Jelinek, Diane F.

Abstract The study of genomic aberrations in chronic lymphocytic leukemia (CLL) has yielded critical information, now routinely used for the prognostication of patients. An unbiased search for genome wide areas of copy number change is needed to identify possible primary genetic events in CLL and to elucidate their relationship to other known cytogenetic factors, such as deletions of 13q14, 11q23 and 17p13 and trisomy 12. Here we report on a study where we used a 60-mer oligo array-based Comparative Genomic Hybridization (aCGH, 44B platform from Agilent Technologies, Palo Alto, CA). We observed the most common genetic aberrations in CLL and in similar frequencies as would be predicted for CLL (e.g. 44% showing deletions of chromosome 13q14). Among those patients, 45% had no apparent other aberration. Of great interest, small bi-allelic deletions of chromosome 13, which predominantly involved the DLEU1 and DLEU2 locus, were detected in 4 patients (16%). Deletions of 11q23 were observed in 6 cases (24%), and while some involved the ATM locus one did not, casting doubt on the precise gene deregulated by these deletions. Large deletions of the whole 17p-arm were detected in three cases (12%). Trisomy 12 was present in only two cases (8%). No other trisomies were detected and also small regions of copy gain were uncommon. Monosomy of chromosomes 19, X and Y were present in three separate cases. Large deletions of chromosome 2p were seen in two cases, but an additional case had a small deletion involving the ALK gene. This case also had a breakpoint at chromosome 5 involving the NMP1 locus, suggestive of a t(2;5). A recent study (Mayr et al., Blood 2006) indicates that chromosomal translocations in CLL are present in 33% and were associated with a dismal prognosis. In our analysis we detected several breakpoints indicative of translocations. Detailed study is necessary to elucidate possible molecular events associated with these translocations. Other small regions of DNA copy loss were found at several locations throughout the genome. Regions of interest are shown in the table. In summary we show that multiple areas of recurrent genomic gain and loss can be identified in clonal cells of CLL and should lead to further insights into the understanding of disease pathogenesis. Chromosome Location Gene(s) of interest Loss 6q24.3 LATS Loss 10q23.3 LG11 Loss 10q24.2 PAX2, WNT8B, TLX1, POLL, NFKB2 Loss 11p15.4 PPFIBP2 Loss 12p13 TNFRSF1A and 7 Loss 15q22.1 TCF12 Loss 19p13.3 BAX, RUVBL2 Loss 20q11.2 TP53INP2 Loss 20q13.1 WFDC13

Abstract Immunoglobulin (Ig) light chain (LC)-associated amyloidosis (AL) is a fatal plasma cell (PC) disorder characterized by the overproduction of Ig light chains that deposit in an abnormal conformation as amyloid throughout the body. Lambda LC are involved in amyloid deposition 2–3 times more often than kappa LC, and certain LC Ig variable genes are more frequently involved than others and influence clinical presentation and outcome. AL is a potential complication of any immunoglobulin clonal process, but is most often pathologically associated with minimal clonal expansion of PC, as seen in monoclonal gammopathy of undetermined significance. It is much less commonly observed in patients with multiple myeloma (MM). Because of this, and to our knowledge, there are no cell lines that have been established from AL patients, an experimental tool that would be of great value in studying amyloid formation and the biology of amyloid producing PC. In this study, we have established two cell lines from a 50 yr old female initially diagnosed with AL. Upon initial diagnosis, the BM aspirate consisted of 27% IgG λPC with a PC labeling index (PCLI) of 1.9% and a κ to λratio of <0.1. Amyloid was present in periosteal vessel walls and in the fat aspirate. The first cell line, ALMC-1, was established from BM mononuclear cells isolated from the diagnostic aspirate. The patient received a peripheral blood stem cell transplant (PBSCT) 2 months later, but relapsed within 100 days post-PBSCT with symptomatic myeloma. At relapse, the patient’s BM aspirate consisted of 70% λ+ PC and a PCLI of 20%; the second cell line, ALMC-2 was established from this aspirate. IgVL and IgVH analysis revealed that both cell lines expressed identical sequences and used IgVλ 6–57*01, IgλJ3*02, and IgλC3*03: both used the IgVH VH3–21 gene and the extent of somatic mutation was approximately 4%. Both cell lines produce significant FLC, and studies are currently underway to characterize in vitro amyloid production. We next used fluorescence in situ hybridization (FISH) to identify the genetic defects in this patient’s tumor population before and after transition to symptomatic MM. The initial BM aspirate at time of diagnosis of AL revealed approximately 30% of the PC had clear evidence of c-MYC gene amplification. In the subsequent draws as well as in both cell lines, 100% c-MYC amplification was identified, consistent with clonal selection. FISH analysis also revealed that 40% of cells had p53 deletion upon initial diagnosis, whereas the subsequent samples and cell lines were 100% for p53 deletion. All cells also had an IgH translocation that did not involve any commonly observed chromosome partners. Lastly, we have characterized the cytokine responsiveness of both cell lines. Although some differences are observed between ALMC-1 and ALMC-2, IL-6 and IGF-1 stimulated growth in both cell lines to varying degrees and both lines expressed autocrine IGF-I. In summary, our initial characterization of ALMC-1 and ALMC-2 predicts that these unique cell lines will prove to be an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology. Insight gained from this model system may eventually have an impact in the clinical arena by providing a better understanding of this incurable disease.

