Faculty Bibliography

Background B cells are selected at multiple developmental checkpoints for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: either insufficient or hyperactive signaling (e.g. from an autoreactive BCR) results in cell death. Acute lymphoblastic leukemia (ALL) is the most frequent type of cancer in children and typically arises from pre-B cells, a large fraction of which are autoreactive. In ~25% of patients, ALL is driven by an oncogenic tyrosine kinase (e.g. BCR-ABL1 in Ph+ ALL) and defines the ALL subgroup with the worst clinical outcome. Ph+ ALL cells invariably develop resistance against tyrosine kinase inhibitors (TKI). Here we tested the hypothesis that inherent mechanisms of negative selection to eliminate autoreactive clones with hyperactive pre-BCR signaling are still active in transformed pre-B cells and identified a potential therapeutic target for ALL patients. Results The BCR-ABL1 oncogene mimics a constitutively active pre-BCR and an incremental increase of pre-BCR downstream signaling (ITAM overexpression) was indeed sufficient to induce cell death in Ph+ ALL, but not in normal pre-B cells with low baseline signaling strength. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. Patient-derived Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1, CD300A and LAIR1. However, ITAM containing activation receptors like CD79B was absent on the cell surface, and there was point or frame-shift mutation for both CD79A and CD79B. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1 at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or rela!

PURPOSE: Five-year overall survival (OS) for children with B-cell precursor acute lymphoblastic leukemia (B-ALL) exceeds 90% with risk-adapted therapy. Age, initial WBC count, genetic aberrations, and minimal residual disease (MRD) are used for risk stratification. Intrachromosomal amplification of a region of chromosome 21 (iAMP21; three or more extra copies of RUNX1 on an abnormal chromosome 21) is a recently identified recurrent genomic lesion associated with inferior outcome in some studies. We investigated the impact of iAMP21 in a large cohort treated in contemporary Children's Oncology Group (COG) ALL trials. PATIENTS AND METHODS: Fluorescent in situ hybridization for specific genetic aberrations was required at diagnosis. MRD was measured by flow cytometry at end induction. Outcome was measured as event-free survival (EFS) and OS. RESULTS: iAMP21 was found in 158 (2%) of 7,793 patients with B-ALL age >/= 1 year; 74 (1.5%) of 5,057 standard-risk (SR) patients, and 84 (3.1%) of 2,736 high-risk (HR) patients. iAMP21 was associated with age >/= 10 years, WBC less than 50,000/muL, female sex, and detectable MRD at day 29. Four-year EFS and OS were significantly worse for patients with iAMP21 and SR B-ALL, but iAMP21 was not a statistically significant prognostic factor in HR patients. There was no interaction between MRD and iAMP21. Among SR patients, day 29 MRD >/= 0.01% and iAMP21 were associated with the poorest EFS and OS; absence of both was associated with the best outcome. CONCLUSION: iAMP21 is associated with inferior outcome in pediatric B-ALL, particularly SR patients who require more intensive therapy and are now treated on HR COG ALL protocols.

The B cell-specific transcription factor BACH2 is required for affinity maturation of B cells. Here we show that Bach2-mediated activation of p53 is required for stringent elimination of pre-B cells that failed to productively rearrange immunoglobulin VH-DJH gene segments. After productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends BACH2-mediated negative selection through B cell lymphoma 6 (BCL6)-mediated repression of p53. In patients with pre-B acute lymphoblastic leukemia, the BACH2-mediated checkpoint control is compromised by deletions, rare somatic mutations and loss of its upstream activator, PAX5. Low levels of BACH2 expression in these patients represent a strong independent predictor of poor clinical outcome. In this study, we demonstrate that Bach2+/+ pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53 and do not initiate fatal leukemia in transplant-recipient mice. Chromatin immunoprecipitation sequencing and gene expression analyses carried out by us revealed that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other cell cycle checkpoint-control genes. These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.

