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Abstract 2833 Introduction: In chronic lymphocytic leukemia (CLL), clonally expanded CD5+ B lymphocytes eventually overwhelm healthy immune cells, hindering normal immune function. To determine mechanisms fueling this expansion, gene expression data were gathered by microarray analysis of cells from CLL patients. Samples were grouped based on Ki-67 expression, an indicator of proliferation. To determine mechanisms correlating with B-cell proliferation and impacting on CLL B-cell biology, microarray profiles were compared using Gene Set Enrichment Analysis (GSEA) [Subramanian A, et al. PNAS 2005]. Methods: Samples were analyzed for intracellular expression of Ki-67 by flow cytometry and divided into 2 groups based on Ki-67 expression (cutoff at 5%). RNA was then purified from CD5+CD19+ CLL cells and gene expression microarray assays were performed using Illumina HumanHT12 beadchips. GSEA was carried out using a library of signatures by Dr. Louis Staudt [Shaffer AL, et al. Immunol Rev 2006] containing 305 gene sets encompassing 13, 564 genes biased towards hematopoietic signatures. Results: Of 61 cases, 14 were Ki-67high and 47 were Ki-67low. When time-to-first-treatment (TTFT) was compared between the groups, Ki67high patients had significantly shorter TTFT (2.76 yrs) compared to Ki-67low patients (23.46 yrs; P Discussion: The observed GSEA profiles in Ki-67high patients correlated with gene signatures biased towards BCR signaling, signal transduction, and hematopoietic cancer, consistent with the Ki-67high group containing more (recently) proliferating cells influenced at least in part by BCR signaling. The profiles also suggest that additional cells (T lymphocytes and dendritic cells) may be involved. It is notable these gene sets were not observed for CLL patients subgrouped by IGHV mutation status or by CD38, and that these other subsets did not show as pronounced a distinction by GSEA profiling. Disclosures: No relevant conflicts of interest to declare.

Abstract 3888 In B-CLL, mutations in the IGHV genes encoding the BCR are correlated with disease aggressiveness: patients with unmutated BCR (U-CLL) typically have a worse prognosis than those with mutated BCR (M-CLL). Evidence that many U-CLL express BCR with specificity toward apoptotic cell antigens suggests that dying cells provide pro-survival signals for U-CLL clones. Thus progression of U-CLL may depend both on mechanisms that ensure the clone's survival and those that promote cell death. A single nucleotide polymorphism (SNP) within the p53 gene at codon 72 significantly affects p53 function: p53-72R (CG C=Arg) is substantially better at inducing apoptosis than p53-72P (CC C=Pro). Although p53-72P is linked with worse outcomes in a number of malignancies, p53-72R has been linked to heightened B-CLL incidence (Leuk Res. 2006;30:1113–8) and to diagnosis as U-CLL (Br J Haematol. 2011;153:533–5). Pro-apoptotic p53-72R may be fostering the apoptotic self antigen needed for BCR signaling in U-CLL. MDM2 is a ubiquitinase with a major role in keeping p53 levels low. Following DNA damage, p53 is phosphorylated by ATM and protected from MDM2. A SNP within the promoter affects MDM2 RNA/protein levels: SNP309G > SNP309T. In some B-CLL cohorts, the SNP309G allele was associated with disease progression (Eur J Haematol. 2010;85:251–6; Blood 2010;115:4191–7). We investigated a cohort of 93 Caucasian B-CLL patients from the New York City metropolitan area for expression of the above p53 and MDM2 SNPs. We examined whether the B-CLL-expressed SNPs differ from Caucasian controls in the same geographic area (Lupus 2009;18:61–6). Additionally, we examined whether U-CLL and M-CLL patients in this cohort vary in SNP expression and assessed whether the above SNPs influence disease progression, as determined by time to first treatment (TTFT) and overall survival (OS). Isolated genomic DNA underwent PCR and pyrosequencing for the p53 SNP at codon 72 (rs1042422) and the MDM2 SNP309 (rs2279744) using primers selected by PSQ Assay Design. Primers used for p53: forward = Biotin-5'-GAAGACCCAGGTCCAGATGA and reverse = 5'-CCGGTGTAGGAGCTGCTG, resulting in an 82 bp PCR product; sequencing = 5'-GGTGCAGGGGCCACG. Primers used for MDM2: forward = 5'-ATTTCGGACGGCTCTCGC and reverse = Biotin-5'-CTAGTGACCCGACAGGCACCT, resulting in a 147 bp PCR product; sequencing = 5'-GGGCTGCGGGGCCGCT. Pyrosequencing was done on a PSQ HS96A instrument as per manufacturer's guidelines using PyroMark Gold Q96 Reagents. Our cohort of B-CLL was not significantly different from controls in distribution of the p53 SNP. However, there was a higher proportion of MDM2 SNP309G/G in B-CLL compared to controls (CLL=31%; normal=16%; p=0.0238). A p53-72R/R genotype was evident in a lesser proportion of U-CLL than M-CLL, albeit the difference was not statistically significant (n=80, p=0.1860). On the basis that U-CLL with p53-72R/R genotype might be favored if the clone underwent a deletion of p53-augmenting ATM, we compared the p53-72 SNP genotype of U-CLL ± FISH evidence of ATM deletion (11q22.3) (patients with 17p13 p53 deletion were excluded). Interestingly, the p53-72R/R genotype was significantly greater in U-CLL patients with ATM deletion than those without (n=27; p=0.0002). Comparison of p53-p72 SNP to TTFT showed trends in agreement with Majid, et al. (Br J Haematol. 2011;153:533–5). B-CLL patients with p53-72P/P had a shorter TTFT (P/P=41 months; R/R=97 months; n=88). However, due to limited power, this did not reach significance (p=0.1531). Also consistent with the above-cited study, the single M-CLL with p53-72P/P had the shortest TTFT, while M-CLL with p53-72R/R had the longest (n=48; p Comparison of MDM2 SNP309 to TTFT in all studied B-CLL patients suggested that those with SNP309G have a shorter TTFT than those with SNP309T (G/G=44 months; T/T+G/T=111 months), but the differences did not reach significance (p=0.1048). However, when considering mutation status, U-CLL with SNP309G/G had the shortest TTFT (U-CLL G/G=33 months; n=42; p=0.0079). With our cohort, there was no statistically significant correlation between the p53 or MDM2 SNPs and CD38 level or OS. Taken together, our study suggests that the genotype of these functionally relevant SNPs may influence B-CLL subtype and disease progression. Disclosures: No relevant conflicts of interest to declare.

Abstract 1774 Introduction: Chronic Lymphocytic Leukemia (CLL) follows a variable, difficult to predict course. Biomarkers (IGHV mutations, CD38 and ZAP-70) have improved prognostication but accuracy is only ∼80%, not sufficient to initiate preemptive therapy. To address this, we have incorporated another molecular biological parameter, hypervariably expressed genes (HVEGs) from global gene expression profiles (GEPs) [Dozmorov, 2011], along with IGHV mutation status to develop a more robust method of prognostication. Methods: RNA was purified from B cells of 65 CLL patients and 25 normal subjects. GEPs were defined with Illumina HumanHT12 beadchips and analyzed to identify HVEGs fluctuating coordinately in CLL patients but not expressed in normal circulating human B cells. A Cox proportional hazards regression model was used to compare expression levels with clinical outcome. Results: Unmutated CLL (U-CLL) and mutated CLL (M-CLL) samples were sorted based on the averaged normalized gene expression level in the largest HVEG cluster consisting of 45 genes. Samples were split into subgroups based on high and low expression of these genes. Four subgroups were identified: U-CLL with high (U-HVEhi), U-CLL with low (U-HVElo), M-CLL with high (M-HVEhi), and M-CLL with low (M-HVElo) expression of HVEGs. When these subgroups were analyzed for time-to-first treatment (TTFT), the findings were remarkably different. Using a Cox proportional hazards regression model we found that for M-CLL the presence of low levels of HVEGs denoted a subgroup with a prolonged median TTFT compared to M-CLL patients with high levels of HVEGs (23.46 yrs in M-HVElo versus 9.05 yrs in M-HVEhi; P=0.0075). This association was reversed in U-CLL, where low levels of HVEGs pinpointed a subgroup with a shortened median TTFT compared to U-CLL patients with high levels of HVEGs: median TTFT was 8.01 yrs in U-HVEhi and decreased by 50% to 3.12 yrs in U-HVElo (P=0.0151). Compared to the TTFT for M-HVElo, the TTFT for U-HVElo was dramatically shorter (3.12 vs. 23.46 years; P Discussion: Combining the HVEG approach with IGHV mutation status enabled us to define intra-group heterogeneity, and thereby improve prognostication for apparently homogeneous subgroups of U-CLL and M-CLL patients. This combination was superior to IGHV mutations alone in that it pinpointed a subset of M-CLL patients with shorter TTFTs and a subset of U-CLL patients with longer TTFTs, indicating that the combined approach selects those patients that IGHV mutation analysis misclassified. Therefore, combining IGHV mutation status and HVEG expression provides a more precise indicator with potentially valuable clinical implications. The data suggest that the HVEGs are responsible for the different outcomes in the two groups and could provide mechanistic insights into key aspects of CLL B-cell biology as well as therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Abstract 942 Background: Emerging evidence suggests that microRNAs (miRNAs) are critical in cancer and adult leukemia by functioning as tumor suppressors and/or oncogenes. Zhang et al identified 32 pediatric acute myeloid leukemia (AML)-specific miRNA patterns by analysis of bone marrow (BM) samples (1). They also established potential miRNAs as biomarkers for predicting CNS-relapse in pediatric acute lymphocytic leukemia (ALL). Altered miRNA expression disrupts normal hematopoiesis and might play a role in niche-induced oncogenesis. Dysfunction of mesenchymal stromal cells induces formation of myeloid sarcomas that infiltrate in the surrounding tissues (2). Previously, we described that mesenchymal stem cells (MSC) of the BM microenvironment participate in leukemic stem cell regulation in an in vivo model of the childhood AML stem cell niche(3). These human MSC niches, created in ectopic bioengineered 3D scaffolds, supported leukemogenesis in NOD/SCID mice. Pediatric AML engrafted at 1 month in the MSC-coated scaffolds in the mice and was retained in the niche up to 4 months, after which distant seeding to murine BM, liver and spleen occurred. The bioengineered niche created a sanctuary for quiescent leukemia cells and at 4 months the AML cells exited the niche and spread hematogenously, mimicking leukemia relapse. Analysis of miRNA patterns in our leukemia niche model could provide novel directions for individual risk-adapted therapy in childhood leukemia. Objective: To analyze miRNA expression patterns of pediatric AML after exposure to the niche microenvironment at different time points. Design and Method: miRNAs were obtained from primary CD34+ selected AML cells at (d0) Day0= no niche exposure, (1Mo) 1 month =niche engraftment, (4Mo) 4 months=hematogenous spread with leukemic exit from the niche. miRNAs were isolated from single cell suspensions with the mirVana miRNA isolation kit (Ambion) and analyzed on an Ilumina MicroRNA Expression Profiling single Beadchip (#RNA probes = 1145). Results: 498/1145 miRNAs expression profiles were selected with a detection p value < 0.00001. Out of 498 miRNAs expressed in the leukemic niche model, 23 were previously described as AML-specific miRNAs (2). 10/23 miRNAs were significantly upregulated and 13/23 were downregulated. Pediatric AML-specific miRNAs – miR100 and miR125b had high expression profile at baseline, but were down regulated upon contact with the niche. AML miR195 and miR193a had low expression at baseline, but miR195 was upregulated on engraftment while miR193a only upregulated at the time of hematogenous spread (niche exit). CNS-relapse in ALL might represent a physiological mechanism of leukemic exit of dormant cells from the niche sanctuary. Consistent with this notion, the same expression profile that was found in CNS-relapse in ALL patients (miR198 up – miR551a downregulated) was seen in our model when AML cells became invasive and exited the niche at 4 months. Conclusion: (1) Zhang et al. 2009, PloSOne, 4, p1 (2) Raaijmakers et al. 2010, Nature, 464, p852 (3) Vaiselbuh et al, 2010, Tissue Eng Part C Methods, June 29 (ePub) Disclosures: No relevant conflicts of interest to declare.

Abstract 2435 Introduction: Chronic Lymphocyte Leukemia (CLL) is characterized by the slow clonal expansion of B-lymphocytes, which eventually overwhelm healthy immune cells, therefore hindering normal function. In order to determine the underlying mechanisms for this uncontrolled proliferation, gene expression data was gathered through cDNA microarray analysis of the B-cells from CLL patients. The data was split into two groups depending on expression levels of Ki67, a strong marker for cellular proliferation. In our study, high Ki67 expression only showed in 60% of unmutated CLL patients and higher in CD38+ CLL B cells. The expression of individuals with high levels of Ki67 was compared to the expression profiles of individuals with low levels of Ki67. This allowed for the determination of differences in gene expression correlated to an increase in proliferation of B-cells. Methods: 1) The RNA were purified from CLL B cells which were isolated by RosetteSep Human B cell Enrichment Cocktail (StemCell Technologies) during 4 hours after blood drawn from patients. Microarray assay was performed on Illumina HumanWG-6 expression beadchips. 2) The samples were devided to 2 groups according Ki67 surface expression, 9 samples with Ki67high (>5%) and 26 samples with Ki67low ( Results: 1) Time to first time treatment (TTFT) were compared between Ki67high and Ki67low patients. Ki67high patients had a significantly shorter TTFT (2.76 yr) compared to Ki67low patients (23.46 years; P Conclusion and discussion: Ki67 is a nuclear protein which upregulated in G1, S, G2, and M phases of the cell cycle but is absent from resting cells (G0 phase1). We reported that Ki67+ cell is enriched in CD38+ cells regardless of the percentage of CD38+ cells in a patient's CLL clone2. To analyze gene expression profile in Ki67high and Ki67low groups will determine the possible interactions responsible for the proliferative nature of B-cells in CLL. Misregulation of the NF-κB complex has been implicated in the development of various immunological diseases as well as cancer, therefore identifying it as a candidate for further research in CLL development and treatment. Disclosures: No relevant conflicts of interest to declare.

The transforming events that lead to B-cell chronic lymphocytic leukemia (B-CLL) are unknown, and although genetic abnormalities appear to promote disease progression, none of these is seen in every patient. Therefore, we have applied a highly sensitive and specific method to probe, in greater depth, for genetic changes that can occur in B-CLL, and to explore if these correlate with disease development or progression. Utilizing ROMA to measure changes in gene copy number, we screened the entire genome of over two dozen B-CLL cases to identify characteristic and novel chromosomal changes. DNA from leukemic cells of patients was compared to the normal DNA from their unaffected neutrophils and to an unrelated, normal human control. By comparing normal DNA from each patient to an unrelated normal, we avoid inadvertently mistaking common copy number variation between people as possible candidates for lesions in B-CLL. ROMA was performed on both 85,000 and 390,000 probe microarrays enabling us to screen for far smaller lesions than are detectable by routine techniques, as well as resolve regions of interest, possibly pinpointing important genes in the etiology and progression of B-CLL. Data were also obtained by using the Illumina platform. These two methods (ROMA and Illumina), while not identical, are largely in agreement. We have observed virtually all the major cytogenetic imbalances previously reported, and in many cases to a higher resolution. Although there are fewer lesions present in B-CLL than in more advanced stages of solid cancers, many early stage cancers exhibit only a few common lesions. The most frequent deletion and often the sole abnormality found in B-CLL occurs at chromosome 13q13.4. Two micro-RNAs, miR-15a and miR-16-1, have been implicated in the smallest region of this deletion. ROMA analysis has further delineated the deletions on 13q, suggesting that there may be two epicenters, one encompassing these micro-RNA genes and one encompassing a neighboring region that does not span miR-15a or miR-16-1. A novel finding is the existence in certain patients (5/26) of numerous single probe deletions or amplifications. Since we compare B-CLL to the normal DNA from the same patient, and to a known control, even single probe events can be relevant. Many B-CLLs display single probe aberrations and these events are currently under investigation. In addition, we have observed two patients with major genomic instability, as evidenced by large and multiple regions that change copy number. The analysis of large numbers of B-CLL cases is essential to identifying and understanding the ongoing progression of genetic lesions and the role they play in clinical outcome. Initial results clearly indicate the need to obtain more data to identify critically mutated genes within the leukemic cells that cause them to become more aggressive. The identification and characterization of these genetic changes may be used as stratification tools for new diagnostic and therapeutic approaches to this currently incurable disease.