Abstract Background: The prognosis of patients with early Rai and Binet stage CLL is variable. Recently developed prognostic tests (mutation status, ZAP-70, chromosome evaluation by FISH) require significant technologic expertise and are not available worldwide to most patients with CLL. Smudge cells are ruptured CLL cells appearing on a blood smear. Using a proteomic approach, we recently discovered that the cytoskeletal protein vimentin is expressed at higher levels in CLL cells with unmutated IgVH and that vimentin expression is a marker of poor prognosis in early stage CLL (Blood, 2005;106:707). Hypothesis: Since vimentin plays an important role in the cytoskeleton and lymphocyte rigidity, we hypothesized that smudge cell formation is inversely correlated with vimentin expression and that the percentage of smudge cells may predict prognosis in CLL. Methods: We blindly reviewed the blood smears archived at the time of diagnosis of patients with untreated, early stage CLL. A total of 200 lymphocytes and smudge cells were counted per slide and results reported as percentage of total lymphocytes (intact and smudged). The percentage of smudge cells was correlated with vimentin expression measured by flow cytometry, IgVH mutation status, time to treatment (TTT), and survival. Results: Blood smears of 75 patients were reviewed. The median percentage of smudge cells was 27% (range, 4–72%). The percentage of smudge cells did not correlate with lymphocytosis (r=0.04, p=0.73) and did not change overtime for serial evaluations for individual patients (p=0.61). The percentage of smudge cells appeared to correlate inversely with vimentin expression (r = − 0.627; p = 0.0017). The percentage of smudge cells was higher in patients with mutated IgVH than in patients with unmutated IgVH (median 31% vs. median 13%, p=0.02). The percentage of smudge cells as a continuous variable correlated with both a prolonged TTT (p=0.0023) and overall survival (p=0.04). Patients with less than 30% smudge cells had a median TTT of 72.7 months. The median TTT in patients with ≥30% smudge cells was not reached (p=0.0011), Figure 1. Conclusion: A lower percentage of smudge cells is associated with unmutated IgVH status and with worse clinical outcome in early stage CLL. The percentage of smudge cells does not vary based on the magnitude of the lymphocytosis, but is patient specific and remains constant over time. Evaluation of the percentage of smudge cells on blood smears could be a universally available prognostic test for patients with early stage CLL. Figure Figure

Abstract Chronic Lymphocytic Leukemia (CLL) is primarily characterized by the presence of leukemic B lymphocytes resistant to the process of apoptosis. Causes for this resistance include aberrant expression of multiple anti-apoptotic proteins, perhaps including Tcl-1 (1). Tcl-1 is expressed during normal development of both B and T cells and activates Akt to provide a survival signal through phosphorylation and inactivation of apoptotic pathway components. Overexpression of Tcl-1 in the B cells of transgenic mice results in a B-cell leukemia phenotype typical of human CLL, suggesting a primary role for Tcl-1 in CLL tumorigenesis. A recent report showed that Tcl-1 protein expression in CLL cells correlated with other biological parameters, but no data were available as to its clinical relevance (2). We therefore examined Tcl-1 expression in relation to clinical outcome using 51 peripheral blood mononuclear cell samples from a Phase II clinical trial testing the combination of pentostatin, cyclophosphamide, and rituximab in previously untreated CLL patients (Kay et al., ASH 2006). Tcl-1 protein expression was measured by immunoblot using GAPDH as a normalization control across lanes and lysate from a single Tcl-1 transgenic mouse spleen as a normalization control across immunoblots. All samples were run at least in duplicate and digital quantitation was performed using a ChemiDoc instrument (BioRad). Tcl-1 expression in patient cells was calculated in comparison to Tcl-1 expression in the mouse lysate, arbitrarily set to 1.0. Data were analyzed as a continuous measure as well as by specific cutoffs. Higher Tcl-1 expression was associated with failure to achieve a minimal residual disease-negative state after treatment. Specifically, Tcl-1 expression (measured as a continuous variable) was associated with residual disease measured by post-treatment CD5+/CD19+ cell percentages (continuous variable as well as dichotomized by cutpoint of 1.0% for CD5+/CD19+; p=0.03 for both comparisons). For the dichotomized Tcl-1 expression with the cutpoint of 0.275 (second tertile), this variable was marginally significantly associated with whether or not post-treatment CD5+/CD19+ was less than 1.0%. Using this same cutoff, there was also a suggestion that patients with reduced Tcl-1 were more likely to achieve a complete response. Finally, progression-free survival was shorter in patients with increased Tcl-1 expression, although these data did not reach statistical significance in this sample set. Tcl-1 < 0.275 Tcl-1≥ 0.275 p value CD5/CD19≥ 1% 15 10 0.046 CD5/CD19 < 1% 16 2 CR 17 3 0.064 No CR 18 13 Median PFS (mos.) 36.5 17.1 0.137 Statistically significant associations with other parameters including Rai stage, IgVH mutation status, ZAP-70 expression, CD38 expression, and interphase cytogenetics categories were not detected in this sample set. These data further support the clinical relevance of the Tcl-1 mouse model to human CLL, and provide preliminary evidence that Tcl-1 protein expression correlates with clinical outcome of CLL patients treated with chemoimmunotherapy. If validated in larger studies, Tcl-1 expression may serve as a novel prognostic factor.

Abstract BACKGROUND: B-Chronic Lymphocytic Leukemia (CLL) is still uncurable but very powerful new tools are available with the use of chemoimmunotherapy (CIT). Purine nucleoside-based regimens that incorporate rituximab have generated very high levels of overall responses (OR) with significant percentage of those complete responses (CR) in previously untreated CLL. Here we report and update our experience with a phase 2 pentostatin-based CIT regimen for previously untreated CLL as conducted at 2 medical centers. We also studied the association of outcome based on risk stratification parameters and achievement of minimal residual disease. METHODS: Building on prior work of pentostatin in CLL by us (Kay ASH, 2004) and others, we initiated a trial of combined pentostatin (P)(2 mg/m2), cyclophosphamide (C)(600 mg/m2) and rituximab (R)(375 mg/m2) for symptomatic, previously untreated patients (n=65). This PCR regimen is given on a 21-day, 6-cycle schedule. However, the initial cycle of treatment uses thrice weekly rituximab as described by us earlier. In brief, this was rituximab at 100 mg/m2 on day 1, 375 mg/m2 on days 3 and 5 of the first week only. Prophylactic Sulfamethoxazole/Trimethoprim and Acyclovir were given to all patients for 1 year starting on the first cycle of therapy with PCR. All patients were risk stratified using CD38, ZAP-70, immunoglobulin heavy chain variable region gene (IgVH) and FISH panel assessments at entry. RESULTS: These patients were characterized as mostly in high-risk categories. Of 64 evaluable patients, 34 (53%) were high Rai risk (stage 3–4), 71% were non mutated for the IgVH gene, 34% were CD38+ and 34% were ZAP-70+. Thirty patients (52%) had one FISH anomaly, and 21 (36%) had complex FISH defects. Thirty-eight patients (58%) had grade 3+ hematologic toxicity but minimal transfusion needs, and no major infections. NCI Working Group Criteria Responses occurred in 58 (91%) with 26 (41%) complete responses (CR), 14 (22%) nodular partial responses (nodular PR), and 18 (28%) partial responses (PR) patients. Outcome for all 64 patients demonstrates a median progression-free survival of 32.6 months. Importantly, no high risk factor (i.e., age, FISH, IgVH status, CD38+, ZAP-70+) except for del (17p) defect (n=3) precluded attaining a CR or NPR. In contrast, we found this regimen was equally effective in young vs. elderly (>70 yrs) patients and in del(11)(q22.3) vs. other favorable prognostic FISH factors. Examination of outcome among CR and nodular PR patients for PFS by flow cytometry status (negative vs. positive, i.e., ≤ 1 % CD5+/CD19+ vs. ≥ 1 % CD5+/CD19+) demonstrated improvement in progression free survival for patients who attained flow cytometry negativity (p = 0.009). Conclusion: This novel regimen of pentostatin, cyclophosphamide and rituximab for previously untreated CLL demonstrated significant clinical activity despite poor risk-based prognoses with minimal toxicity in terms of bone marrow suppression and/or infections. The additional feature of this approach is the ability to have durable responses for all age groups and even CLL patients with a del(11)(q22.3).

