VPREB1 Deletions Occur Independent of Lambda-Light Chain Rearrangement and Predict Worse Outcome In Pediatric Acute Lymphoblastic Leukemia (ALL)

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.