Citations

Abstract Abstract 2406 The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. Disclosures: Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Abstract Abstract 2817 Poster Board II-793 Abnormal plasma cells (PC) present in patients with multiple myeloma (MM) and its precursor condition, monoclonal gammopathy of undetermined significance (MGUS), characteristically possess multiple chromosomal abnormalities. Moreover, both stages of disease exhibit considerable intratumor heterogeneity, which often becomes even more complex during disease progression. The precise mechanism(s) underlying this process remains unknown. However, we hypothesize that DNA double-strand breaks (DSBs) and compromised repair of these deleterious lesions may underlie intratumor heterogeneity and clonal evolution in the monoclonal gammopathies. In this regard, H2AX, a member of the H2A family of histones, plays a particularly important role in the DSB response and prevention of cancer. Immediately following DSB formation, one or more of the PI3K-like kinases become activated and rapidly phosphorylate H2AX on a conserved serine residue. Phosphorylated H2AX (γH2AX) is then rapidly recruited to the DSB site and is readily detectable as DNA damage foci by immunohistochemistry. The precise function of γH2AX has yet to be determined, however, it is hypothesized that γH2AX may recruit DNA repair proteins to the DSB site and may aid in keeping severed DNA ends in place in order to avoid erroneous end joining. Despite the functional uncertainty of γH2AX, the presence of γH2AX nuclear foci serves as an excellent indicator of DSBs. Therefore, the goal of our study was to assess MM cells for evidence of DSBs. We began our studies using a panel of 8 human MM cell lines. Of note, the number of foci was found to vary among the MM cell lines and to vary from cell to cell with the number of γH2AX foci per cell ranging from 0 to 28. The presence of γH2AX in these cells was also confirmed via flow cytometry and western blotting. We also wished to determine if primary MM and MGUS PCs displayed evidence of DSBs. Among primary patient samples, freshly isolated PCs from 13/18 MM patients and 1/3 MGUS patients exhibited evidence of γH2AX foci. Taken together with the MM cell line data, the number of γH2AX foci was found to increase across the disease spectrum of MGUS to MM patient sample to MM cell line. Endogenous γH2AX foci have previously been detected in a variety of tumor cell lines. Although these foci have been hypothesized to derive from multiple factors, the extent of phosphorylation has been shown to be associated with the number of chromosomal aberrations as well as the phase of the cell cycle. In general, S and G2/M phase cells tend to demonstrate higher levels of H2AX phosphorylation, which is most likely due to doubling of histone content during the cell cycle and the fact that chromatin condensation during DNA replication can also trigger H2AX phosphorylation. Thus, it remained possible that the γH2AX displayed by the cell lines simply reflected cells in the S phase of the cell cycle. To address this possibility, we labeled cells with BrdU and then measured levels of γH2AX in cells in the G1, S and G2/M phases of the cell cycle. However, we observed nearly equal levels of γH2AX in G1 and S phase cells suggesting some level of γH2AX foci was independent of DNA replication. These results were also consistent with our observation that there is no correlation between the plasma cell labeling index and the number of γH2AX foci in CD138+ plasma cells isolated from 18 MM patients. Thus, endogenous γH2AX in MM cells does not appear to be primarily attributed to cycling cells and may be indeed reflective of DSBs. Finally, to further demonstrate that the γH2AX foci genuinely reflected sites of DSBs, we performed double staining for γH2AX foci and 53BP1, a protein that is known to be recruited to DSB sites following DNA damage. Results revealed clear colocalization of γH2AX and 53BP1 in both MM cell lines and MM patient samples. Given that DSBs can lead to genomic instability and tumor progression, our observations that primary MGUS and MM PCs display evidence of DSBs at isolation are intriguing and suggest a mechanism whereby clonal evolution occurs in the monoclonal gammopathies. The presence of a higher frequency of γH2AX foci in MM cell lines is consistent with their derivation from MM patients with aggressive disease. Collectively, these studies suggest MGUS/MM PCs may display an impaired ability to repair DNA damage and studies designed to examine this possibility are underway. Disclosures: Dispenzieri: Celgene: Research Funding.