Abstract Abstract 2883 CLL-like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotypic features and cytogenetic abnormalities with CLL and is generally perceived as its premalignant state. The World Health Organization has set a consensus cut-off of 5×109/L circulating B cells to discriminate between what constitutes ‘disease’ and what not. However, the clonal size within MBL is extremely variable. High-count (HC), clinical MBL is associated with absolute lymphocytosis and progresses to CLL requiring treatment at a rate of 1–2% per year, whereas low-count (LC) MBL is found in the general population through high-sensitivity techniques and carries a risk of progression that is limited if any. Given the high frequency of CLL-like MBL in the general population, it is important to understand the underlying mechanisms and also identify biological markers endowing malignant potential that may distinguish between the different forms. To this end, we performed a detailed immunogenetic profiling of 334 CLL-like MBL cases (78 LC and 256 HC) for a total of 355 productive VDJ rearrangements (including double rearrangements), 91 from LC MBL and 264 from HC MBL. We also compared the immunoglobulin (IG) gene repertoires of MBL to 544 CLL Rai Stage 0 (CLL-0) that were part of an IG sequence dataset of 7424 CLL cases previously analyzed by our group. LC and HC MBL had distinct IG gene repertoires, with over-representation of the IGHV1–69 and IGHV4–34 genes in HC and the IGHV4–59/61 genes in LC MBL, respectively (p<0.001). The HC MBL repertoire exhibited clear similarities to CLL-0 in terms of IGHV gene usage (similar frequencies of IGHV1–69 and IGHV4–34). Regarding somatic hypermutation, no differences were identified between LC MBL versus HC MBL versus CLL-0, in that the frequency of mutated rearrangements (<98% identity to the germline) was overall similar (LC MBL: 72.5%, HC MBL: 76.1%, CLL-0: 75%). In this respect, all the aforementioned subgroups differed significantly (p<0.001) from the frequency previously reported by us in CLL where only 55% of rearrangements carried mutated IGHV genes. We finally analyzed the expression of stereotyped B cell receptor (BcR) IGs, identified through a cluster analysis of the MBL sequences together with all CLL sequences from our cohort and 5494 non-CLL IG sequences retrieved from public databases. Overall, only 6/91 (5.5%) LC MBL rearrangements could be clustered with other sequences in subsets with stereotyped BcRs. Two of these six LC MBL cases were clustered together with IG sequences from various entities, including CLL, other lymphomas and autoimmune diseases; thus, they were considered to carry ‘public’ BcR stereotypes. In contrast, HC MBL showed a significantly (p=0.0002) higher frequency of ‘CLL-specific’ BcR stereotypes versus LC MBL, with 60/264 (22.7%) HC MBL cases carrying stereotyped VH CDR3s. This frequency was comparable to the one observed in CLL-0 (20.2%). Notably, a gradation was observed in the frequency of BcR IG stereotypy depending on the absolute count of CLL-like cells, starting with 5.5% in LC MBL, raising to 22.7–20.2% in in HC MBL/CLL-0 and, finally, peaking at 30.4% in the entire CLL cohort as previously reported. Altogether, these findings suggest that rather than a true premalignant condition, LC MBL may merely reflect immune senescence or result from persistent antigen stimulation. On the other hand, HC MBL appears to be a continuum with Rai stage 0 in the evolution to clinically overt CLL, being maybe one step behind where it requires either additional genetic hits or simply extra time to cross the numerical border that discriminates it from CLL. Hence, the identification of molecular genetic markers that may predict progression of HC MBL/CLL-0 into full-fledged CLL is strongly warranted. Disclosures: Shanafelt: Genentech: Research Funding; GlaxoSmith Klein: Research Funding; Teva/Cephalon: Research Funding; Celgene: Research Funding.

