Tschumper, Renee C.

Abstract Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with >30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p<0.0001). The median percent abnormal nuclei for pts in groups 3 and 2 was 72.5% and 68.5%, respectively. Median % abnormal nuclei for group 3 was not significantly different than the other FISH groups. Treatment status was available on 147 pts, where the proportion of pts who had treatment in each group was as follows: group 1, 15/66; group 2, 2/22; group 3, 4/21; and group 4, 9/38. Due to limited sample size and heavy censoring, any analysis on time to treatment is preliminary; however, these early analyses suggest group 4 pts have a lower median time to treatment (9 yrs) compared to groups 3 and 1 (12.3 and 13 yrs, respectively). Conclusions: This study has generated new information about the 13q- anomaly in B-CLL. First, known prognostic markers for B-CLL pts with 13q-x2 are not significantly different than for pts with 13q-x1 or 13q-x1/13q-x2. Second, 13q-x1 and/or 13q-x2 occurring with other unfavorable FISH anomalies have an unfavorable prognosis; i.e. potential benefits of 13q- are trumped when it is observed with unfavorable FISH anomalies. Thus, patients with any form of 13q- alone may have indolent disease while patients with 13q- and unfavorable FISH anomalies should be considered to be in a more aggressive phase.

Abstract B cell-activating factor (BAFF) is known to regulate normal B cell development and homeostasis primarily by signaling through the high affinity receptor, BAFF-R, one of three BAFF binding receptors (BBRs). BAFF also binds two other receptors, BCMA and TACI with lesser affinity. We have recently shown that normal peripheral blood (PB) B cells express high levels of prebound soluble BAFF, which is lost upon B cell activation. Because of BAFF’s activity on normal B cells, we have been interested in the roles of BAFF and BBRs in B cell chronic lymphocytic leukemia (B-CLL). We and others have demonstrated that BAFF promotes primary CLL B cell survival and that serum BAFF levels are elevated in some patients. Although CLL B cells are known to express BBRs, a comprehensive and quantitative analysis of BBR levels and CLL B cell capacity to bind BAFF has not yet been done. We began this study by characterizing the level of soluble BAFF bound to freshly isolated CLL B cells, measured by both western blot analysis and flow cytometry. To assess receptor occupancy, cells were incubated with or without exogenous BAFF before assessing anti-BAFF reactivity and changes in median fluorescence intensity (ΔMFI; defined by dividing the MFI of the anti-BAFF antibody by the MFI of the isotype matched control antibody) were calculated. Normal B cells have higher detectable levels of bound BAFF with a ΔMFI ranging from 16 to 35 (mean=22.2). Upon addition of exogenous BAFF, the ΔMFI range increased to 27–96.6 (mean=49.1; n=8). Thus, despite evidence of prebound BAFF, clearly not all BBRs were occupied on normal PB B cells. By contrast, the levels of prebound BAFF on CLL B cells were significantly lower with a ΔMFI ranging from 1 to 13.1 (mean=2.7; n=36). Of note, 10/36 patients did not exhibit increased anti-BAFF reactivity upon incubation with exogenous BAFF (mean fold induction=0.8) whereas 26/36 patients displayed a mean fold induction of anti-BAFF reactivity of 3.5. These observations prompted us to next quantitate CLL B cell BBR expression. All patient CLL B cells expressed BAFF-R but at significantly lower levels than observed in normal B cells (p=0.0009). When CLL patients were categorized into IGHV mutated (M; n=22) and unmutated (UM; n=24), UM patients were observed to express higher levels of BAFF-R (ΔMFI =8.9) than M patients (ΔMFI =5.24). Regarding TACI, we previously demonstrated that normal memory B cells uniformly express TACI (ΔMFI =12.7; n=10) and there is a small population of activated naïve B cells that express TACI at lower levels (ΔMFI =8.3; n=10). In our CLL cohort, 14/22 M patients were TACI+ (ΔMFI =7.0) and 19/24 UM patients were TACI+ (ΔMFI =4.7). Finally, whereas normal PB B cells completely lack BCMA expression, 7/22 M and 4/22 UM patients expressed BCMA. Thus, using the BBR profile and analysis of expression levels relative to normal PB B cells, the following subgroups of B-CLL can be defined: BAFF-R+; BAFF-R/TACI+; BAFF-R/BCMA+; BAFF-R/TACI/BCMA+. It remains to be determined if these BBR profiles correlate with aspects of clinical disease. In addition, given the putative importance of BAFF in this disease, it is interesting to note that in general, CLL B cells display overall lower levels of prebound BAFF. Current studies are focused on determining whether this reflects CLL B cell activation status, increased competition for BAFF, and/or reduced levels of BBR expression.

