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Background Alemtuzumab is a monoclonal antibody used in the treatment of CD52-expressing B-cell malignancies, including Waldenstrom's macroglobulinemia (WM). Recent studies demonstrate high levels of alemtuzumab activity in relapsed/refractory disease. One potential target of alemtuzumab is bone marrow mast cells (BMMCs), which provide growth and survival signaling for WM lymphoplasmacytic cells. Patients and Methods We therefore examined BMMCs (FcɛRI + , CD117 + ) from WM and other mast cell (MC) disorders for expression of CD52. Results We identified cell surface antigen expression by multicolor flow cytometric analysis and found CD52 expressed on human mast-derived cell line–1 (HMC-1) and LAD2 MC lines, on BMMC from 13 of 15 patients with WM, and on BMMCs from 4 of 4 patients with systemic mastocytosis (SM). None of 4 healthy donors expressed CD52. Reverse-transcriptase polymerase chain reaction analysis confirmed CD52 expression in the HMC-1 and LAD2 MC lines, in BMMCs from 14 of 15 patients with WM, and 3 of 3 patients with SM. CD52 transcripts were also detected in BMMCs from 6 of 6 healthy donors, despite the absence of CD52 cell surface expression. Importantly, we observed high levels of alemtuzumab-mediated, antibody-dependent, cell-mediated cytotoxicity against LAD2 MCs and BMMCs from patients with WM and SM. Conclusion These studies demonstrate that CD52 is widely expressed on human MCs and WM bone marrow lymphoplasmacytic cells and provide the preclinical rationale for the use of alemtuzumab in the treatment of WM and possibly other MC-related disorders.

Familial clustering of B-cell disorders among Waldenström's macroglobulinemia (WM) patients has been reported, though the frequency and any differences in disease manifestation for familial patients remain to be defined.We therefore analyzed clinicopathological data from 257 consecutive and unrelated WM patients. Forty-eight (18.7%) patients had at least one first-degree relative with either WM (n = 13, 5.1%), or another B-cell disorder including non-Hodgkin's lymphoma (n = 9, 3.5%), myeloma (n = 8, 3.1%), chronic lymphocytic leukemia (n = 7, 2.7%), monoclonal gammopathy of unknown significance (n = 5, 1.9%), acute lymphocytic leukemia (n = 3, 1.2%) and Hodgkin's disease (n = 3, 1.2%). Patients with a familial history of WM or a plasma cell disorder (PCD) were diagnosed at a younger age and with greater bone marrow involvement.Deletions in 6q represented the only recurrent structural chromosomal abnormality and were found in 13% of patients, all non-familial cases. Interphase FISH analysis demonstrated deletions in 6q21-22.1 in nearly half of patients, irrespective of familial background.The above results suggest a high degree of clustering for B-cell disorders among first-degree relatives of patients with WM, along with distinct clinical features at presentation based on familial disease cluster patterns. Genomic studies to delineate genetic predisposition to WM are underway.

The preclinical evaluation of investigational agents for Waldenström macroglobulinemia (WM) has been limited by the lack of in vivo models that enable the use of explanted patient cells. We describe here a novel in vivo model of human WM in severe combined immunodeficient (SCID) mice implanted with human fetal bone chips (SCID-hu mice) into which WM cells from patient bone marrow are engrafted directly into the human bone marrow (huBM) microenvironment. WM cells in SCID-hu mice produced human monoclonal paraprotein (immunoglobulin M [IgM] and/or κ or λ chain) detectable in mice sera. Immunohistochemical analysis of human bone retrieved from SCID-hu mice showed infiltration with CD20+, IgM+, and monotypic light chain+ lymphoplasmacytic cells. Mast cells were observed to be associated with the infiltrate in these sections. Treatment of SCID-hu mice bearing WM with rituximab induced tumor regression, associated with a decrease in serum paraprotein. This model, therefore, recapitulates the in vivo biology of WM and allows the study of novel investigational drugs targeting WM cells in the huBM milieu. (Blood. 2005;106:1341-1345)

We report two patients with severe thrombocytopenia and life-threatening bleeding that were successfully managed with recombinant activated factor VII (rFVIIa). The first was a 75-year-old male with Waldenström's macroglobulinemia. During a therapeutic course with fludarabine, he developed severe autoimmune thrombocytopenia resistant to conventional treatment, followed by persistent uncontrollable nasal bleeding. Platelet transfusions failed to increase the platelet count and control the hemorrhage. When hemoglobin levels fell below 8.5 g/dL and the patient's clinical condition got much worse, a single dose of 4.8 mg rFVIIa (90 microg/kg) was given as an i.v. bolus. Ten minutes after the rFVIIa injection, nasal bleeding stopped, the patient's clinical condition progressively improved, and splenectomy could be carried out uneventfully 2 days later. The second patient, a 52-year-old female, was under treatment for pre-B lymphoblastic leukemia. She developed severe thrombocytopenia, secondary to chemotherapy, complicated by massive gastrointestinal bleeding. Despite intensive treatment with platelet transfusions, hemorrhage continued and her condition deteriorated rapidly. She was then given an i.v. bolus injection of 4.8 mg rFVIIa, which resulted in cessation of hemorrhage and dramatic improvement of her clinical status. No adverse effects from the treatment with rFVIIa were observed. In conclusion, rFVIIa appears to be an attractive alternative for controlling hemorrhage in patients with severe thrombocytopenia, especially when platelet transfusions are unavailable or ineffective.