Abstract Abstract 470 Multiple myeloma (MM) is an incurable and fatal neoplasm characterized by the accumulation of clonal plasma cells (PC) that results in significant end organ and tissue damage. MM is preceded by either of two premalignant asymptomatic stages, monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), both of which are identified by the presence of clonally expanded abnormal PC populations. While MGUS and SMM patients' abnormal PC populations may remain stable for years, both have a life-long significantly increased risk of progressing to MM (1% and 10% per year, respectively). For this reason, a better understanding of the molecular events prompting malignant progression and increased accuracy in identifying those patients that have begun to transition to MM is urgently needed. In this study, we identified a novel MM marker, CD147 (also known as extracellular matrix metalloproteinase inducer (MMP), or EMMPRIN), that is not only over-expressed on MM PCs as compared to its premalignant counterparts, but whose increased expression correlates with the level of abnormal PC proliferation. To our knowledge, we are the first to demonstrate a role for CD147 in MM. CD147 has been shown by others to display a variety of activities and those with potential relevance to MM include stimulation of increased MMP production and angiogenesis, and playing a critical role in glycolysis via facilitation of excess cellular lactate transport. Our initial experiments revealed that MM PCs overexpress CD147 mRNA relative to MGUS PCs. Flow cytometric analysis corroborated these data and demonstrated variable expression of CD147 across the disease continuum ranging from no expression to bimodal or uniform expression. Indeed, there was a significant difference between CD147 expression on MGUS and SMM PCs compared to that on MM PCs (p=0.02 and 0.005, respectively). We next determined whether CD147 had a signaling role in these cells. Using the natural CD147 ligand, cyclophilin B (CypB), we showed that addition of CypB to either human MM cell lines (HMCLs) or CD138+ patient PCs resulted in increased PC proliferation as measured by [3H]-thymidine incorporation. In a complementary manner, addition of CD147 antibodies significantly inhibited proliferation without an effect on cell viability. By western blot analysis we further demonstrated that CypB-mediated CD147 activation leads to MAPK phosphorylation. Next, we isolated CD147+ and CD147- MM cells from patients whose tumor cells bimodally expressed this marker and assessed the response of each subset to IL-6 and CypB. The CD147+ subset was almost solely responsible for the proliferative response in all cases examined. In addition, we cultured bone marrow mononuclear cells from CD147 bimodally expressing MM patients overnight with bromodeoxyuridine before performing cell cycle analysis on the CD147+ and CD147- MM PC populations. Remarkably, the CD147+ PCs were greatly enriched for cells in the S and G2/M phases of the cell cycle, whereas the CD147- PCs resided almost entirely in the G0/G1 phase. In the final set of experiments, we employed siRNA knockdown strategies using HMCLs to definitively test the role of CD147 in MM cell proliferation. Indeed, IL-6 induced proliferation was significantly compromised following CD147 down regulation, which was not attributed to increased apoptosis. However, IL-6 mediated phosphorylation of MAPK remained robust suggesting that the IL-6 signaling pathway overall was not compromised in these cells. Finally, cell cycle analysis demonstrated that CD147 down regulation resulted in a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a decrease in the number of cells in the G2/M phase of the cell cycle, as compared to cells transfected with control siRNA. In conclusion, our data suggest that the CD147 molecule plays a critical role in the biology of malignant MM PCs, particularly as it concerns MM cell proliferation, and may thus serve as a useful and attractive target for reducing the proliferative compartment of this disease. Ongoing studies are investigating additional roles for MM cell CD147 expression, e.g., its role in MMP induction in the tumor microenviroment. Disclosures: No relevant conflicts of interest to declare.

Abstract BACKGROUND: CD49d (a.k.a. alpha 4 integrin) plays a critical role in leukocyte trafficking, activation, survival, and facilitates interactions between leukocytes and stromal cells via VCAM-1 and fibronectin. We and others have previously reported that CD49d gene expression in CLL B-cells correlates with CD38 expression (Durig Blood101:2748; Pittner Leukemia19:2264). We have also confirmed CD49d protein expression correlates with CD38 protein expression on CLL B cells (Pittner Leukemia 19:2264). Notably, one small study reported that CD49d expression may relate to overall survival among CLL patients independent of CD38 status (Zucchetto, Leukemia 20:523), although the relationship to other prognostic markers, such as ZAP-70 and cytogenetic abnormalities, was not evaluated. METHODS: We measured CD49d expression by flow cytometry in 130 patients with CLL (NCI 1996 criteria), accrued to observational studies at Mayo Clinic, between 1994 – 2006. CD49d expression was measured by 2 color flow cytometry using antibodies specific for CD19 (BD Biosciences) and CD49d (BD Pharmingen). CD49d expression was compared to level of CD38 expression, ZAP-70 expression, and degree of IgVH gene mutation (all treated as continuous variables). We also evaluated the relationship between CD49d expression and time to treatment (TTT) and other prognostic parameters, using the previously published 30% threshold to classify CD49d expression (Zucchetto, Leukemia 20:523). Finally, we evaluated the relationship between CD49d expression and in vitro sensitivity to fludarabine (1 um x 24 hrs) and chlorambucil (1um x 24 hrs) in a subset of 70 patients. RESULTS: The percent of B-cells expressing CD49d varied from 0.3 to 99.4% among the 130 patients tested (median expression= 6.1). The level of CD49d strongly correlated with the expression of ZAP-70 (r=0.34; p<0.0001) and CD38 (r=0.65; p<0.0001), as well as the degree of IgVH gene mutation (r=−0.29; p=0.006). The relationship between CD49d expression and other prognostic parameters using the previously published 30% threshold to classify CD49d expression is shown in Table 1. The median time to treatment for patients with low CD49d expression was 14.9 years compared to 5.4 years for those with high CD49d expression (p=0.008) High CD49d expression was correlated with in vitro resistance to chlorambucil (r=0.27, p=0.03) but not fludarabine (r=0.20, p=0.09). CONCLUSION: CD49d expression varies on CLL B-cells and relates to other prognostic parameters and TTT as well as in vitro sensitivity to chlorambucil. Since CD49d facilitates interactions between CLL B-cells and stromal cells, the association of this marker with poor prognostic features and in vitro drug resistance may relate to enhanced stromal nuturing of leukemic cells with higher CD49d expression. Additional studies are needed to determine whether CD49d expression may be clinically useful as a prognostic test or to predict response to treatment in patients with CLL. CD49D low CD49D high P value % ZAP70+ 33.0 75.0 <0.0001 % IgVH unmutated 24.3 55.6 0.02 %CD38+ 12.8 63.9 <0.0001 FISH <0.0001 %with 17p- or 11q- or 6q− 9.2 25.0 % with +12 5.8 43.8 %with normal or 13q− 85.1 31.3 Current Stage 0.17 0 30.9 16.7 I-II 67.0 77.8 III-IV 2.1 5.6

Abstract Mutation status of the immunoglobulin heavy chain variable region (IgVH) in B cell chronic lymphocytic leukemia (B-CLL) is a critical prognostic tool. Although patients with unmutated (UM) IgVH genes exhibit an overall shorter survival than those with mutated (M) IgVH genes, considerable heterogeneity in clinical progression exists among UM B-CLL patients. The goal of this study was to evaluate UM CLL patients (n=215) in a large B-CLL cohort for Ig V, D, and J gene usage and relevant clinical parameters to identify Ig molecular features in addition to UM vs. M status that have prognostic value. Consistent with the literature, the most commonly expressed IgVH gene in our UM B-CLL cohort was VH 1–69 (69/215). We first evaluated D and J usage in VH 1-69 vs. non-VH 1–69 UM patients. The factors that were significantly different between VH1–69 vs. non-VH 1–69 cohorts were JH6 usage (p=0.0014), D3–3 usage (p=0.0025), and the combination of JH6 and D3–3 usage (p=0.0002). We then examined potential associations between patient time to treatment (TTT) and specific IgVH molecular features. Although there was a trend that VH 1–69 patients exhibited a shorter TTT than non-VH 1–69 patients, the association did not reach statistical significance (p=0.06). When all UM patients were instead grouped on the basis of D and J usage, JH6 usage was not significantly associated with TTT, but D3–3 usage, irrespective of VH or JH usage, significantly correlated with shorter TTT (p=0.005). Of interest, when JH6 patients were excluded from the analysis, differences in TTT between those with and without D3–3 usage were particularly pronounced (p=0.011). We next explored whether a specific D3–3 reading frame (RF) is associated with TTT. Within the group of D3–3 patients, we evaluated differences in TTT between those with RF 2 (n=38) vs. RF 3 (n=19) but did not study RF1 patients due to small numbers (n=6). Comparison of D3–3/RF 3 patients (n=19) with all other UM patients (n=190), did not reveal a significant difference in TTT, however, there was a significant difference (p=0.012) in TTT between D3–3/RF 2 patients (n=38) and all other UM patients (n=171). Rai risk was still the best overall prognostic factor, and was the only significant factor (p<0.0001) in the multivariable (MV) setting for TTT using the entire UM cohort. However, when we categorized patients by both Rai risk (low vs. intermediate/high) and D3–3 usage, the most pronounced differences for TTT were observed based on D3–3 usage in the low Rai risk = 0 patients. Thus, the D3–3 cohort within the Rai 0 risk group displayed a significantly shorter TTT than did the non-D3–3 Rai 0 risk cohort (p=.0006). In model-building approaches in the MV setting using both the stepwise and score statistic methods for Rai 0 patients, D3–3 usage was the only significant factor for TTT (p=0.0004) even when RF 2 usage, JH6 usage, and absolute mutation percentage were also considered. These data illustrate for the first time the clinical importance of D gene usage and D gene RF in disease progression in early stage UM B-CLL. Moreover, this study adds further support to the notion that there is selection for specific Ig receptors in B-CLL and that the antigenic specificity of certain receptors may be associated with aggressive disease. Finally, these data demonstrate that the prognostic utility of IgVH mutation testing goes beyond simply determining categorical IgVH mutation status.

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 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 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 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.