Abstract Purine nucleoside combination therapy, with and without immunotherapy has become the standard treatment approach to CLL. Past approaches, other than stem cell transplantation, have not been successful in curing the disease. Inherent resistance of CLL precursors, impaired immune surveillance (T-cells, NK cells) and microenvironmental abnormalities may all play a role in the ability to effectively eradicate leukemia. PCR has been successfully employed for previously treated (Lamanna 2006) and previously untreated (Kay 2007) patients with CLL. In addition, CA has been added to FCR (Wierda 2006) to treat relapsed/refractory CLL disease. We designed a study to evaluate PCR followed by CA for relapsed/refractory CLL. This ECOG study includes, in addition to response criteria, molecular correlates (FISH anomalies and CpG karyotype), risk parameters analysis, including CD38, ZAP-70 and IgVH mutational status. We assessed the baseline and sequential changes in the angiogenic status of patients by measuring plasma VEGF, bFGF, thrombospondin and their immune status via T-cell repertoire. From September 2004 to September 2006, 32 patients with relapsed/refractory CLL were enrolled in the protocol. The protocol combined 2 regimens (PCR and CA). Step 1, PCR, was planned for 6 monthly cycles. Steps 2 and 3 consisted of short term CA (4 weeks) or standard CA (18 weeks) depending upon Step 1 response. There were 3 ineligible, 1 not evaluated, and 28 evaluable patients. FISH anomalies revealed that 8 patients had a homozygous or heterozygous 13q-defect, 9 had complex FISH defects (more than one FISH anomaly), 5 patients had FISH anomalies that included del(17p13.1), 4 patients had FISH anomalies that included 6q-and 3 had FISH anomalies that included 11q-. CD38 was positive in 10 patients, 11 were ZAP-70 positive and 12 of 19 patients tested had unmutated type clones. The planned 6 cycles of Step 1 (PCR) was administered to 14/32 patients (44%), the others receiving 0–5 cycles. All responses were considered partial responses (11/29; 37.9%), with 2 patients (6.9%) demonstrating progressive disease. However, patients receiving the planned 6 cycles of PCR, achieved a partial response of 71% (10/14) with 2 cases of tumor lysis syndrome. Toxicity (Step 1-PCR) was predominately myelosuppressive (Grade 1–4). A case of chronic idiopathic polyneuropathy and a case of “paraneoplastic pemphigus” were reported. There were 4 deaths. CMV reactivation was diagnosed in 2 patients, neither of which was fatal. During Step 2 (CA), short-term administration in one patient led to grade 3 skin toxicity, which limited administration. During long-term administration of CA (PR, PD, SD) (n=7), infectious complications occurred in 6 patients all of whom developed grade 4 lymphopenia. The median survival for the entire group was 15 months. In conclusion, a significant number of heavily pre-treated and poor prognosis patients with CLL will respond to PCR. Further analysis will determine whether a significant number of patients will achieve a higher level of response and longer survival after CA administration. Studies of association of outcome with the risk parameters are ongoing and will be presented with the clinical response data.