Aim : Primary lymphomas of endocrine glands are extremely rare. Our study adds more data to the few published series regarding the incidence, clinical characteristics, management and overall survival (OS) by comparing the various diffuse large B-cell endocrine lymphomas. Moreover, it contributes to a better understanding of these neoplasms and provides concepts for future research. Methods : We retrospectively evaluated the clinical profile and the patterns of outcome among patients who were treated in our center with the diagnosis of aggressive, B-cell, primary endocrine lymphoma. Results : Between May 1980 and December 2011, 450 patients were diagnosed as primary extranodal non-Hodgkin lymphomas. Among them, 18 cases (4%) were primary testicular lymphoma (PTL), 8 cases (2%) were primary thyroid lymphoma (PTHL) and 4 cases (1%) were primary adrenal lymphoma (PAL). The therapeutic approaches employed were variable, including mainly chemotherapy in combination with radiotherapy and surgery. The median OS for the patients with PTL and PAL was 27 and 6 months, respectively. Better outcome was observed in patients with PTHL for whom the median OS has not been reached yet, whereas the PAL group had the worst prognosis. Conclusions : The discrepancies in the outcome among endocrine lymphomas could be partly attributed to their biologic variability, which might be determined by the initial site involved. We conclude that treatment decisions should be made according to a multi-disciplinary approach to avoid unnecessary surgery. Existing treatment strategies for PTL and PAL fail to provide long-term survival, rendering the application of novel therapeutic approaches essential.

Abstract 2909 Unlike the established role of acquired genetic events and bone marrow microenviroment in the pathogenesis of multiple myeloma (MM), the phenotype and function of cells enriched in tumor-propagating activity and their relationship to the phenotypic architecture in MM are controversial. Because the immunoglobulin heavy (IgH) chain class-switched myeloma plasma cell (PC) is the final event of a linear, B cell lineage developmental process, it was suggested that myeloma cell growth is sustained by a minority of cells more immature than the PC. We combined multicolour flow-cytometry and sorting with patient-specific quantitative PCR (qPCR) to dissect the myeloma clonal organisation in the bone marrow (BM) and peripheral blood (PB). In a cohort of 30 patients we show that MM comprises at least four hierarchically organised, clonally-related sub-populations which, although phenotypically distinct, share the same oncogenic chromosomal abnormalities as well as IgH chain complementarity region 3 area sequence. We found rare CD19+ clonotypic cells with phenotype either of resting memory cell (CD19+CD38-IgD-CD27+/−) or plasmablasts (CD19+CD38++CD319+CD138-) and CD19-CD38hiCD319+CD200+CD56+ clonotypic cells, comprising CD138- (∼3% of the clone, termed Pre-PC), CD138low and CD138+ PC. The clonal populations resemble their normal counterparts and exist in nearly logarithmic incremental frequencies in the BM. Using dynamic mathematical models and a Bayesian approach a Pre-PC->PC transition was predicted, outside the linear developmental process. Following intravenous injection of purified clonotypic populations into sub-lethally irradiated NSG mice, we found that both PC (in 9/12 of the injected mice, 75%) and Pre-PC (4/16, 25%) can engraft, but not the CD19+ clonotypic cells (0/10). Of note, upon engraftment both PC and Pre-PC regenerate the original CD19- hierarchy of the human BM, as predicted by mathematical modelling. In addition, Pre-PC are more quiescent and unlike PC, preferentially localize at extramedullary niches, such as the liver and spleen of the engrafted animals. Therefore, the myeloma-propagating activity is the exclusive property of a population characterized by its ability for bi-directional transition between the dominant PC and the low frequency Pre-PC. To gain insights into the molecular mechanisms underpinning this reversible phenotypic transition we used gene expression profiling of highly purified BM Pre-PC and PC (n=9). Differential expression analysis and principal component analysis clearly separate Pre-PC from PC, with 7 of 9 samples following the same expression pattern in hierarchical clustering. Functional annotation analysis using DAVID shows that Pre-PC are enriched in epigenetic regulators, including histone methyl-transferases (belonging to the Polycomb repressive complex 2 or Trithorax MLL activating complex) and de-methylases, histone acetyl-transferases and de-acetylases as well as several members of SWI/SNF chromatin remodeling complex, suggesting that epigenetic plasticity underpins the phenotypic diversification of myeloma-propagating cells. Finally, to study the clinical importance of the myeloma clonal organisation, with emphasis to clinical drug resistance, we prospectively assessed the size of the different phenotypes in paired, pre- and post-treatment BM samples (n=8). We show that in all cases a higher proportion of Pre-PC than PC persisted after treatment suggesting that Pre-PC are clinically more drug-resistant than PC (median 10.3-fold, range 4.4–332, p=0.008). Thus, clinical drug resistance in MM is linked to reversible, bi-directional phenotypic transition of myeloma-propagating cells, likely under the orchestration of epigenetic regulators. These novel biological insights have important clinical implications in relation to assessment of minimal residual disease and development of alternative therapeutic strategies in MM. Disclosures: No relevant conflicts of interest to declare.

Waldenström's Macroglobulinemia (WM) is a low grade lymphoma with a prolonged course and a median survival exceeding 7 years. However, there are patients who die of WM early during the course of the disease while a significant proportion of WM patients can survive ≥10 years. The characteristics of these two groups of patients may differ and their identification may augment the choice of risk adapted treatment strategies. The aim of our study was to identify and characterize patients with short survival due to WM as well as those with survival exceeding 10 years, and to compare their characteristics in order to evaluate clinical factors associated with poor or with good outcomes, based on data from a large database with long follow up. The analysis included 492 patients that have been entered in the prospectively maintained database of the Greek Myeloma Study Group, who fulfill the criteria form symptomatic WM requiring therapy. The median follow up of all the patients in the database is 10 years. For the first part of the analysis we included 292 patients who have at least 10 years of follow up (thus, they started therapy at least 10 years ago, before 2006). Among them, we identified 101 (34.5%) patients who survived ≥10 years, and 13% who died due to WM within As a validation of the previous results, we evaluated the presence of the above clinical characteristics in patients with survival 65 in 63%, hgb4 mg/L were the two most important predictors of early WM-related death. When age >65 years was also included as a predictor, patients with 0, 1, 2 or 3 of the above factors had 3-year WM-related death rate of 3%, 4%, 16% and 25% (p In conclusion, 34.5% of patients with WM survive ≥10 years, but 10%-13% die of WM within < 3 years from initiation of treatment. These patients with high risk disease are older with higher tumor bulk and increased LDH. However, by applying only the presence of serum albumin < 4 gr/dl, b2 microglobulin ≥4 mg/L and age >65 years we can identify patients at very low risk of early death as well as patients with significant risk of early death and short survival. This simple staging system may also outperform IPSSWM. Figure 1 Figure 1. Disclosures Kastritis: Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Kyrtsonis:Genesis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Katodritou:Genesis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Delimpasi:Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

BACKGROUND - AIM Ruxolitinib can be effectively used for the treatment of splenomegaly or constitutional symptoms in patients with myelofibrosis. Its main side effect is myelosuppression which leads to dose modifications. Real-life data from the use of ruxolitinib in every-day practice were analysed to assess efficacy, safety, dose modifications or reasons for treatment discontinuation. PATIENTS - METHODS A retrospective observational study was conducted in patients with primary or secondary myelofibrosis, treated in 13 sites. Statistical analysis was done with non-parametric tests using SPSSv17 software. A total of 116 patients, diagnosed between 1993 and 2016 were included in the analysis. The male:female ratio was 2:1, median age at diagnosis was 63 years (range 27 to 82) and median duration of ruxolitinib therapy was 15.3 months. The study expands over 664.2 patient-years of observation, including 176.7 patient-years while on ruxolitinib. RESULTS The diagnosis, revisited according to the WHO 2008 criteria, was primary myelofibrosis in 56%, post-essential thrombocythemia myelofibrosis in 22% and post-polycythemia vera myelofibrosis in 22% of patients. At the beginning of ruxolitinib treatment, the international prognostic scoring system (IPSS) risk category was low (6.2%), intermediate-1 (26.5%), intermediate-2 (39.8%), high (23%). Constitutional symptoms were present in 48% of patients at diagnosis and in 73% of them at the onset of ruxolitinib therapy. Median spleen size was 5cm (centimeters) below costal margin at diagnosis and 12cm at onset of ruxolitinib. Ruxolitinib starting dose was 5mg (16%), 10mg (18%), 15mg (31%), 20mg (35%) twice daily (BID), while the final dose was Ruxolitinib was permanently discontinued in 29% of patients, due to progressive disease in 6.4%, transformation to acute leukemia in 2.7% and death in 8.2% of patients. Side effects (including anemia, thrombocytopenia, investigator decision, patient preference) was the reason for treatment discontinuation in 5.5% and lack of response in 2.7% of patients. Efficacy (reduction in constitutional symptoms and spleen size) and hematological toxicity (values of platelets, white blood cell counts, hemoglobin) of ruxolitinib was assessed after 1, 6 and 12 months of treatment (table 1). Thrombocytopenia (PLT Constitutional symptoms after 1 month were significantly less frequent with increased doses of ruxolitinib, whereas there was no statistically significant difference among the various doses at 6 and 12 months of treatment. Platelet counts were significantly lower in patients taking 5mg BID at 6 months of treatment (p=0.02), and they also tended to be lower in 1 and 12 months (p=0.07, p=0.08 respectively). White blood cells and hemoglobin values did not differ significantly among the various doses of ruxolitinib in either time point. Table 1. Results at 1, 6, 12 months of treatment, according to different doses. CONCLUSIONS Ruxolitinib can effectively improve splenomegaly and alleviate constitutional symptoms in patients with myelofibrosis. The symptoms are relieved slower with the low dosage, whereas the main side effect is thrombocytopenia. It can however be maintained within safety limits (grade < 3), with dose modifications without hampering efficacy. Table Table. Disclosures No relevant conflicts of interest to declare.

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p Disclosures Terpos: Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), a new marker reflecting osteoclast activity, and osteoprotegerin (OPG) were measured in 121 patients with multiple myeloma (MM) at diagnosis, and in 63 of them during pamidronate administration, to define their correlation with the extent of bone disease and disease activity in MM. Radiographic evaluation of the skeleton, measurement of other markers of bone remodelling, including N-terminal cross-linking telopeptide of type-I collagen (NTX), bone alkaline phosphatase and osteocalcin and of markers of disease activity (beta2-microglobulin, paraprotein, interleukin-6 (IL-6), were also performed. Levels of TRACP-5b were increased (p

Backgound: The assessment of disease burden is often difficult in patients with Waldenstrom’s macroglobulinemia (WM) who receive rituximab due to the induction of an IgM flare, and following the removal of serum IgM by plasmapheresis. Soluble CD27 (sCD27) is a tumor necrosis factor family member secreted by WM cells which is strongly correlated with serum IgM levels and clinical responses in patients with WM. As such, we attempted to delineate its potential role in WM patients experiencing a rituximab-induced IgM flare and following plasmapheresis. Patients and Methods: sCD27 levels were serially measured by serum-based ELISA in 8 patients who ultimately demonstrated a response to therapy, and in whom a rituximab-mediated IgM flare was observed, as well as in 3 WM patients undergoing plasmapheresis. Results: Among the 8 patients who experienced a rituximab-mediated IgM flare, IgM levels rose from 3515 to a peak of 5270 mg/dL (P = .008), while sCD27 levels decreased from 174.1 to 155.9 U/mL (P = .012), with a decline observed in all patients. Among 3 patients undergoing plasmapheresis, IgM levels declined from a median of 6940 to 4770 mg/dL (P = .031), while median sCD27 levels remained without significant change (P = .317). Conclusion: sCD27 is a faithful marker of disease burden and is unaffected by the rituximab-induced IgM flare, as well as plasmapheresis in WM. The use of this marker may aid in correctly predicting clinical outcome in patients undergoing treatment with rituximab and/or plasmapheresis in WM.

Purpose Nucleoside analogs (NAs) are considered as appropriate agents in the treatment of Waldenström macroglobulinemia (WM), a lymphoplasmacytic lymphoma. Sporadic reports on increased incidence of transformation to high-grade non-Hodgkin's lymphoma and development of therapy-related myelodysplasia/acute leukemia (t-MDS/AML) among patients with WM treated with NAs prompted us to examine the incidence of such events in a large population of patients with WM. Patients and Methods We examined the incidence of these events in 439 patients with WM, 193 and 136 of whom were previously treated with and without an NA, respectively, and 110 of whom had similar long-term follow-up without treatment. The median follow-up for all patients was 5 years. Results Overall, 12 patients (6.2%) either developed transformation (n = 9; 4.7%) or developed t-MDS/AML (n = 3; 1.6%) among NA-treated patients, compared with one patient (0.4%) who developed transformation in the non-NA treated group (P < .001); no such events occurred among untreated patients. Transformation and t-MDS/AML occurred at a median of 5 years from onset of NA therapy. The median survival of NA-treated patients who developed transformation did not differ from other NA-treated patients as a result of effective salvage treatment used for transformed disease. However, all NA-treated patients who developed t-MDS/AML died at a median of 5 months. Conclusion These data demonstrate an increased incidence of disease transformation to high-grade NHL and the development of t-MDS/AML among patients with WM treated with NAs.

Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P < .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency–human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoma characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic cells. The induction of unfolded protein response (UPR) genes (“physiologic” UPR) enables cells to differentiate into professional secretory cells capable of production of high amounts of endoplasmic reticulum (ER)–processed proteins, such as immunoglobulins. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected, called proapoptotic/terminal UPR. We show that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 μg/mL to 1 μg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrate that ER stress inducer synergizes with other agents used in the treatment of WM. These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

In the past years, new developments have occurred both in the understanding of the biology of Waldenstrom Macroglobulinemia (WM) and in therapeutic options for WM. WM is a B-cell disorder characterized primarily by bone marrow infiltration with lymphoplasmacytic cells, along with demonstration of an IgM monoclonal gammopathy. Despite advances in therapy, WM remains incurable, with 5-6 years median overall survival of patients in symptomatic WM. Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. The main therapeutic options include alkylating agents, nucleoside analogues, and rituximab. Studies involving combination chemotherapy are ongoing, and preliminary results are encouraging. No specific agent or regimen has been shown to be superior to another for treatment of WM. As such, novel therapeutic agents are needed for the treatment of WM. In ongoing efforts, we and others have sought to exploit advances made in the understanding of the biology of WM so as to better target therapeutics for this malignancy. These efforts have led to the development of several novel agents including the proteasome inhibitor bortezomib, and several Akt/mTor inhibitors, perifosine and Rad001, and immunomodulatory agents such as thalidomide and lenalidomide. Studies with monoclonal antibodies are ongoing and promising including the use of alemtuzumab, SGN-70, and the APRIL/BLYS blocking protein TACI-Ig atacicept. Other agents currently being tested in clinical trials include the PKC inhibitor enzastaurin, the natural product resveratrol, as well as the statin simvastatin. This report provides an update of the current preclinical studies and clinical efforts for the development of novel agents in the treatment of WM.

The serum IgM level has been utilised as a marker of tumor progression and to assess response to therapy in patients with Waldenstrom macroglobulinemia (WM). However, there are many limitations to the IgM protein level. The objective of this study was to evaluate the association of known tumor burden markers and prognostic factors with serum free light chain (sFLC) in 98 patients with WM. We demonstrated that sFLC measurement accurately differentiated IgM-MGUS compared with WM reflecting a measurement of tumor burden. In univariate and multivariate analysis, median sFLC at the cut-off at 60 mg/L was higher for WM patients with low hemoglobin and high beta2M, when we applied the WM-IPSS cut-offs, but showed no association with IgM level. This study demonstrates that sFLC is a new marker in WM disease. Further analysis is required to prospectively study the role of sFLC in monitoring response to therapy and as a prognostic marker in WM patients.

Purpose: Resveratrol (3,4',5-tri-hydroxy-trans-stilbene) is an antioxidant constituent of a wide variety of plant species including grapes. It has gained considerable attention because of its anticancer properties, as shown in solid and hematologic malignancies. Whether resveratrol could inhibit proliferation or induce cytotoxicity in Waldenström's macroglobulinemia (WM) was investigated. Experimental Design: We studied resveratrol-induced inhibition of proliferation and induction of cytotoxicity in WM cell lines, WM primary tumor cells, IgM-secreting cells, and peripheral blood mononuclear cells. The mechanisms of action and different signaling pathways involved were studied using Western blot and gene expression profile analysis. Resveratrol activity was also evaluated in the bone marrow microenvironment. We finally investigated whether or not resveratrol could have any synergistic effect if used in combination with other drugs widely used in the treatment of WM. Results: Resveratrol inhibited proliferation and induced cytotoxicity against WM cells, IgM-secreting cells, as well as primary WM cells, without affecting peripheral blood mononuclear cells; down-regulated Akt, extracellular signal-regulated kinase mitogen-activated protein kinases, and Wnt signaling pathways, as well as Akt activity; induced cell cycle arrest and apoptosis; and triggered c-Jun-NH2-terminal-kinase activation, followed by the activation of intrinsic and extrinsic caspase pathways. Lastly, adherence to bone marrow stromal cells did not confer protection to WM cells against resveratrol-induced cytotoxicity. Furthermore, resveratrol showed synergistic cytotoxicity when combined with dexamethasone, fludarabine, and bortezomib. Conclusion: Our data show that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in this disease.

The nuclear factor-κB (NF-κB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-κB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-κB activity. We demonstrated that perifosine and bortezomib both targeted NF-κB through its recruitment to the promoter of its target gene IκB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-κB pathway.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. We demonstrate up-regulated Akt activity in WM, and that Akt down-regulation by Akt knockdown and the inhibitor perifosine leads to significant inhibition of proliferation and induction of apoptosis in WM cells in vitro, but not in normal donor peripheral blood and hematopoietic progenitors. Importantly, down-regulation of Akt induced cytotoxicity of WM cells in the bone marrow microenvironment (BMM) context. Perifosine induced significant reduction in WM tumor growth in vivo in a subcutaneous xenograft model through inhibition of Akt phosphorylation and downstream targets. We also demonstrated that Akt pathway down-regulation inhibited migration and adhesion in vitro and homing of WM tumor cells to the BMM in vivo. Proteomic analysis identified other signaling pathways modulated by perifosine, such as activation of ERK MAPK pathway, which induces survival of tumor cells. Interestingly, MEK inhibitor significantly enhanced perifosine-induced cytotoxicity in WM cells. Using Akt knockdown experiments and specific Akt and PI3K inhibitors, we demonstrated that ERK activation is through a direct effect, rather than feedback activation, of perifosine upstream ERK pathway. These results provide understanding of biological effects of Akt pathway in WM and provide the framework for clinical evaluation of perifosine in WM patients.

Waldenstrom’s macroglobulinemia (WM) is a distinct clinicopathological entity resulting from the accumulation, predominantly in the bone marrow, of clonally related lymphocytes, lymphoplasmacytic cells and plasma cells which secrete a monoclonal IgM protein (Figure 1). This condition is considered to correspond to the lymphoplasmacytic lymphoma (LPL) as defined by the Revised European American Lymphoma (REAL) and World Health Organisation classification systems. Most cases of LPL are WM, with less than 5% of cases made up of IgA, IgG and non-secreting LPL.

Our better understanding of the complex interaction of multiple myeloma (MM) cells with their bone marrow microenvironment and the signaling pathways that are dysregulated in this process has resulted in a dramatic increase in the therapeutic agents available for this disease. A number of these new agents have demonstrated significant activity in patients with MM. Over the past 5 years, three drugs have received approval from the US Food and Drug Administration for therapy in MM--bortezomib, thalidomide, and lenalidomide. To date, the choice of therapy for MM is not individualized according to the biologic characteristics of the disease, but future studies should enable us to identify patients who may benefit most from certain therapeutic interventions, and thus develop individualized therapy for MM. In this review, we will present some of the treatment algorithms currently developed for patients with MM and focus on established advances in therapy, specifically with thalidomide, bortezomib, and lenalidomide. We will also discuss some of the emerging novel therapeutic agents showing promise in phase I/II clinical trials in MM.

Multiple myeloma is a plasma cell neoplasm with a median survival of 3 to 5 years. Recent advances have improved patient outlook, but the disease remains incurable. Therefore, continued efforts to develop new therapies that target aberrant signaling pathways are needed. The phosphatidylinositol 3-kinase pathway regulates apoptosis, cell cycle regulation, and tumor proliferation. This pathway is constitutively activated in multiple myeloma and its inhibition induces apoptosis. Advances in understanding the signaling cascades mediating proliferation and survival of multiple myeloma cells have markedly improved the treatment of this disease. In this article, we review the role of the phosphatidylinositol 3-kinase/Akt pathway in the pathogenesis of multiple myeloma and the potential therapeutic implications of targeting this pathway in the treatment of multiple myeloma.

The presence of valine (V) at position 158 of FcgammaRllla (CD16) is known to improve clinical response to rituximab in indolent non-Hodgkin lymphoma (NHL). Little is known about the basic mechanisms for this observation. We examined natural killer (NK) cells from healthy donors representing the FcgammaRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F) for gene transcript and cell surface CD16 expression, rituximab binding, and rituximab-dependent NK cell-mediated cytotoxicity. We observed higher levels of FcgammaRIIIa transcripts among individuals with the FcgammaRIIIa-158 V/V versus V/F or F/F genotype (P < .001); increased cell surface CD16 expression by quantitative flow cytometry on NK cells from individuals expressing at least one valine at FcgammaRIIIa-158 versus F/F (P = .029); as well as augmented rituximab binding and rituximab-mediated, antibody-dependent cellular cytotoxicity (ADCC). These results suggest that individuals expressing at least one valine at FcgammaRIIIa-158 might, in part, have better clinical outcomes due to increased CD16 expression, rituximab binding, and rituximab-mediated ADCC.

To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKα. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics. [Cancer Res 2007;67(8):3777–8]

A significant impairment in understanding the biology and advancing therapeutics for Waldenstrom's macroglobulinemia (WM) has been the lack of a representative cell line and animal model. We, therefore, report on the establishment of the BCWM.1 cell line, which was derived from the long-term culture of CD19(+) selected bone marrow lymphoplasmacytic cells isolated from an untreated patient with WM. BCWM.1 cells morphologically resemble lymphoplasmacytic cells (LPC) and propagate in RPMI-1640 medium supplemented with 10% fetal bovine serum. Phenotypic characterization by flow cytometric analysis demonstrated typical WM LPC characteristics: CD5(-), CD10(-), CD19(+), CD20(+), CD23(+), CD27(-), CD38(+), CD138(+), CD40(+), CD52(+), CD70(+), CD117(+), cIgM(+), cIgG(-), cIgA(-), ckappa(-), clambda(+), as well as the survival proteins APRIL and BLYS, and their receptors TACI, BCMA and BAFF-R. Enzyme-linked immunosorbent assay studies demonstrated secretion of IgMlambda and soluble CD27. Karyotypic and multicolor fluorescence in situ hybridization studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements. Inoculation of BCWM.1 cells in human bone marrow chips implanted in severe combined immunodeficient-hu mice led to rapid engraftment of tumor cells and serum detection of human IgM, lambda, and soluble CD27. These studies support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the severe combined immunodeficient-hu mouse model may be used as a convenient model for studies focused on both WM pathogenesis and development of targeted therapies for WM.

The treatment of multiple myeloma has seen significant changes from the time of the initial use of cytotoxic agents such as melphalan, to the introduction of high-dose chemotherapy and stem cell transplantation, and most recently the era of novel targeted agents. These new drugs have rapidly become the mainstay of therapy of this disease and transformed the treatment paradigm, leading to improvements in survival and quality of life. Existing therapeutic options include agents such as thalidomide, bortezomib and lenalidomide, either used alone or in combination with standard agents, including glucocorticoids, and in conjunction with high-dose chemotherapy supported with stem cell transplantation. Several other targeted agents have demonstrated exciting preclinical activity, and are presently being tested in early Phase I and II clinical trials. This review summarizes the role of novel therapeutic agents in multiple myeloma, and the promising effect of multiple new agents in development.

Background: Polymorphisms in FcγRIIa and FcγRIIIa receptors are associated with responses to the CD20-directed immunoglobulin G1 (IgG1) monoclonal antibody rituximab among patients with indolent lymphoma. At odds with the aforementioned clinical observations has been the finding that IgG1 binding is impacted by polymorphisms in FcγRIIIa but not FcγRIIa. One possibility for this discrepancy might involve linkage of polymorphisms between FcγRIIa and FcγRIIIa. Materials and Methods: As such, we performed allelespecific polymerase chain reaction and directed sequencing of the genomic DNA coding region of FcγRIIA and FcγRIIIA for 52 healthy individuals. Results: Two common polymorphisms were observed for FcγRIIA (at positions 27 and 131) and FcγRIIIA (at positions 48 and 158). Importantly, we observed linkage among polymorphisms within and between FcγRIIa and FcγRIIIa, including the expression of histidine at FcγRIIa-131 and valine at FcγRIIIa, both of which are associated with enhanced responses to rituximab. The results of these studies demonstrate that there is wide linkage within and between polymorphisms in FcγRIIa and FcγRIIIa and might provide an explanation for why polymorphisms at FcγRIIa are associated with rituximab responses despite a lack of impact on IgG1 binding. Conclusion: Knowledge of such linkages could facilitate the development of diagnostic tests aimed at identifying patients who might be more suitable for treatment with rituximab and possibly other therapeutic antibodies.

Waldenström macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase Cβ (PKCβ) regulates cell survival and growth in many B-cell malignancies. In this study, we demonstrate up-regulation of PKCβ protein in WM using protein array techniques and immunohistochemistry. Enzastaurin, a PKCβ inhibitor, blocked PKCβ activity and induced a significant decrease of proliferation at 48 hours in WM cell lines (IC50, 2.5-10 μM). Similar effects were demonstrated in primary CD19+ WM cells, without cytotoxicity on peripheral blood mononuclear cells. In addition, enzastaurin overcame tumor cell growth induced by coculture of WM cells with bone marrow stromal cells. Enzastaurin induced dose-dependent apoptosis at 48 hours mediated via induction of caspase-3, caspase-8, caspase-9, and PARP cleavage. Enzastaurin inhibited Akt phosphorylation and Akt kinase activity, as well as downstream p-MARCKS and ribosomal p-S6. Furthermore, enzastaurin demonstrated additive cytotoxicity in combination with bortezomib, and synergistic cytotoxicity in combination with fludarabine. Finally, in an in vivo xenograft model of human WM, significant inhibition of tumor growth was observed in the enzastaurin-treated mice (P = .028). Our studies therefore show that enzastaurin has significant antitumor activity in WM both in vitro and in vivo, providing the framework for clinical trials to improve patient outcome in WM.

Waldenstrom's macroglobulinemia (WM) is an incurable B-cell malignancy. Interestingly, an unusual response activity was observed in 5 patients with WM that appeared related to their use of sildenafil, a phosphodiesterase inhibitor used to treat erectile dysfunction. One patient demonstrated a complete remission, and 4 other patients demonstrated less dramatic, but also unexpected, responses associated with sildenafil. In view of these findings, WM tumor cells were culture-sorted with sildenafil at pharmacologically achievable levels and apoptosis was demonstrated in tumor cells from all 5 patients. These studies suggest that sildenafil may be an active agent in the treatment of WM.

Interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK/osteoprotegerin (OPG) plays a dominant role in osteoclast activation and possibly in plasma cell survival in multiple myeloma (MM). We measured soluble RANKL (sRANKL), OPG, and bone remodeling markers in 121 patients with newly diagnosed MM to evaluate their role in bone disease and survival. Serum levels of sRANKL were elevated in patients with MM and correlated with bone disease. The sRANKL/OPG ratio was also increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity. The sRANKL/OPG ratio, C-reactive protein (CRP), and β2-microglobulin were the only independent prognostic factors predicting survival in multivariate analysis. We generated a prognostic index based on these factors that divided our patients into 3 risk groups. The low-risk group had a 96% probability of survival at 5 years, whereas the intermediate-risk and the high-risk groups had probabilities of survival of 52% and 0%, respectively. Not only do these results confirm for the first time in humans the importance of sRANKL/OPG in the development of bone disease, they also highlight the role of this pathway in the biology of plasma cell growth as reflected by its influence on survival.

While a familial predisposition may exist in up to 20% of patients with Waldenström's Macroglobulinemia (WM), the precipitating cause of this B-cell malignancy remains unknown in most patients. In previous studies, an association between hepatitis C virus (HCV) infection and WM has been suggested as etiological. This relationship has been the subject of debate, however, with some studies demonstrating increased incidence of HCV infection among WM patients and other studies showing no such association exists. This discordance might be attributable to the analytical method used, HCV antibody detection, which might be ineffective in patients with immunosuppression. We therefore analyzed the prevalence of HCV in a large population of WM patients utilizing both an HCV antibody detection immunoassay as well as qualitative polymerase chain reaction assay to directly detect HCV presence in serum samples. None of 100 randomly tested WM patients in this study tested positive for HCV by either analytical method. Our results therefore demonstrate a lack of association between HCV and WM.

WM is a disease of the elderly with a protracted course in many patients. There are limited data which indicate that several WM patients die due to causes which are not directly related to their underlying malignancy. However, the realization and the estimation of the contribution of unrelated mortality in WM are important for the design of treatment strategy in patients of advanced age. To our knowledge there are no such data published for WM patients. Thus, we analyzed the outcomes of 408 patients with symptomatic WM who received therapy within the centers of the Greek Myeloma Study Group in order to assess disease related survival. In this analysis unrelated death was considered to be a competing risk event. Causes of death other than WM, treatment toxicity or myelodysplasia/transformation were considered as unrelated deaths. Median age of patients was 68 (28-92) years; 21% were >75 years and 9% were ≤50 years of age. Patients who started therapy after 2000 were older (median age 70 vs. 65 years before 2000, p75 years (vs. 13% before 2000). In terms of ISSWM stage, more patients had high and intermediate risk disease after 2000 (41% & 42% vs. 25.5% & 38% before 2000, p Median follow up for all patients was 5.5 years (9 years in the pre-2000 and 4.5 years in the post-2000 group) and 52% of patients have died (77% in the group before 2000 and 40% in the group after 2000). However, 23% of deaths were considered unrelated to WM. Thus, 5-year and 8-year overall survival (OS) was 70% and 54% respectively, with a median OS of all patients of 8.8 years. When we performed survival analysis with unrelated deaths as competing risk, then 5-year risk of WM-related death was 21.4% (95% CI 17-26%) and of unrelated death was 7.6% (95% CI 5-10.5%), while 8-year WM-related death rate was 32% (95% CI 27-37%) and unrelated death 11.5% (95% CI 8-15%). Because older patients are at higher risk of unrelated deaths we performed an age-specific analysis. The median survival of patients >75 years was 5.3 vs. 9.7 years for patients ≤75 years (p75 years, the 5-year death rate due to WM was 22% (95% CI 13-32%) vs. 21% (95% CI 16-26%) for patients ≤75 years (p=0.193), while the 5-year unrelated death rate was 17% (95% CI 10-27%) and 5.1% (3-8%), respectively (p75 years) >40% of deaths are unrelated to WM, while WM-specific death rates were similar for patients >75 or ≤75 years. In patients ≤50 years there were no WM-unrelated deaths. We then evaluated the prognostic significance of IPSSWM, which discriminated 3 groups with 5-year overall survival of 86%, 68% and 51% for low, intermediate and high risk groups, respectively (p In conclusion, this is the first analysis in a large cohort of patients with symptomatic WM in which WM-unrelated death is treated as a competing risk. Many patients of advanced age die of causes unrelated to WM and this fact should be taken into account in the evaluation of long term outcomes and the design of clinical trials in patients with WM, especially since more patients of advanced age are diagnosed and treated for WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3032 The treatment of Waldenström's Macroglobulinemia (WM) has changed over the last decades, mainly since the introduction of nucleoside analogues and of rituximab in the management of this disease. Furthermore, novel agents such as bortezomib have been recently introduced. Several analyses in multiple myeloma indicated that the outcome of these patients has significantly improved over the last decade as a result of the introduction of novel agents. However, such data are not available for patients with WM. Thus, we performed an analysis in order to investigate whether the outcome of patients with WM has improved over the last years, compared to that of patients who started treatment before new drugs, such as rituximab became widely available, especially as part of the frontline treatment. We analyzed the database of the Greek Myeloma Study Group which includes 345 patients, who started treatment after January 1985: 130 patients initiated treatment before 1/1/2000 (Group A) and 215 patients started treatment after 1/1/2000 (Group B). More patients were males in both groups (54% in group A vs. 63% in group B, p=0.084), but patients in group B were older (median age 70 years vs. 65 years in group A, p=0.001). Patients in both groups started treatment mainly due to anemia (40% and 43% in groups A and B, respectively). Similar percentages of patients in groups A and B had hemoglobin ≤11.5 g/dl (74% vs. 77%, p=0.57), platelets ≤100,000/ml (12% vs. 14%, p=0.643), albumin ≤3.5 g/dl (44% vs. 50%, p=0.306) and elevated LDH (≥250 IU/L) (21% vs. 17%, p=0.422). The median serum M-peak levels were also similar (3.95 g/dl vs. 3.75 g/dl, p=0.485) while 5% and 7% of patients in groups A and B had a serum M-peak >7 g/dl (p=0.491). However, more patients in group B had serum beta2-microglobulin above 3 mg/dl (62% vs. 42%, p=0.004). Thus, according to the International Prognostic Scoring System (IPSS) for WM, 30%, 48%, and 22% had low, intermediate and high risk disease in group A and 15%, 42% and 43% in group B, respectively (p Disclosures: No relevant conflicts of interest to declare.

INTRODUCTION: The procurement of NHC, for use as controls in hematologic research, is often problematic. The once-conventional source of a voluntary bone marrow [BM] aspirate is now widely prohibited, forcing researchers to purchase these specimens from only a few retailers worldwide. Having to both compensate a donor and generate a profit, the current cost of healthy, human BM is, on average, $40/mL or $4000 for a typical experiment [3 Donors X 50mL]. These specimens are also plagued by several other shortcomings, including: long and erratic lead times, legal restrictions which prohibit age-matching, and variability in their preanalytical handling due to shipping. More than 750,000 joint replacements are now performed annually in the United States alone, and as part of the preparation for these procedures, femoral BM is commonly aspirated and discarded; however, this phenomenon is largely overlooked as a source of controls due to the excess adipocytes found in adult long bones. As such, we sought to develop a method for the acquisition of NHC from the intramedullary aspirates routinely discarded during TJA. METHODS: In collaboration with the Department of Orthopedics and IRB at the Massachusetts General Hospital, Boston, we obtained the discarded intramedullary aspirates of 50 patients undergoing primary, elective TJA of either the hip or knee [Ratio-1:1]. Eligible patients were free of active leukemia or lymphoma and a history of pertinent illness. Aspirates were collected via an in-line sputum trap and standard medical suction just prior to the placement of the femoral prosthesis, heparinized [50 U/mL] and stored at 4oC for < 3 hours before being processed. A novel, hydrophobicity-based methodology was developed to remove the bone, reticulocytes, and excess adipose which dominate these specimens, while sparing the mononuclear cell compartment [MNC]. