ALBERT

Description: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3244 Poster Board III-181 Recent genome-wide profiling studies have revealed that childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent microdeletions, including the cell cycle regulator CDKN2A, the B-cell differentiation genes PAX5, EBF1 and IKZF1 (Ikaros) and the anti-proliferative gene B-cell translocation gene 1 (BTG1). In a previous study, we have shown that BTG1 is an important determinant of glucocorticoid sensitivity (Van Galen et al. Blood/ ASH Annual Meeting Abstracts, 2008). In the present study we have characterized these cases in more detail and elucidated the frequency of recurrent lesions in BTG1 deletion cases. Using locus-specific MLPA screening of an unselected cohort of 305 precursor B-ALL cases, we identified 26 microdeletions (8.5%). All deletions encompassed BTG1 only. We were able to genomically profile 22 diagnosis samples using Affimetrix SNP6.0 arrays. Of these, 12 did not develop a relapse during a minimal of 4,5 years of follow up. The mean number of CNVs was 29.6 of which 10.3 gains and 22.5 losses (median size 512 kb and 115 kb respectively). BTG1 deletions were generally focal, varying in size from 104 kb to 1,4 Mb. In all but one patient the breakpoints at the 5' end of the deletion tightly clustered and subsequent fine-mapping using qPCR revealed that this breakpoint cluster was located within intron 1 of the BTG1 gene. At the 3'end of the deletion, four breakpoint clusters could be identified. Analysis of the copy number variation (CNV) profiles showed that patients with a BTG1 deletion more often harbored a deletion in IKZF1 compared to an unselected cohort of pre-B ALL cases (27% vs 7%, chi-square p=0.042). In contrast, recurrent CNVs like PAX5, EBF1 and CDKN2A/B occur in similar frequencies (23%, 9% and 32% vs 17%, 0% and 23% respectively). In addition, the BTG1 deletion cases that developed into a relapse showed significantly more often a deletion in CDKN2A/B compared to the BTG1 deletion cases that did not develop a relapse (60% vs 8%, p=0.02). Together, these data indicate that pediatric precursor-B ALL carrying BTG1 deletions have distinct genomic profiles, showing increased frequencies of deletions in IKZF1 and CDKN2A. Disclosures No relevant conflicts of interest to declare.

Description: Interactions between T cells and antigen-presenting cells (APCs) are the first step in the induction of an adaptive immune response. Here, we show that CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) are actively recruited to the antigen-induced dendritic cell (DC)–T-cell contact zone. Moreover, ALCAM-blocking antibodies interfere with DC–T-cell conjugate formation, demonstrating that CD6-ALCAM binding is essential for stable T-cell–APC contact. We now demonstrate that besides their role in establishing initial contacts, CD6-ALCAM interactions are also required during the proliferative phase of the T-cell response; the presence of CD6-blocking antibodies or recombinant ALCAM-Fc proteins results in a strong and sustained inhibition of T-cell proliferation. Furthermore, simultaneous crosslinking of CD6 and CD3 induces enhanced proliferation and transcriptional activity to a similar level as observed after CD3 and CD28 co-crosslinking, demonstrating that CD6 is an important costimulatory molecule. The stability of ALCAM-CD6 binding, which contrasts with transient homotypic ALCAM-ALCAM interactions, further supports the long-lasting effects observed on T-cell proliferation. Taken together, we demonstrate that CD6 and ALCAM form a key adhesive receptor-ligand pair that is not only involved in early DC-T-cell binding but also in sustaining DC-induced T-cell proliferation long after the initial contact has been established.

Description: Abstract 3458 Poster Board III-346 Background By genome wide profiling we have found that about 10 % of pediatric pre-B ALL cases contain a (single copy) deletion of the B cell translocation gene 1 (BTG1) gene. BTG1 belongs to a family of potential tumor suppressor genes, which include BTG2, BTG3, TOB and TOB2. Proteins encoded by members of this gene family have been implicated in the induction of growth arrest or apoptosis in a variety of model systems. Moreover, BTG1 associates with and regulates the activity of the arginine methyl transferase PRMT1, a coactivator of nuclear receptor-mediated transcription. Hence we hypothesized that loss of BTG1 function, for instance due to deletion, may affect glucocorticoid induced therapy responses in ALL. Results Using RNA interference, we find that loss of BTG1 expression decreases sensitivity of pre-B ALL cells to the apoptosis-inducing effects of synthetic GCs about 10,000 fold (Figure). This acquired GC resistance is accompanied by a greater than 10 fold reduction in GR protein expression as well as a (near complete) loss of GC-induced gene expression. Conversely, re-expression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression. By chromatin immunoprecipitations using anti PRMT1 antibodies we show that PRMT1 is recruited to the GR gene promoter in a BTG1-dependent manner, consistent with a role for this arginine methyl transferase in the regulation of GR-mediated gene expression. Conclusions Together, our results demonstrate the importance of the BTG1/PRMT1 complex in regulating GR-mediated gene expression and reveal how deregulation of the this complex can give rise to GC resistance. Targeting of these coactivators as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC based therapies in ALL as well as other hematological malignancies. Disclosures No relevant conflicts of interest to declare.