Abstract Abstract 1814 Multiple myeloma (MM) is an incurable hematologic disorder characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Like normal B cells and PCs, MM cells express a distinctive immunoglobulin (Ig) molecular fingerprint resulting from heavy and light chain gene rearrangements and unique mutations introduced by the somatic hypermutation (SHM) process. Detailed examination of the Ig molecule may offer insight into the etiology of B cell malignancies as well as the ontogeny of malignant transformation and ongoing genetic evolution. Because prior studies of the Ig fingerprint of MM cells have been limited in size and scope, our current understanding of MM Ig repertoire is minimal. We used massively parallel sequencing of normal and malignant PCs to interrogate: 1) DNA versus RNA (cDNA) as starting material for studies of Ig repertoire; 2) the Ig repertoire of normal and malignant PCs; and 3) intraclonal heterogeneity in MM. Bone marrow PCs (BMPCs) from an untreated MM patient and from a normal control subject were isolated by magnetic bead separation. DNA and RNA were isolated from purified cells and amplified in a multiplex PCR reaction with primers designed to capture the entire IGHV region including the heavy chain complementarity determining region 3 (HCDR3) necessary for defining clonal cells. Using Roche 454 GS-FLX Titanium chemistry and a GS FLX Titanium Genome Sequencer, over 30,000 sequence reads were obtained from each sample. Following a detailed algorithm to minimize Taq and sequencing related errors and removal of non-Ig or non-productive Ig sequences, the resulting sequences were grouped based on HCDR3 amino acid identity. Each unique HCDR3 represented one member of the repertoire and all the sequences with the same HCDR3 defined the clone size. This measurement showed that there was nearly a five-fold higher level of unique HCDR3s in normal BMPCs when the starting PCR template was DNA vs. cDNA. Thus, DNA is clearly a better starting template for repertoire analysis using deep sequencing methods. Thorough analysis of the MM DNA sample identified PCs expressing 37 different IGHV genes, and predictably, the vast majority of sequence reads (83%) had exact nucleotide identity in the IGHV and HCDR3 region (IGHV3-74 with 18 mutations in the IGHV region) thereby defining the dominant MM clone. There were also ∼5000 additional sequences that either shared an identical HCDR3 region but varied within the IGHV3-74 gene sequence (n=3975) or were identical within the IGHV3-74 sequence yet varied within the HCDR3 (n=862). These sequences were further analyzed, with sequences not present 10 or more times or not found in both the 5' and 3' directions eliminated from further scrutiny. This process revealed a total of 64 putative subclones with single point mutations in the IGHV or HCDR3 region (10–56 copies of each unique sequence representing a total of 1583 clonally related sequences). When we used a more stringent subclone frequency of ≥0.1%, 22 subclones remained. Of note, conventional Ig repertoire analysis would require sequencing ≥1000 colonies to detect MM subclones present at this frequency. Finally, 11 subclones displayed a significant number of IGHV gene nucleotide differences from the dominant MM clone despite exhibiting an identical HCDR3 region. Notably, a number of these distinguishing IGHV gene mutations are silent and thus may preserve the antigenic specificity of these clonally related cells. These subclones failed our frequency criteria but the type and quality of deviations from the clonal sequence suggest that they are not artifact and warrant further investigation. Notably, all of the putative subclones were also found in the MM cDNA cohort providing further evidence as genuine subclones. The very large number of identical MM clonal sequences is consistent with the idea that MM is a post germinal center malignancy that can no longer undergo SHM. However, we present clear evidence of MM-related subclones albeit relatively small in number. The subclone sequence deviations discovered may reflect on-going genetic evolution independent of the formal SHM mechanism. Alternatively, these subclones may indeed reflect “sister” cells generated during the original germinal center reaction that resulted in the malignant clone. In summary, our studies demonstrate the extraordinary potential of this methodology to track clonal evolution in MM and its precursor conditions over time. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: 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. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

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.