Timm, Michael

Abstract Background The presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. Methods We evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. Results There were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs. We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. Conclusion The utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Abstract Abstract 4995 Background: Multiple myeloma results from an accumulation of monoclonal nonproliferating plasma cells arising from a small subpopulation of proliferating myeloma cells. In an effort to optimize detection of the human myeloma cell growth fraction and obviate the need for slide review, a novel FC strategy was developed combining elements of light chain restriction, surface antigen expression, and ploidy analysis. Methods: Bone marrow cells from 41 patients with plasma cell proliferative disorders were lysed with ACK and resuspended in 3% BSA. Cells were stained using a 6-color assay with anti-CD45, anti-CD38, anti-CD138, and anti-CD19. Cells were washed and 100 ul Caltag solution A was added for 15 min. Cells were washed and 100 ul of Caltag solution B, anti-kappa and anti-lambda were added for 10 min. Cells were washed and 100 ul PBS and 3 ul RNAse are added for 15 min. Cells were washed and 400 ul of a 1:1000 solution of 3uM DAPI in Tris 0.1% NP-40 was added. Cells were incubated at 4°C for 45 minutes before running on a FACSCanto instrument for ploidy determination. All patients were analyzed both by flow cytometry and the slide based plasma cell labeling index. Results: Forty-one patients were studied; 34 demonstrated a proliferative fraction and 7 had too few plasma cells for analysis after therapy. Of the 34 patients, 6 had MGUS/SMM, 5 newly diagnosed MM, 7 amyloid, and 16 were treated MM. The mean percent proliferating cells were 1.1% (range 0 – 8.6%) with PCLI and 1.4% (range 0.1 – 12.7%) by flow. The correlation between PCLI and flow gave a RSquare value of 0.54. Twelve patients with a PCLI of 0% had a flow proliferation between 0.1 – 1.4% (mean 0.49%). Treated patients received lenalidomide, dexamethasone, bortezomib, and/or autologous transplantation. All 16 treated myeloma patients with adequate plasma cells had a flow proliferation between 0.2 – 12.7% (mean 2.2%). Conclusion: Flow cytometry offers a useful way to detect the proliferative myeloma component at diagnosis and after treatment. The continued presence of proliferating myeloma cells after treatment may explain why most patients relapse and offers another important marker to monitor and cell population to target in patients with active disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract Background Prior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. Methods We evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results There were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). Conclusion Quantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Abstract Malignant B-cells characteristically home to the bone marrow and lymph nodes. However, the mechanisms by which cells are recruited into and mobilized from the bone marrow/lymph nodes into the peripheral blood are not well understood. Chemokines such as SDF-1 and it receptor CXCR4 play a central role for lymphocyte trafficking and homing. Downstream activation of the PI3K pathway by chemokines has been implicated in the migration of many cell types. We recently demonstrated that CXCR4 expression correlates significantly with disease progression in CLL. Patients with Rai stage 4 had a higher expression of CXCR4 when compared to patients with Rai stage 0. The expression of CXCR4 was not found on CLL B cells resident within the lymph nodes implying that there may have been downregulation of CXCR4. In this study, we explore the molecular mechanisms involved in homing and migration of B-CLL cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of CLL cells in response to SDF-1. Boyden chamber in vitro migration assays were used with primary CLL peripheral blood cells and the MEK1 CLL cell line. In addition, we expressed fluorescent YFP-CXCR4 in the cells and used confocal microscopy to visualize changes in the subcellular location of the fluorescent CXCR4 before and after SDF-1 stimulation. Inhibitors of the PI3K pathway such as the PI3K inhibitor LY294002 (10uM, one hour pretreatment) and the mTOR inhibitor, rapamycin (200nM overnight) were used. SDF-1 induced a dose dependent migration of primary CLL cells and MEK1 cells, indicating functional CXCR4 receptors. MEK 1 cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping and internalization subcellularly. Pretreatment of the cells with PI3K pathway inhibitors led to inhibition of migration with LY294002 but not with rapamycin. Confocal microscopy demonstrated abrogation of subcellular localization in the presence of LY294002 but not with rapamycin with the addition of SDF-1. These results demonstrate that CLL cells express functional CXCR4 receptora and that the PI3K pathway is essential for SDF-1 dependent CLL migration. In addition, we demonstrate that the CXCR4 receptor is internalized in response to SDF-1 stimulation indicating that CLL cells downregulate the CXCR4 receptor once they home to the bone marrow or lymph nodes where there is abundant SDF-1 in the microenvironment. This leads to the confinement of the cells in the microenvironment preventing further migration of the cells outside of the bone marrow/lymph nodes. Supported in part by CA97274

Abstract Malignant plasma cells characteristically home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and mobilized from the bone marrow into the peripheral blood (PB) are not well understood. In this study, we explore the molecular mechanisms involved in homing and migration of plasma cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of MM cells in response to SDF-1. CXCR4 surface expression was determined by FACS analysis (PE CXCR4, Pharmingen) using samples from bone marrow and peripheral blood of patients diagnosed with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and primary systemic amyloidosis (AL). All results were expressed as percent expression in gated CD38+ and CD45+ cells. Boyden chamber in vitro migration assays were performed using Kas6/1MM cells. In addition, live confocal microscopy was used to visualize changes in the subcellular location of YFP-fluorescent CXCR4 before and after SDF-1 stimulation. Cells were pretreated with 10mM LY294002 or 200nM rapamycin to assess the effect of inhibition of PI3K or mTOR on CXCR4 subcellular localization. Immunoblotting was performed to confirm inhibition of the PI3K pathway. Comparisons between groups was performed using Mann-Whitney testing. The median CXCR4 expression is described in table 1. There was a significant difference between expression of BM CXCR4 in MM and MGUS (p=0.02). SDF-1 induced dose dependent migration of Kas 6/1 cells indicating a functional CXCR4 receptor. MM cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping, internalization subcellularly, and production of pseudopodia in response to SDF-1. This process was abrogated with pretreatment with LY294002 and rapamycin. These data demonstrate for the first time that the surface expression of CXCR4 is markedly elevated in the peripheral blood as compared to the bone marrow. Once in the bone marrow, and with the presence of excess SDF-1, the receptor becomes internalized and downregulated. In contrast to MM, CXCR4 expression on plasma cells in the bone marrow of normal and MGUS patients was higher. In addition, we demonstrate that the process of CXCR4 subcellular localization is abrogated by the administration of PI3K inhibitors LY294002 and rapamycin. These data suggest that CXCR4 expression is required for MM cells to circulate, and that downregulation occurs in the BM leading to immobilization of the cells. Future clinical trials using CXCR4 inhibitors, or inhibitors of PI3K and mTOR to prevent the homing and migration of MM cells into the bone marrow may be explored. This was supported in part by CAP50 CA97274. CXCR4 expression N=45 Median CXCR4% expression of total plasma cells Range MM BM (n=23) 27 4.3–90.1 MM PB (n=5) 91 60–95.5 MGUS BM (n=6) 65 41–78.6 AL BM (n=7) 46 17.5–71.6 Normal BM (n=4) 59 6.3–69.5 Kas6/1 cell line 100 -

Abstract Background: Epigallocatechin-3-gallate (EGCG) is a polyphenol extracted from green tea that has been studied for cancer prevention and treatment. Green tea and its extracts have been an integral part of traditional medicines from Asia and are thought to have considerable medicinal value. EGCG has been studied in many tumor types and in vitro studies have been encouraging. It is currently in Phase I/II clinical trial in the treatment of chronic lymphocytic leukemia. Here we examined the activity of EGCG in the setting of myeloma and its potential mechanisms of action. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of EGCG was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Fresh myeloma cells were obtained from bone marrow aspirates and subjected to ACK lysis to remove red cells. These cells were cultured in RPMI1640 with or with out EGCG and apoptotic rates were determined by Apo 2.7 expression on gated plasma cells. Flow cytometry was used for determining the phosphorylation state of various signaling proteins. Anti-angiogenic activity of EGCG was measured using a human in vitro angiogenesis kit (Angiokit). Results: EGCG treatment of myeloma cell lines resulted in dose- and time-dependent cytotoxicity to the cells. EGCG was cytotoxic to several myeloma cell lines, including IL-6 dependent cell lines KAS-6/1 and ANBL-6 as well as drug resistant cell lines MM1.R, Dox40 and LR5. The effect was maintained when the cells were grown in co-culture with marrow stromal cells, indicating the ability to overcome microenvironment mediated resistance. EGCG induced apoptosis of myeloma cell lines, apoptotic cells being measured as Annexin (+)/PI (−) cells by flow cytometry. It induced a dose-dependent apoptosis of freshly isolated myeloma cells with increased effect seen on the CD45+ myeloma cells compared to CD45- cells, reflecting increased activity on the proliferative fraction. Down regulation of Akt phosphorylation was seen following treatment of myeloma cells with EGCG. Using the Angiokit, we were able to demonstrate inhibition of tubule formation by EGCG at concentrations lower than those cytotoxic to myeloma cells. It was also able to overcome the angiogenic activity of VEGF. Conclusion: We demonstrate potent anti-myeloma activity of EGCG in vitro. In vivo studies in animal models of myeloma are ongoing. We demonstrate, as in other cell lines, inhibition of Akt pathways as a potential mechanism of action of EGCG. Ongoing studies are exploring other mechanisms. Most importantly, we demonstrate anti-angiogenic activity of this compound in the setting of myeloma, a finding in concordance with the inhibition of VEGF activity seen with other tumor systems.

