ALBERT

Description: Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most significant genes was osteonectin, also known as secreted protein, acidic, rich in cysteine (SPARC). This gene encodes a matricellular glycoprotein with diverse functions in cell-matrix interactions. SPARC has been identified as a target of epigenetic silencing in pancreatic cancer and addition of exogenous SPARC to cancer cell lines induces growth arrest and apoptosis. To determine if leukemia cell lines could be used as a model to study the basis for SPARC silencing and its role in cell growth and survival we measured SPARC expression in AML cell lines with rearranged and germline MLL genes. By real-time quantitative reverse transcriptase PCR (Q-RT-PCR), the cell lines THP-1 (MLL-AF-9) and ML-2 (MLL-AF6) expressed SPARC mRNA levels 40 to 1000 fold lower than Kasumi-1 (t(8;21)) and KG1a. By Western blot, SPARC was easily detectable in Kasumi-1 and KG1a but undetectable in the MLL rearranged lines. Bisulfite sequencing revealed extensive methylation of CpG dinucleotides in the promoter region and first exon of SPARC in THP-1 and ML-2 but a complete lack of methylation in KG1a. Treatment with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine (DAC) restored SPARC expression to nearly normal levels in THP-1 cells by Q-RT-PCR and Western blot, suggesting that promoter methylation is critical to the silencing of this gene. To determine if SPARC was contributing to the growth inhibitory effect of DAC, cells were cultured in varying concentrations of exogenous purified SPARC. Concentrations of SPARC that reduced the growth of ML-2 and THP-1 by 30 to 40% had no effect on the growth of KG1a cells. We conclude that SPARC, a putative tumor suppressor that is epigenetically silenced in pancreatic cancer, is also silenced by promoter methylation in AML with MLL rearrangements. These studies suggest that SPARC expression may constitute a reliable pharmacodynamic endpoint for clinical studies of chromatin remodeling agents in patients with MLL rearranged AML. Whether SPARC is a direct target of MLL and how MLL rearrangements are related to SPARC silencing are the subject of future studies.

Description: Fms-like tyrosine kinase 3 (FLT3) mutations are associated with unfavorable outcomes in children with acute myeloid leukemia (AML). We used DNA microarrays to identify gene expression profiles related to FLT3 status and outcome in childhood AML. Among 81 diagnostic specimens, 36 had FLT3 mutations (FLT3-MUs), 24 with internal tandem duplications (ITDs) and 12 with activating loop mutations (ALMs). In addition, 8 of 19 specimens from patients with relapses had FLT3-MUs. Predictive analysis of microarrays (PAM) identified genes that differentiated FLT3-ITD from FLT3-ALM and FLT3 wild-type (FLT3-WT) cases. Among the 42 specimens with FLT3-MUs, PAM identified 128 genes that correlated with clinical outcome. Event-free survival (EFS) in FLT3-MU patients with a favorable signature was 45% versus 5% for those with an unfavorable signature (P = .018). Among FLT3-MU specimens, high expression of the RUNX3 gene and low expression of the ATRX gene were associated with inferior outcome. The ratio of RUNX3 to ATRX expression was used to classify FLT3-MU cases into 3 EFS groups: 70%, 37%, and 0% for low, intermediate, and high ratios, respectively (P 〈 .0001). Thus, gene expression profiling identified AML patients with divergent prognoses within the FLT3-MU group, and the RUNX3 to ATRX expression ratio should be a useful prognostic indicator in these patients. (Blood. 2004;104:2646-2654)

Description: Abstract 2568 Background: L-asparaginase (L-ASP) is an important component of multi-agent chemotherapy for treatment of Acute Lymphoblastic Leukemia (ALL) in children and young adults. The pegylated E. coli derived form, Oncaspar® (PEG-ASP), is most commonly used because of its longer half-life and lower immunogenicity compared to the native enzyme; however, clinical hypersensitivity reactions still occurs in 10–30% of patients (pts) requiring its discontinuation. Asparaginase Erwinia Chrysanthemi (Erwinaze™) is an L-ASP derived from a different bacterium and is immunologically distinct from the E. coli L-ASP. We conducted a compassionate use trial of Erwinaze in pts with ALL and hypersensitivity to native E. coli or PEG-ASP to collect safety information. Patients and Methods: Pts of any age with ALL or lymphoblastic lymphoma (LBL) who developed a Grade ≥2 clinical hypersensitivity reaction to PEG-ASP or native E. coli ASP (Elspar®) were eligible. Pts with a history of pancreatitis, previous allergic reaction to Erwinaze, or pregnant, were excluded. The study was IRB approved at each institution and pts/family provided informed consent/assent. Safety information on Erwinaze-related adverse events (AEs) were captured on Case Report Forms (CRFs) submitted to the Sponsor when the pt completed his/her entire Erwinaze treatment plan. AEs may have also been reported directly by the investigational sites. Results: Between February 2006 and November 2011, 1,368 pts were treated with Erwinaze. CRFs were received in 893 pts and 47 additional AEs were received from patients for which a CRF was not obtained. The average age was 9.6 years (range 1–66). The majority of patients (63.5%) were male. Of the pts for which CRFs were received (893); 77.6% were able to conclude their Erwinaze treatment. Discontinuation was due to allergic reaction in 8.8%; other AEs 4.7%; at the physician or patient discretion 7.5%; and missing information in 1.3%. Anaphylaxis was reported in 8 pts (0.9%) and Grade ≥2 clinical hypersensitivity reactions in 130 (13.8%). The incidence of pancreatitis was 3.9%, hemorrhagic or thrombosis abnormalities 2.4%, hyperglycemia 3.6% and elevation in liver enzymes 3.5%. There were 18 deaths on study; 11 disease progression, 3 intracranial hemorrhage, 4 other individual reports. Conclusion: This is the largest study of pts treated with Erwinaze to determine the toxicity profile. Erwinaze was well tolerated with no unexpected toxicities identified beyond those associated with L-Asp treatment. This compassionate use trial permitted the continuation and completion of asparaginase treatment in 77.6% of pts with hypersensitivity reactions to E. coli formulations. Final results will be available for presentation. Disclosures: Plourde: Jazz Pharmaceuticals: Employment, Equity Ownership. Mercedes:Jazz Pharmaceuticals: Employment. Corn:EUSA Pharma: Employment.

Description: Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012).  Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy and kappa chain (IGH and IGK), as well as T-cell receptor beta, delta and gamma.  The assay was performed on genomic DNA isolated from cells from the bone marrow, cerebrospinal fluid, or testes.  The test was first done at the time of relapse to identify the malignant clonotype, which was monitored at subsequent time points. The patients were ineligible for clinical trials and concurrently underwent MRD testing using flow cytometry. The sequencing assays were performed to show feasibility of the approach. Results  Patient one was a 14 y/o ALL relapse patient who was not in morphologic remission after standard re-induction therapy. The malignant clonotype was identified on a bone marrow aspirate from relapse; follow-up MRD tests were done using both flow cytometry and deep sequencing five times throughout salvage therapy with 5-aza-2'-deoxycytidine, suberoylanilide hydroxamic acid and high dose cytarabine over 75 days; the last two MRD data points showed 0.6% and 6% by ClonoSIGHT MRD and 0.4% and 1.3% by flow cytometry MRD. Morphologic remission with count recovery was used as the criteria to direct this patient to SCT. Patient two was a 9 y/o with ALL, for whom MRD was used to test for relapsed disease in multiple tissues.  This patient experienced three isolated testicular relapses (M1 marrow and no CNS involvement) at the time of each relapse. The ClonoSIGHT assay was used on tissue from a testicular biopsy to identify the malignant clone(s).  Testing of the bone marrow and cerebrospinal fluid did not detect the malignant clones in those sites. This patient underwent therapeutic orchiectomy and 4-week systemic re-induction resulting in a fourth complete remission and now is under evaluation for consolidation therapy with a SCT. A third patient was an 8 y/o with a combined bone marrow and testicular ALL relapse, who was in morphologic remission in the marrow after re-induction therapy and testicular radiotherapy. Prior to undergoing SCT the patient had negative MRD by flow cytometry but had 0.008% MRD using the ClonoSIGHT MRD assay.  The fourth patient was a 15-yo with ALL relapse at 9 years from first remission, treated with a four-drug re-induction and Berlin-Frankfurt-Münster based consolidation and maintenance therapy.  This patient was MRD negative by both flow cytometry and ClonoSIGHT MRD at end of re-induction as well as end of consolidation and remains in remission. Conclusions We have shown the feasibility of using sequencing-based tests for monitoring MRD in children with relapsed ALL in medullary (bone marrow) and extramedullary compartments (testes and CSF).  Further studies are needed to establish the prognostic value of MRD detected by the ClonoSIGHT assay in both medullary and extramedullary sites that are below the limit of detection of PCR and flow cytometry. These sequencing-based tests may provide a useful tool to develop risk stratification schemas for drug development in relapsed childhood ALL. Disclosures: Weng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Description: Abstract 1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain 〉1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient's reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results: We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient's clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions: We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Description: Abstract 3544 Background: In pediatric de novo AML, cytarabine (Ara-C)-based induction therapy results in above 80% complete response (CR) rates but nearly half of those who achieve an initial remission relapse die of their disease. Accurate prediction of initial response to chemotherapy at the time of diagnosis as well as identification of those at high risk of relapse despite initial remission would allow for patient specific therapy and improved clinical outcome. SCNP is a multiparametric flow cytometry-based assay that provides simultaneously quantitative measurements of extracellular surface markers as well as changes in intracellular signaling proteins in response to extracellular modulators at the single cell level (Kornblau et al. Clin Cancer Res 2010). In a previous study, we used this assay to define two distinct classifiers associated with response to standard induction therapy and risk of relapse in diagnostic bone marrow mononuclear cells from pediatric patients (pts) with non-M3 AML (ASH 2010;116:Abstract 954). This study is intended to confirm the validity of the pre-specified response to induction therapy classifier in an independent set of AML pediatric pts. Methods: The SCNP-based response classifier developed using 53 AML cryopreserved samples from patients enrolled on POG (now COG) trial 9421 was comprised of a combination of three SCNP readouts that measure apoptosis, MAPK signaling, and PI3K signaling and had a bootstrapped out-of-bag estimated Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.84 (95% CI 0.67– 0.96). The classifier was tested on 68 cryopreserved samples (20 non–responders (NR) and 48 CRs) from patients enrolled on COG trials AAML0531 (samples from patients randomized to Ara-C, Daunomycin and Etoposide [ADE] induction therapy) and AAML03P1 (samples from patients treated with ADE plus Gemtuzumab Ozogamicin induction therapy). The primary hypothesis was that the prediction of induction response by the continuous score from the pre-specified classifier would yield an AUROC significantly greater than 0.5. Results: The primary objective of the study was met with an AUROC of 0.66 (n=68) p=0.042 (see table). The primary analysis used an NR classification that combined patients with either induction failure (n=14) or induction death (n=6). A pre-specified analysis in which induction deaths were removed resulted in an AUROC of 0.70 (n=62) p=0.021, suggesting that the underlying disease biology may be different for induction death vs. induction failure. In this study, WBC and cytogenetics risk groups were associated with induction response while age, gender and FLT3-ITD status were not. In a multivariate analysis of induction response that included WBC, cytogenetics and the pre-specified continuous SCNP classifier score, only cytogenetic risk group (p=0.001) and SCNP score (p=0.017) remained significant. Exploratory analyses excluding induction deaths suggest that the relationship between the SCNP score and induction response is strong among patients with an intermediate cytogenetic classification (n=23) (AUROC=0.88, p=0.002), while no relationship (AUC=0.48, p=0.959) is seen in those patients with a poor cytogenetic classification (n=17). Among the three SCNP signaling nodes contributing to the score, the node measuring drug-induced apoptosis performs most consistently across the training and validation sets. Conclusion: This study is the first validation of a SCNP-based classifier that predicts response to induction Rx. It shows that performing quantitative SCNP under modulated conditions can serve as the basis for developing biologically based tests in leukemia that offer new insights into the individual patient's disease biology. Additionally, this technology could prove useful in guiding alternative therapeutic strategies. Disclosures: Gayko: Nodality Inc.: Employment, Equity Ownership. Westfall:Nodality Inc.: Employment, Equity Ownership. Purvis:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Hackett:Nodality Inc.: Employment, Equity Ownership. Cleary Cohen:Nodality Inc.: Consultancy, Equity Ownership.

Description: 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.

Description: Background Despite successful remission induction, a significant proportion of children with acute myeloid leukemia (AML) experience relapse. In our previous AML02 study, adjusting treatment intensity according to levels of minimal residual disease (MRD) after remission induction has contributed to improved outcome. Because relapse still occurred in a proportion of patients who achieved MRD-negative status after remission induction therapy, we searched for presenting features associated with outcome in this patient cohort. Objectives To identify presenting features predictive of relapse among children with newly diagnosed AML who achieve MRD-negative status after remission induction therapy. Methods Among patients enrolled in AML02 who were MRD-negative (