Abstract Abstract 2928 Multiple myeloma (MM) is caused by the clonal expansion of malignant plasma cells (PCs) producing high levels of monoclonal immunoglobulin (Ig) or M-protein. However, ∼2% of all patients with monoclonal gammopathies (MG) have more than 1 M-protein, a condition termed biclonal gammopathy or double monoclonal gammopathy (DMG). These patients can express M-proteins with different heavy chain (HC) isotypes and different light chains (LCs) or different HCs that are LC-matched. Reports suggest that ∼87% of DMGs with different HCs have matched LCs, but without molecular analysis at the nucleotide level, clonal relationships are unknown. Thus, DMG patients may have either two independent malignant PC clones that coincidentally express the same type of LC or the two clones are related and reflect previous or ongoing class switch recombination (CSR). This latter variant would offer novel insight into the origin and differentiation of the malignant clone. Of note, prior reports of HC-dissimilar/LC-matched DMGs are limited to the analysis of single patients and with few exceptions; do not include molecular analysis of the variable regions in Ig HC or LC genes at the nucleotide level. In this study, we interrogate a cohort of MM patients with known HC-dissimilar/LC-matched DMGs for the frequency of clonal relatedness. The cohort was identified by searching our MG database for patients with: 1) HC-dissimilar/LC-matched Igs as determined by immunofixation; 2) >10% bone marrow (BM) PCs; and 3) no transplant prior to BM collection. Eight patients (7 MM and 1 smoldering MM/primary amyloid) were identified for further molecular evaluation. All 8 cases expressed IgG and IgA HCs. Three patients expressed kappa LC (K-LC) and 5 expressed lambda LC (L-LC). To determine if the IgG and IgA M-proteins were clonally related in each patient, we performed Ig HC variable gene (IGHV) PCR with sense primers to all IGHV genes and an antisense primer specific to IgG or IgA. In all 8 patients, a single productive IGHV gene was determined with IgG and IgA. In 2/8 patients, the IgG and IgA HC variable regions were clearly distinct and therefore genuinely biclonal. Of note, PCs from 6/8 patients used the same IGH V-D-J genes in both IgG and IgA transcripts. In 5 of these 6 patients, the entire IgG and IgA variable region and HCDR3 were identical at nucleotide and amino acid (AA) levels. Of interest, one patient had common, as well as distinguishing IGHV region somatic mutations (SHM) between the IgG and IgA transcripts. The HCDR3 regions (including N nucleotides) were highly homologous but differed by 7 nucleotides which resulted in 3 AA differences between the 2 clonally related sequences. While these are compelling data, it remained possible that the malignant PCs in these patients simultaneously expressed IgG and IgA as a result of long nuclear transcripts. To test this possibility, we used immunofluorescence (IF) and verified the presence of both IgG and IgA expressing PCs in all cases. Parallel analysis of both K-LC and L-LC variable gene by PCR was done on the 6 patients with the same IGHV gene in both IgG and IgA transcripts. In 3/6 patients, there was a single somatically mutated LC identified corresponding to the LC detected by immunofixation. These results were also confirmed by IF. The remaining 3 patients appeared to express more than 1 LC rearrangement. Taken together these results confirm that HC-dissimilar/LC-matched DMG can present with various phenotypes at the molecular level. We describe the largest cohort to date documenting the frequency of DMG patients that can be classified as expressing 2 identical HCs with either a single LC or >1 LC based on molecular analysis. The former case may reflect an original progenitor cell that divided with one daughter cell subsequently experiencing further CSR yet the LC remained untouched. In the case where the HCs were slightly different, there may have been CSR with additional SHM of a daughter cell, or the molecular differences may reflect random somatic mutations occurring independent of germinal center reactions. These results suggest that HC-dissimilar/LC-matched DMG may occur more often than thought. Larger studies tracking M-protein levels over time accompanied by cytogenetic analyses are needed to determine if the two clonally-related class switch variants in DMG patients exhibit similar biological properties. This analysis would offer new insight into the genesis of this unique subtype of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract The existence of stereotyped B cell receptor immunoglobulins (BcR IG) in chronic lymphocytic leukemia (CLL) strongly implicated antigen selection in disease ontogeny. We have previously shown that the stereotyped fraction encompasses ~30% of all CLL and includes multiple subsets with distinct BcR IG configuration and variable size. Eventually, certain major subsets emerged as distinct clinical entities, exemplified by subset #2 (IGHV3-21/IGLV3-21, ~2.5-3% of all CLL, mixed somatic hypermutation (SHM) status) of a particularly aggressive clinical course, thus, sharply contrasting subset #4 (IGHV4-34/IGKV2-30, ~1% of all CLL, mutated IGHV genes, M-CLL), a prototype for indolent disease. Here, taking advantage of a multi-institutional cohort of 21,123 CLL IG rearrangements, almost three times the size of the largest previous study, and the availability of validated, purpose-built immunoinformatics methods, we reappraised BcR IG stereotypy especially focusing on major subsets and the degree of their sequence similarity to related minor subsets. Stereotypy discovery was performed with ARResT/Teiresias, while stereotypy assignment to existing subsets previously deemed as major was performed with ARResT/AssignSubsets (http://bat.infspire.org/arrest/). In the present study, a subset was characterized as major if representing > 0.2% of the cohort (i.e. at least 50 cases). Minor subsets closely related to major ones (termed satellite) were identified applying the following criteria: (i) usage of IGHV genes from the same phylogenetic clan; (ii) VH CDR3 length difference ranging from -2 to +2 compared to the respective major subset; (iii) shared VH CDR3 sequence motif; and, (iv) -2 to +2 difference in the offset of the VH CDR3 motif compared to the respective major subset. In total, 7378/21123 (34.9%) IG sequences were grouped into subsets with stereotyped VH CDR3, with the previously characterized 19 major subsets accounting collectively for 2594 sequences (12.3%) of the cohort: of these, 12 included cases with unmutated IGHV genes (U-CLL), 6 concerned M-CLL and 1 (subset #2) included cases with mixed SHM status. Four additional subsets exceeded 50 cases, and, thus, were also considered as 'major'. These results reinforce the notion that not all CLL will end up being stereotyped but rather that a plateau for stereotypy exists at ~1/3 of the cohort. Subset #2 was the largest subset (n=572, 2.7%), while subset #1 (IGHV clan I (IGHV1,5,7 subgroups)/IGKV1(D)-39) was the most frequent subset within U-CLL (n=515, 2.4%) and subset #4 the most common M-CLL subset (n=192, 0.9%), hence displaying remarkable consistency regarding their frequency in all cohorts published since the pioneering studies. Altogether, Teiresias and AssignSubsets gave concordant results for previously identified major subsets, illustrating the validity of our approach. Satellite subsets were sought for individually for each major subset. In general, few satellite subsets were identified, most of which concerned U-CLL major subsets. That notwithstanding, notable cases of satellite subsets were exemplified by major subset #1 and its satellite subset #99 from which it differed only in VH CDR3 length (13 aminoacids in subset #1 versus 14 in subset #99); interestingly, both subsets displayed equally aggressive clinical course. Another example concerned subset #8 (IGHV4-39/IGKV1(D)-39, U-CLL), an aggressive subset with very high risk for Richter's transformation, that, except for a one-aminoacid difference in VH CDR3 length, was otherwise identical to satellite subset #215, also displaying clinical aggressiveness. Overall, our results confirm that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease subgroups amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Most major subsets display unique sequence motifs, however satellite subsets exist, especially within U-CLL. Considering ever-increasing evidence that major stereotyped subsets may represent distinct disease subgroups, the existence of satellite subsets reveals a novel aspect of repertoire restriction and has implications for refined molecular classification of CLL. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding. Gaidano:Roche: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Niemann:Janssen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Gilead: Consultancy. Langerak:F. Hofmann-LaRoche, Genentech: Research Funding; InVivoScribe Technologies: Patents & Royalties: Royalties are provided to European Network (EuroClonality). Jaeger:Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Kater:Celgene: Research Funding; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding. Hallek:Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Rosenquist:Gilead Sciences: Speakers Bureau. Ghia:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding; Adaptive: Consultancy; Abbvie: Consultancy, Honoraria. Stamatopoulos:Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding.