Abstract Background: The clonal plasma cells in multiple myeloma have relatively low rates of apoptosis. We have previously shown that the apoptotic rates of the plasma cells have prognostic value in these patients. However, several methods are used both for targeting the myeloma cells for study as well as for detection of apoptosis in these cells. It is not clear which method is optimal for studying apoptotic rates of plasma cells from primary patient samples. We examined two different ways of studying the plasma cells, namely CD138 magnetic bead based positive selection of myeloma cells (sorted) and gating on the plasma cells in whole bone marrow (unsorted) using CD38/CD45 expression patterns. Additionally, we compared two different methods of identifying apoptotic plasma cells. Methods: Paired samples processed by two different methods were studied. The bone marrow aspirate samples were initially processed using ACK lysis to remove the red cells. The samples were then studied using flow cytometry (unsorted sample) or subjected to a second step of magnetic bead based separation of myeloma cells based on CD138 expression (sorted sample). Each of the samples was studied using Annexin/PI and Apo 2.7 expression to identify the plasma cells undergoing apoptosis. Plasma cells were identified in the unsorted samples by virtue of their CD38/CD45 expression. Samples were studied at collection and after 48 hours of culture. Results: At collection, the median percentages of cells considered apoptotic by annexin/PI (Annexin pos, PI neg) were higher than that detected by the Apo 2.7 method (Apo 2.7 + cells), for both sorted (6.5% vs. 1.5%) and unsorted cells (26% vs. 10%). However, after 48 hours of culture the percentage of apoptotic cells detected using the Apo 2.7 method were higher than that by Annexin/PI for both sorted (22.5% vs. 14.5%) and unsorted cells (43.5% vs. 10%). When the 48 hr study was compared with the initial study, percent apoptotic cells detected by Apo 2.7 had increased at 48 hrs for both sorted and unsorted cells. However, the percentage of apoptotic cells by annexin/PI had decreased in the unsorted cells at 48 hrs compared to initial study. The sorted samples had an overall increase in apoptosis at 48 hours as measured by annexin/PI. We then looked at the percentage of apoptotic cells detected by either method on initial study and compared the sorted cells and unsorted cells. Using either method, the percentage of apoptotic cells was significantly less (P=0.01; paired t-test) among the sorted cells compared to the unsorted cells. Conclusion: Previous studies have shown that CD138 expression is lost from PCs as they enter apoptosis. Here we show in paired samples that the percentage of apoptotic myeloma cells significantly decreases following CD138 sorting. CD138 sorting should not be used when studying apoptosis of myeloma cells in patient samples. Use of unsorted cells also allows for examination of the effects of interventions on other cell populations as well. When serial studies of apoptosis are done with unsorted samples, we recommend using Apo 2.7 rather than Ann/PI, since the percentage of apoptotic cells by Ann/PI expression actually appear to decrease with time in culture, thus making it difficult to assess effects of intervention such as in drug treatment studies. We hypothesize that this is due to an artificially high level of Annexin positivity at the initial study that might be brought on by non-specific damage of the membranes secondary to cell processing.

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

Abstract Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The isolation of plasma cells have generally been performed by positive selection using plasma cell markers such as CD138 or by negative selection by removing other marrow cells using a cocktail of antibodies. We have previously demonstrated differences in apoptotic rates in plasma cells with and without this enrichment step. Here we have examined the effect of CD138 magnetic bead selection on the surface phenotype of plasma cells by flow cytometry. Methods: Bone marrow aspirates from patients with myeloma (n=12) were first washed, and then lysed with ACK to eliminate red cells. The samples were split with a portion of the cells further processed for CD138 selection. ACK lysed only whole bone marrow (WBM) and sorted cells were stained with CD38, CD45, CD56, activation markers CD71 and CD69, adhesion markers CD49d, CD11b, and CD66, B cell markers CD19 and CD20, and clonality (kappa and lambda). Gates were drawn around the plasma cells and plasma cell subsets on the basis of CD38/45 expression for both sorted and unsorted samples. In addition, an aliquot of the sorted preparation was examined by immunohistochemistry to calculate the purity of the sorted sample. Results: Significant differences were observed in terms of the percentage of plasma cells expressing the different antigens when the cells were selected using CD138. This difference included a greater than 10% difference in expression between the two preparations as well as a change from positive to negative in several cases. There was a substantial loss in the expression of CD20, CD71 and CD11b on plasma cells following CD138 based sorting (Table). Among the other markers, CD49d remains unchanged and changes are variable for the other markers. In addition, in 8 of the 12 cases, there was a nearly complete loss of the CD45 positive subset with a loss of discrimination between CD45 negative and CD45 positive plasma cell subsets in the remaining CD138 sorted preparations (figure: unsorted left, sorted right). Conclusion: In conclusion, the process of CD138 sorting of plasma cells appears to change important markers on the plasma cells and may even eliminate a key subset from further analysis. This should be kept in mind when isolating plasma cells using CD138 positive selection for analysis such as gene expression profiling. Consideration should be given to negative selection using antibodies against other cell types to deplete them. Marker Unsorted % Sorted % CD56 74 67 CD69 11 2 CD71 23 8 CD49d 95 92 CD11b 33 4 CD66 5 1 CD20 16 6 Figure Figure

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure