAs) in T-ALL, we compared all D. rerio genes found in any CNA in our 14 zebrafish cancers to a cohort comprised of 61 published primary human T-ALLs analyzed by aCGH. In these D. rerio CNAs, we identified 764 genes with human homologues. Of these, we found significant overlap (62%) with genes in CNAs from the human T-ALL dataset. In addition, 10 genes recurrently altered (>3 samples) in zebrafish T-ALL were also seen in CNAs from 5 or more human T-ALL cases, suggesting a conserved role for these loci in T-ALL transformation across vertebrate species. In addition to studying primary T-ALL, we also conducted iterative allo-transplantations with 3 zebrafish malignancies. This technique selects for particularly aggressive disease, increasing engraftment rates and also resulting in shorter time to death of recipient animals in successive transplant rounds. Because these passaged cancers show more malignant behavior in vivo, this procedure models refractory and relapsed T-ALL. In these 3 serially-passaged cancers, 55% of the original CNAs were preserved after iterative transplantation, demonstrating the clonal relationship between the primary and passaged leukemia. In addition, 101 CNAs were acquired during passaging of the 3 T-ALLs. Genes in these loci may underlie the enhanced malignant behavior of these neoplasias. We also compared genes found in CNAs from passaged zebrafish malignancies to human aCGH results from 50 T-ALL patients who failed induction, went on to relapse, or had already relapsed. Again, many genes (n=76) were present in both datasets. Four genes altered in 2 of 3 D. rerio samples were also found in >4 cases from the human dataset. Collectively, these results suggest that zebrafish and human T-ALL are similar at the genomic level, with several homologous genes commonly gained or lost by cancers from both organisms. Genes from recurring CNAs in disease samples from both species may have particular relevance, as these candidates likely have conserved roles in T cell oncogenesis or T-ALL disease progression. As the comprehensive list of genes in CNAs from human T-ALL cases is vast and heterogeneous (comprising 15,724 unique genes from just 75 clinical samples), zebrafish T-ALL models may permit a more expedient prioritization of these candidates for further investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 273 VPREB1 deletions occur in 1/3 of pediatric ALL (pALL) patients, but have been considered a normal consequence of λ light chain rearrangement due to VPREB1's location among λ chain variable region genes. Recently, VPREB1 deletions were reported in high risk pALL samples with BCR-ABL1-like gene expression (Den Boer 2009) and VPREB1 under-expression was found in a 38-gene signature associated with relapse (Kang 2010). We characterized VPREB1 deletions to determine if they are part of normal λ light chain rearrangement or represent a bona fide copy number alteration (CNA) that predicts worse outcome in pALL. We first used the genome-wide Molecular Inversion Probe (MIP) 330K Cancer Panel (Affymetrix) to identify VPREB1 deletions in a cohort of 52 pALL patients from the University of Utah (n=47 Precursor B-cell (Pre-B), 4 Precursor T-cell (Pre-T), 1 relapse Pre-B ALL). We found focal VPREB1 deletions in 25% (n=12/48) of Pre-B ALL and no (n=0/4) Pre-T ALL patients. We also identified a distinct and often homozygous deletion (14 kb in length) located ≂f 78 kb upstream of VPREB1 that could represent a potential enhancer region; this possible enhancer deletion was seen in 16.7% (n=8/48) of Pre-B and none of the Pre-T ALL patients. 50% of Pre-B ALL samples had at least one deletion of B-cell developmental gene (SPI1, IKZF1, BCL11A, TCF3, EBF1, PAX5, FOXP1, LEF1, VPREB1, BLNK), and of these, half (n=12/24) had deletion only of VPREB1 or its possible enhancer. We next sorted blood leukocytes from 10 healthy volunteers for quantitative PCR (qPCR) analysis on κ V-J junction (deletion=attempted κ rearrangement), λ V-J junction (deletion=attempted λ rearrangement), LOC96610 and PRAME (distal to VPREB1, among λ-light chain variable genes), VPREB1, and VPREB1 enhancer (proximal to VPREB1, among λ-light chain variable genes). As predicted for normal light chain rearrangement, we observed no deletions in monocytes; 100% of κ-sorted B-cells had κ V-J deletions without lambda deletions; and 100% of λ-sorted B-cells had both κ V-J and λ V-J deletions. We also analyzed 10 mature B-cell (Burkitt) lymphoma samples and observed VPREB1 deletions only in lambda-expressing samples with both κ and λ V-J deletions, and deletions always extended contiguously downstream to λ chain V-J region. We re-analyzed 30 Pre-B ALL samples (Utah cohort) and 11 Pre-B ALL cell lines by qPCR. Results validated MIP CNA patterns and differed from normal sorted B-cell and mature B-cell (Burkitt) results, displaying several types of rearrangements: 1) No light chain rearrangements with no VPREB1/enhancer deletion (n=5/30 [17%] clinical samples and 2/11 [18%] Pre-B cell lines), 2) Normal light chain (κ or κ/λ) rearrangement with no VPREB1/enhancer deletion (n=5/30 [17%] samples and 6/11 [55%] cell lines), 3) Normal light chain (κ or κ/λ) rearrangements with contiguous VPREB1/enhancer deletion (n=1/30 [3%] samples and no cell lines), 4) λ-light chain rearrangement with non-contiguous VPREB1/enhancer deletion (n=13/30 [43%] samples and 2/11 [18%] cell lines), and 5) No λ light chain rearrangement and focal VPREB1/enhancer deletion (n=6/30 [20%] samples and 1/11 [9%] cell lines). In the Utah Pre-B ALL cohort (n=47), VPREB1/enhancer deletions were significantly associated (Fisher's Exact Test, 2-tailed) with Hispanic ethnicity (p= 0.013), relapse (p= 0.027), death (p=0.026), and Day 14 M2 Marrow (5-25% blasts; p=0.038). Finally, we examined the publicly available St. Jude Children's Research Hospital ALL dataset (NEJM 351:533) and found VPREB1 under-expression associated with higher LC50 (resistance) for DNR (p=0.04) and VCR (p=0.04), but not PRED or ASP. In summary, we have shown VPREB1 deletions occur outside the context of normal light chain rearrangement. Unlike physiologic λ rearrangements, VPREB1 deletions do not always extend to the λ V-J junction in pALL. We also identified a focal, homozygous deletion just proximal to VPREB1 that may affect a possible enhancer region for this B-cell developmental gene. VPREB1/enhancer deletions were the only B-cell developmental gene lost in 25% of our cohort, perhaps defining a new Pre-B ALL subtype. Finally, deletion of VPREB1 (and its possible enhancer) predict worse clinical outcome and VPREB1 down-regulation correlates with in vitro drug resistance. Further studies of VPREB1's function in pediatric ALL may improve our understanding of leukemogenesis and could further refine clinical risk stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract 3123 Although cure rates are high for pediatric Burkitt lymphoma (BL), a subset of patients relapses and succumbs to the disease. BL is characterized by translocation of the MYC gene with an immunoglobulin gene, but secondary changes including gain of 1q, gain of 13q, loss of 13q, loss of 17p, and others have been described by both conventional cytogenetic and oligo array CGH approaches. Secondary changes may contribute to the clinical heterogeneity of BL as evidenced by the fact that loss of 13q is associated with a worse prognosis in pediatric BL (Poirel et al., Leukemia 23:323, 2009). However, high resolution, genome-wide copy number analysis has not yet been reported in pediatric BL. The objective of this study was to identify copy number alterations (CNAs) in pediatric BL using a genome-wide approach, and to examine the relationship between CNAs and clinical parameters including outcome. After institutional review board approval, we identified 30 pediatric BL patients treated at Primary Children's Medical Center (n=25, Salt Lake City, UT) and Penn State Hershey Medical Center (n=5, Hershey, PA) with available formalin-fixed, paraffin-embedded (FFPE) diagnostic biopsy specimens. Age, site, and gender data were available for all specimens, and 22/24 of the Utah patients were treated according to the COG 5961 protocol with full clinical information and follow-up available. DNA was isolated from FFPE biopsies containing at least 80% tumor. In addition, germline DNA was isolated from negative staging bone marrow clot sections on Utah patients (n=25) to serve as a pooled normal reference and to provide germline copy number variation data on individual patients. Tumor and paired normal DNA was submitted for Molecular Inversion Probe (MIP) assay (330K Cancer Panel, Affymetrix, Santa Clara, CA). The Nexus Copy Number (BioDiscovery, El Segundo, CA) software package was used to analyze the MIP data with the following stringent call criteria: SNPRank segmentation, 5.0E-4 significance threshold, 1000 kb maximum contiguous probe spacing, minimum of 5 probes per segment, gains ≥ 2.7 copy number value, and loss ≤ 1.3 copy number value. Patients included 23 males and 7 females with a mean age of 8.0 years (range 2 – 18). At presentation, lactate dehydrogenase (LDH) levels ranged from 443 – 13851 U/L and uric acid levels ranged from 1.5 – 13 mg/dL. Patients were Murphy stage I (n=1), II (n=9), III (n=13), and IV (n=2). A total of three patients relapsed (one stage II patient and two stage III patients) and three died (the two stage III patients that relapsed and a different stage II patient who did not have a complete clinical response). 27 of the 30 tumor samples and 22/25 paired normals had adequate DNA for MIP analysis. The 3 tumor samples without adequate DNA were clinically similar to the others. We identified a total of 103 CNAs (defined as change seen in the same cytoband in 1 or more patients), which included 63 gains and 40 copy number losses. 23/27 cases (85%) had at least one gain or loss. We identified 21 recurrent CNAs (same cytoband affected in 2 or more patients), which included 14 gains and 7 losses. We found gains of 1q in 10/27 patients (37%), gains of 13q in 4/27 patients (15%) and losses of 17p in 3/27 patients (11%). Despite a relatively small sample size, deletion of 17p13 was significantly associated with relapse (p=0.041). To our knowledge, this is the first report of high-resolution genome-wide copy number analysis of pediatric BL. Furthermore, we show for the first time that FFPE archived materials can be used for high resolution gene copy number analysis in lymphomas. We identified CNAs in 85% of the pediatric BL cases, which include previously described changes of 1q, 13q, and 17p. Although sample size limited statistical power, deletion of the 17p13 locus was associated with relapse. We plan to extend these studies in a larger sample of patients to evaluate the potential prognostic significance of both the 17p13 locus deletion as well as additional recurrent CNAs. Disclosures: No relevant conflicts of interest to declare.

Abstract 275 T-cell receptor (TCR) gene rearrangements occur in 50–80% of childhood precursor B-cell ALL and have been used for minimal residual disease (MRD) detection. However, TCR rearrangement has never been examined for its contribution to disease classification and outcome. We used Molecular Inversion Probe technology (Affymetrix) to analyze precursor B-cell ALL samples (n=48) from the University of Utah for genome-wide copy number changes, and evaluated the relationship between focal TCR deletions (indicating rearrangement) and clinical features. Copy number deletions were detected by Nexus Copy Number (BioDiscovery, Inc.) and were based on 5 consecutive probes having a value of 25% blasts; p=0.019). TCR-δ deletion was also significantly associated with Day 28 MRD Positivity (p=0.0206). Due to the small sample size and insufficient follow-up, outcome analyses were not done in this cohort. The COG HR-ALL TARGET cohort had the following TCR deletions: TCR-α=53/221 (24%), TCR-β=24/221 (11%), TCR-γ=55/221 (25%), TCR-δ=53/221 (24%), or TCR-‘any'=96/221 (43%). Fisher's Exact test (two-tailed) was used to look for associations between TCR deletions and clinical features; outcome analyses were conducted using the Kaplan-Meier method and the log-rank test. In the TARGET cohort, we found TCR-α, TCR-γ, and TCR-δ deletions were significantly associated with occurrence of relapse and/or death (p=0.0209, p=0.0143, and p=0.0209, respectively). TCR-β deletion was associated with age at diagnosis ≥ 10 yo (p=0.0225), a high risk feature, and Day 29 MRD Positivity (p=0.0084). In the high-risk TARGET validation cohort, 43 of the 78 children (55%) who experienced a relapse or died contained TCR deletions vs. 53 of the 143 children (37%) who were event-free with TCR deletions (p=0.0109). There was a significant difference in event-free survival (EFS) between patients with TCR-‘any' deletions vs. those without the deletions (p=0.013), with the largest difference in survival occurring beyond two years of diagnosis (Figure 1). TCR deletions were uncommon in Hyperdiploid (0/4, 0%), E2A-PBX1 (n=1/25, 4%), and MLL (n=4/19, 21%) subtypes in the TARGET cohort, but were more frequent in TEL-AML1 (n=2/3, 67%) and Other (n=89/170, 52%) subtypes. In summary, both the Utah discovery cohort and the larger COG HR-ALL TARGET validation cohort showed focal TCR gene deletions that were linked to high risk clinical features, as well as associated with poor prognostic attributes such as end-of-induction MRD Positivity. Additionally, the larger TARGET cohort demonstrated that deletion of any TCR region was significantly associated with worse outcome. TCR gene deletions may indicate a more immature state of developmental arrest and could imply initial abortive T-cell lineage development prior to B-cell commitment and transformation. This represents the first study to reveal an association between TCR gene deletion status and clinical outcome in childhood B-precursor ALL, and these findings will need validation in additional pediatric and adult cohorts. Ultimately, focal TCR gene deletions may prove to be a valuable addition to the molecular risk classification schema currently applied to B-precursor ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4117 Background Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, and the handful of studies that investigate ethnic differences in childhood ALL consistently report an increased incidence and worse survival in African-American and Latino children. Studies examining clinical features (high white count) and biological prognostic factors (ploidy, immunophenotype, chromosomal abnormalities) by ethnicity could find no contribution that explained poor outcome among African-Americans. However, investigations exclusively focused on biological factors contributing to worse outcome in Latino children with ALL have been limited. The Latino population in Utah is approximately 10% and Latino children account for 15-20% of new ALL diagnoses treated at Primary Children's Medical Center (PCMC), the only children's hospital located in Utah. As the Latino population continues to expand in Utah and the surrounding states, it will become increasingly important to understand what biological differences, if any, exist between Latino and non-Latino children with ALL. Objective To investigate whether Latino children with ALL in Utah have different clinical and biological features compared to non-Latino children. Methods For this pilot study, we conducted a retrospective chart review of clinical and biological risk factors in 90 pediatric patients (Latino, n=40 and Caucasian, n=50) diagnosed with ALL at PCMC between 1997-2009. We included all Latino children treated during this time and randomly-selected Caucasian children for ∼ 1:1 comparison. Over 50 clinical and biological variables were recorded for each patient and compared with Fisher's Exact Test (2-tailed) and logistic regression models. Results We found no statistical differences between the Latino and Caucasian cohort in our univariate analysis for the following features: age ≥ 10 years old (Latino 27.5% vs. Caucasian 26%), initial WBC > 50 × 10-3/uL (Latino 22.5% vs. Caucasian 14%), NCI-Rome High Risk Group (Latino 37.5% vs. Caucasian 46%), initial hemoglobin < 11.5 gm/dL (Latino 85% vs. Caucasian 80%), initial platelet count < 150 × 10-3/uL (Latino 87.5% vs. Caucasian 80%), precursor B-cell phenotype (Latino 98% vs. Caucasian 86%), slow early response (SER) marrow defined as >5% blasts (M2 or M3) for day 8 (Latino 66.6% vs. Caucasian 57.4%), day 15 (Latino 15.4% vs. Caucasian 15.8%), and day 29 (Latino 2.8% vs. Caucasian 0%), minimal residual disease (MRD) ≥ 0.1% on Day 29 (Latino, n=4 vs. Caucasian, n=7), FISH cytogenetic results (Chr. 4 hyperdiploidy: Latino, n=5 vs. Caucasian, n=9; Chr. 10 hyperdiploidy: Latino, n=2 vs. Caucasian, n=10; Chr. 17 hyperdiploidy: Latino, n=3 vs. Caucasian, n=9; Chr. 21 hyperdiploidy: Latino, n=7 vs. Caucasian, n=16), t(12;21) status (Latino, n=1 vs. Caucasian, n=7), t(9;22) status (Latino, n=2 vs. Caucasian, n=0), hepatomegaly (Latino 10% vs. Caucasian 4%), splenomegaly (Latino 18% vs. Caucasian 16%), combined hepatosplenomegaly (Latino 18% vs. Caucasian 36%), CNS disease defined as ≥ CNS 2 (Latino 12.5% vs. Caucasian 12%), DNA index 1 (Latino 27% vs. Caucasian 27%), patients enrolled in COG clinical trial (Latino 75% vs. Caucasian 75%), and relapse rate (Latino 15% vs. Caucasian 8%). Logistic regression found no differences between the Latino and Caucasian cohort for: age (Latino median = 5 yrs vs. Caucasian median = 4 yrs), platelet count at diagnosis (Latino median = 51 vs. Caucasian median = 56), hemoglobin at diagnosis (Latino median = 8.2 vs. Caucasian median = 8.0), WBC at diagnosis (Latino median = 10.3 vs. Caucasian median = 7.0), and initial absolute blast count (% x WBC count) (Latino median = 3024 vs. Caucasian median = 1680). Conclusion We have performed the first comprehensive evaluation of ethnic variation in clinical and biological features in pediatric ALL in Utah. Surprisingly, we found no significant differences between the Latino and Caucasian patients in the features that we examined (although several variables did show a trend). This lack of difference could be explained by: 1) lack of adequate power, 2) lack of any true difference (our Latino vs. Caucasian relapse rate in Utah was similar), or 3) ethnic variation in molecular biology beyond the level of detection for collected variables. We are now preparing to expand our patient numbers and to interrogate clinical samples from each patient using high-resolution, genome-wide technology as we continue our pilot study. Disclosures: No relevant conflicts of interest to declare.

Background: Acute leukemia is the most common pediatric malignancy, and high risk or relapsed disease accounts for the most frequent need for pediatric hematopoietic stem cell transplant (HSCT). The 5-year Event Free Survival for acute leukemia following HSCT approaches 55%, and we currently cannot predict which children will relapse following transplant. Prognostic determinants for allogeneic HSCT recipients are limited to risk status of underlying disease, type of transplant received (matched sibling donor vs. unrelated donor), and severity of graft vs. host disease (GVHD). Although some clinicians believe that an increase in RRT may actually reflect a better outcome, the prognostic significance of engraftment and regimen related toxicity (RRT) remains to be investigated. Patients receiving similar preparative regimens respond with varying degrees of toxicity which may reflect individual genetic variations and ultimately may determine their treatment outcome. Objective: To determine whether engraftment or RRT correlates with outcome (sustained remission vs. relapse) in pediatric HSCT patients with acute leukemia. Methods: A chart review was conducted on 96 consecutive pediatric allogeneic HSCT recipients with acute leukemia at Stanford University from 1996–2006 treated with TBI/ VP or TBI/CY. The previously validated Common Terminology Criteria for Adverse Events v3.0 (CTCAE) was used to assess stomatotoxicity, hepatoxicity, and nephrotoxicity as indicators of RRT based on a 5-point Likert scale (0=no toxicity, 5=death). Toxicity scores were recorded within a 15 and 30 day period following HSCT to minimize toxicity attributed to GVHD. Time to hematological recovery also was assessed. An independent sample, two-tailed t-test was used to compare children in different outcome groups (relapse vs. sustained complete remission [CR]) for both combined total acute leukemia (TOTAL) and separate subtypes (ALL and AML). Finally, the percentage of patients with CR was calculated above and below the mean for any significant variables. Patients who died of treatment-related mortality (TRM) were removed from the analyses regardless of their remission status to avoid confounding. Results: Data were collected on 96 patients (ALL=53, AML=43). Twenty-one patients with TRM were removed, and the following number of HSCT patients analyzed: 75 TOTAL (CR=53, Relapse=22), 44 ALL (CR=29, Relapse=15), and 31 AML (CR=24, Relapse=7). CR patients took longer time to reach platelet recovery (P100) compared to relapsed pt’s for TOTAL (111 vs. 45 days, p=0.007), ALL (110 vs. 47 days, p=0.055), and AML (112 vs. 40 days, p=0.041). The TOTAL CR group also had higher creatinine-15 day scores (0.70 vs. 0.31, p=0.033) as did the AML CR group (0.83 vs. 0.28, p=0.05). This creatinine difference was no longer significant by Day-30. The percentage of CR patients above the mean P100 time to platelet recovery for each category was: TOTAL 89.5% (n=17/19, mean 97 days), ALL 82% (n=9/11, mean 92 days), and AML 100% (n=8/8, mean 104 days). The percentage of CR patients with creatinine-15 day scores above the mean was: TOTAL 79% (n=23/29, mean score 0.59) and AML 86% (n=12/14, mean score 0.7). The difference in denominators for each group is due to removal of patients without available data for specific variables or death due to TRM. Conclusion: This study represents one of the first attempts to methodically investigate whether engraftment and RTT are related to outcome in pediatric HSCT patients. Our retrospective analysis found delayed platelet engraftment (defined by platelet level of 100K/uL x 3 consecutive days) and higher creatinine-15 day values (less than 1.5 x normal) in leukemia patients who achieved sustained remission vs. those who ultimately relapsed. The difference in RRT scores between children who relapsed and those who did not may reflect differences in bone marrow and kidney exposure to previous chemotherapeutic agents; however, another explanation may be individual genetic variation in drug metabolism which leads to rapid drug clearance with lower RRT and subsequent relapse. Prospective studies are needed to validate our findings, in addition to high-throughput genomic screening to identify biological differences between children with different engraftment and RRT scores.

Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the leading cause of death for children with cancer. Genomic instability events contribute to tumorigenesis and have been used to classify and risk stratify adult and pediatric cancers. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution, and can detect both gene copy number and loss of heterozygosity (LOH) events in clinical samples. Studying pediatric leukemia samples with MIP technology may identify new molecular alterations that could prove useful in risk stratification and discovery of new therapeutic targets for childhood leukemia. Objective: To use MIP technology to identify novel areas of allelic imbalance in childhood leukemia. Methods: DNA was extracted from leukemia blasts at diagnosis (n=45, 23 pre-B ALL, 14 AML, 7 pre-T ALL, 1 Burkitt’s). DNA was also extracted from normal peripheral blood collected at remission to use as paired germline controls. The MIP assay was run with a customized Affymetrix 24K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). DNA required for this assay was limited to 75 ng per sample. Copy number changes and LOH were identified by comparing probe signal intensity between leukemia and normal germline samples. Clinical cytogenetic data (karyotype and FISH analysis) was used as a control to confirm findings from known areas of allelic imbalance. Results: Each leukemia sample had unique patterns of allelic imbalance distributed across all chromosomes. MIPs identified all clinically reported cytogenetic copy number changes for each sample, in addition to areas of allelic imbalance not clinically reported. Samples had recurring areas of copy number changes and LOH events shared by all leukemia types. MIPs detected areas of allelic imbalance in both previously described and novel genes. Areas of recurring genomic deletions included: ATR (3q23), TLX3 (5q35.1), ADRB3 (8p12), CDKN2A (9p21.3), DOCK8 (9p24.3), PAX5 (9p13.2), PTPN11 (12q24.13), C3AR1 (12p13.31), TCRA (14q11.2), AKT1 (q14q32.33). Areas of recurring genomic amplification included: SLC2A9 (4p16.1), RAI14 (5p13.2), CDH12 (5p14.3), PMCHL1(5p14.3), AURKB (17p13.1). These findings are being validated with Quantitative Real-Time PCR. Conclusions: MIPs represent a novel genomic technology that can identify previously unreported gene copy number and LOH events in childhood leukemia. Unique and overlapping areas of allelic imbalance were found in both childhood ALL and AML clinical samples. The shared genomic regions of allelic imbalance between different leukemia types may represent a common molecular mechanism of leukemogenesis that warrants further investigation. Analysis of more childhood leukemia samples through Pediatric Cooperative Groups may help to determine how common these areas of allelic imbalance are in children and whether they are of prognostic significance. Further exploration of these copy number and LOH events may help us to better understand the biology of childhood leukemia and its clinical behavior.

Background: Leukemia is the most common pediatric malignancy, accounting for nearly 40% of all new childhood cancer. The cure rates for pediatric ALL have increased to more than 80% and the cure rates for pediatric AML now approach 50%. Much of this progress can be attributed to cytogenetic and molecular risk stratification with subsequent randomized control trials. Genomic instability events may also serve as relevant prognostic biomarkers. A novel high-throughput genomic technology called Molecular Inversion Probes (MIPs) quantifies genomic instability, gene copy number and allelic imbalances at the highest genomic resolution. MIPs can analyze genetic target sequences in parallel with high specificity and sensitivity. Further classifying leukemic blasts using more precise molecular techniques could prove useful in further risk stratifying and developing new treatment strategies. Objective: To adapt MIPs to characterize and define unique molecular subtypes of childhood leukemia. Methods: DNA was extracted from AML and ALL clinical samples obtained at diagnosis. 400 ng of leukemic DNA was mixed with a previously synthesized MIP cancer probe set (667 probes representing all human chromosomes from exon sequences of 298 cancer genes); each probe contains two unique recognition sequences to targeted genomic DNA and a unique barcode (tag) sequence. A thermostable polymerase and ligase were added, and the mixture was denatured. Probe homology sequences were then annealed to complementary genome sites. Standard PCR reagants were added including one fluorescent-labeled primer and unlabeled primer. The reaction was thermocycled and probes circularized in the nucleotide specific “gap fill” reaction were amplified. The PCR mixture was hybridized to a barcode microarray overnight. The chip was stained and washed, and then interrogated by an Affymetrix scanner via argon laser excitation at 488-nm. ‘Cluster along Chromosomes’ analysis was performed to detect significant amplifications or deletions. Results: Childhood ALL and AML samples had unique patterns of multiple gene deletions. Additionally, overlap was found in several of these deleted genes. Significantly deleted genes in common between ALL and AML samples included: RAP1GA1, HYAL1, HSPA9B, CSF1R, TNF, NOTCH4, FANCE, CCND3, BAG2, PLAGL1, MAD1L1, POLM, POLD2, IGFBP3, PSD, SUFU, CYP17A1, MXI1, HRAS, IGF2, RRM1, BRCA2, TP53, ERBB2, ERCC2, and POLD1. Conclusions: MIPs represent a novel technology for highly sensitive and specific gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of deleted genes in both ALL and AML may represent a common molecular mechanism that requires further investigation. We currently are adapting MIPs for an expanded cancer probe set and are exploring new techniques to determine loss of heterozygosity (LOH) in pediatric leukemia.