Abstract Abstract 2902 Background: Although chronic lymphocytic leukemia (CLL) is a low grade B-cell malignancy, 2–8% of patients (pts) are reported to undergo transformation to diffuse large B-cell lymphoma (DLBCL; Richter's syndrome [RS]). Whether the risk of transformation is related to the underlying biology of CLL or is a consequence of treatment for CLL is uncertain. Also, the role of prognostic markers such as immunoglobulin heavy chain (IGHV) gene mutation status, ZAP-70 and CD38 expression, and genetic abnormalities detected by fluorescence in situ hybridization (FISH) in predicting the development of RS is not well defined. The available data on stem cell transplant (SCT) in pts with RS is based on retrospective case series of selected RS pts and is of uncertain relevance to the majority of CLL pts experiencing RS. Therefore, we conducted a cohort study of newly diagnosed CLL pts to explore the incidence and natural history of RS. Methods: The Mayo Clinic CLL database includes all consenting pts with a pathologic diagnosis of CLL. Information on date of diagnosis, treatment history, and disease-related complications is abstracted from clinical records on all pts and then maintained prospectively. We used this database to identify newly diagnosed pts with CLL from January 2000 through July 2011, who were seen at our institution within 12 months of diagnosis. Pts with biopsy-confirmed RS were included in this analysis. For pts referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included in this study. The Mayo Clinic Institutional Review Board approved the study. Results: Between January 2000 and July 2011, 1641 pts with newly diagnosed CLL were seen at Mayo Clinic within 12 months of diagnosis, and this cohort had a median follow-up of 4.0 yrs (range, 0–12.3 yrs). Of these pts, 37 (2.3%) were diagnosed with RS. The median time to development of RS after a diagnosis of CLL was 1.8 yrs (range, 0–11.7 yrs). The incidence of RS at 2 and 5 yrs post CLL diagnosis were 1.3% and 2.1% respectively (Figure). Pts who developed RS had more advanced (Rai III/IV) stage disease (22% vs. 6%; p<0.001) and higher beta 2-microglobulin levels (median 3.1 mg/dL vs. 2.3 mg/dL; p=0.003) at diagnosis of their CLL compared to pts without RS. Pts who developed RS were also more likely to be positive for ZAP-70 (58% vs. 33%; p=0.03), CD49d (77% vs. 31%; p=0.0004), and CD38 (57% vs. 29%; p=0.001), have unmutated IGHV (76% vs. 44%; p=0.003) and have either del(11q22.3) or del(17p13) on FISH analysis (46% vs. 13%; p<0.0001). RS transformation occurred prior to treatment of CLL in 17 (46%) pts and after treatment of CLL in 20 pts (54%). Treatment for CLL was associated with an increased risk of RS (92% vs 34%; p<0.0001). The odds ratio (OR) of RS was increased for pts who received purine nucleoside analogues (OR=3.0; 95%CI=[1.6, 5.9]; p=0.001), alkylating agents (OR=2.5; 95% CI=[1.3, 4.9]; p=0.004), or both (OR=3.3; 95% CI=[1.7, 6.4]; p=0.0003) for treatment of their CLL. Median overall survival after development of RS was 2.1 yrs. Most pts (29/37, 78%) received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Two (5%) pts underwent an autologous SCT and 5 (14%) underwent an allogeneic SCT. Additionally, 1 (3%) patient received an autologous SCT followed by an allogeneic SCT. By the end of the study period, 17 (46%) pts with RS had died. Conclusion: The overall rate of biopsy-proven RS in this large cohort of pts with newly diagnosed CLL was 2.3% (5 year incidence: 2.1%). Clinical characteristics (Rai stage III/IV) and biologic characteristics of the CLL B-cell (positive expression of ZAP-70, CD38 and CD49d; unmutated IGHV; and either del(11q22.3) or del(17p13) on FISH) at the time of diagnosis were associated with future risk of RS. Exposure to purine nucleoside analogues and/or alkylating agents for treatment of CLL was also associated with increased risk of RS. Disclosures: Zent: GlaxoSmithKline: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Genzyme: Research Funding; Biothera: Research Funding. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) remains a heterogeneous disease despite significant advances in disease classification based upon cellular and molecular markers. Rai stage, immunoglobulin heavy chain variable region mutation status (IGHV), cytogenetic abnormalities, beta 2 microglobulin levels, expression of ZAP-70, CD38 and CD49d provide useful guides alone or in prognostic models for progression risk assessment but variability in clinical outcome in early Rai stage (Rai 0/I) unmutated (UM) IGHV patients persists. This variability in clinical outcome suggests that there are yet-to-be defined biologic/genetic factors that contribute to disease heterogeneity. Long non-coding RNAs (lncRNAs) display a range of diverse activities including regulation of gene transcription, post translational regulation and epigenetic regulation and represent an intriguing prospect for influencing CLL disease progression. LncRNAs have been implicated in diagnosis, prognosis and therapy of various cancers however their role in influencing disease progression in early Rai stage UM CLL has not yet been studied. Methods: To investigate a role for lncRNAs in UM Rai 0/I CLL patients we employed the Arraystar Human lncRNA Microarray v4.0. Using blood samples collected within 3 months of diagnosis, two cohorts of UM Rai 0/I patients were compared: one defined as progressive disease with time to first treatment (TTT) ≤ 2 years after diagnosis (n=34) and one as indolent disease with TTT ≥5 years after diagnosis (n=29). Differentially expressed lncRNAs were identified using Agilent GeneSpring GX Software v12.1. To be considered for further analysis, the difference in lncRNA expression had to have a fold change (FC) ≥ 2.0, a t-test p-value ≤ 0.05 and a false discovery rate (FDR) ≤ 0.05. Results: Over 1100 lncRNAs were found to be differentially expressed between the progressive and indolent UM CLL cohorts. Greater than 150 of these lncRNAs have known associated genes and are well annotated and validated. Among the differentially expressed lncRNAs, several are of particular interest and potentially relevant to CLL disease biology. For example, the lncRNA AL833181 was overexpressed in the progressive group. The associated gene for AL833181 is BCL11A and encodes a zinc finger protein that interacts with BCL6 and may itself serve as a proto-oncogene. Translocations involving BCL11A have been shown to identify a very aggressive subset of CLL (Am J Pathology, 2009). Therefore it is possible this lncRNA may be playing a role in deregulated expression of BCL11A. Within the indolent disease group, CTD-2566J3.1 was overexpressed >10-fold as compared to the progressive disease group. Its associated gene RAD51B is essential for DNA repair, suggesting the possibility that CTD-2566J3 may protect CLL cells from further DNA damage and disease progression. Also overexpressed in the indolent disease group were 2 lncRNAs that are associated with genes that encode proteins involved in the ubiquitin-proteasome system. USP2-AS1 lncRNA is associated with the USP2 gene and encodes an ubiquitin-specific protease required for TNF-alpha induced NF-kB signaling and is a specific deubiquitinase for cyclin D1, a known proto-oncogene. Furthermore, USP2-AS1 may also be linked to MYC. RP11-522I20.3 is associated with the QBLN1gene which encodes a protein that mediates the proteasomal degradation of misfolded or accumulated proteins. Thus, the associated lncRNAs may be acting as post-transcriptional gene silencers. Although CLL is not viewed as an "invasive" cancer, there were 5 lncRNAs overexpressed in the indolent group that are associated with Ankyrin genes which play a role in cell motility, activation and proliferation along with the lncRNA RP11-477D19 that is associated with theTIAM2 gene. TIAM2is a Rac guanine nucleotide exchange factor that can promote invasion and motility of cells. The respective lncRNAs may be down-regulating these genes and prohibiting malignant cell expansion. Conclusions: Our study reveals that there are indeed specific lncRNAs that are expressed at different levels in progressive versus indolent early Rai stage UM CLL and have the potential to impact a number of relevant biological processes and pathways. While these data are preliminary, the lncRNA AL833181 and its associated gene BCL11A may prove to be a potential marker and therapeutic target for aggressive disease. Disclosures Shanafelt: Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; GlaxoSmithkKine: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Hospira: Research Funding.