Children's Oncology Group AALL07P2 tested whether substitution of Erwinia asparaginase 25,000-IU/m2 for 6 doses intramuscularly (IM) given Monday/Wednesday/Friday to children and young adults with acute lymphoblastic leukemia (ALL) and clinical allergy to pegaspargase would provide a 48-hour nadir serum asparaginase activity (NSAA) >/=0.10-IU/mL in at least 70% of patients. AALL07P2 enrolled 55 eligible/evaluable patients. NSAA >/=0.1-IU/mL was achieved in 38/41 patients (92.7%) with samples meeting acceptability criteria 48-hours after dosing and in 38/43 patients (88.4%) 72-hours after dosing during course 1. Among acceptable samples obtained during all therapy courses, 95.8% (252/263) of 48-hour samples and 84.5% (125/148) of 72-hour samples had NSAA >/=0.10-IU/mL. Pharmacokinetic parameters were estimated by fitting the serum asparaginase activity-time course for all 6 doses given during course 1 to a one compartment open model with first order absorption. Erwinia asparaginase administered with this schedule achieved therapeutic NSAA at both 48- and 72-hours and was well tolerated with no reports of hemorrhage, thrombosis, or death, and few cases of grade 2-3 allergic reaction (n=6), grade 1-3 hyperglycemia (n=6), and grade 1 pancreatitis (n=1). Following allergy to pegaspargase, Erwinia asparaginase 25,000-IU/m2 x 6 doses IM Monday/Wednesday/Friday for 2 weeks can be substituted for a single dose of pegaspargase.

Approximately 90% of the 2,000 children, adolescents, and young adults enrolled each year in Children's Oncology Group acute lymphoblastic leukemia (ALL) trials will be cured. However, high-risk subsets with significantly inferior survival remain, including infants, newly diagnosed patients with age >/=10 years, white blood count >/=50,000/microl, poor early response or T-cell ALL, and relapsed ALL patients. Effective strategies to improve survival include better risk stratification, optimizing standard chemotherapy and combining targeted therapies with cytotoxic chemotherapy, the latter of which is dependent upon identification of key driver mutations present in ALL. Pediatr Blood Cancer (c) 2012 Wiley Periodicals, Inc.

Background: The poor prognosis of relapsed ALL warrants the need for new insights into drug resistance mechanisms. We have previously shown that relapsed blasts can be re-sensitized to chemotherapy by the reversal of their gene expression signature using epigenetic agents.[1] Objectives: We hypothesize that aberrant epigenetic mechanisms may play a role in chemoresistance. To assess the degree to which the histone code evolves from diagnosis to relapse, we have used an unbiased, whole genome approach to examine changes in the epigenomic landscape by mapping the genome-wide location of key histone marks in diagnosis/relapse patient pairs with ALL enrolled on Children's Oncology Group protocols. Design/Method: We have performed Chromatin-immunoprecipitation sequencing (ChIP-seq) on two cryopreserved matched diagnosis/relapse pairs using active (H3K4me3, H3K9ac) and repressive (H3K9me3, H3K27me3) histone "mark" antibodies, with non-immunoprecipitated DNA (input) as control. Libraries for each patient pair were multiplexed in a single lane and sequenced in duplicate. 51-cycle single-end sequencing was performed using the Illumina HiSeq2000 Analyzer. Reads were aligned to reference genome using BWA and filtered using Samtools to remove multiple mapping reads. Peaks were called using MACS (v2.0.9) with default settings for histone marks. Results: 94.2% of the sequence reads passed filter (PF) and 96.4% of PF reads had quality score of more than 30. Using a p-value<0.00001, we identified 83,380 and 74,453 peaks (enriched regions) for activating and, 37,483 and 46,143 peaks for repressive histone marks at diagnosis and relapse respectively; suggesting that relapsed blasts are enriched with repressive marks and depleted of activating marks compared to those at diagnosis. Sixty-six genes had shared peaks for various histone marks between the two patients. Upon analyzing the top most differentially expressed transcripts at relapse from our previous cohort[2],64% genes showed concordant histone modificatio!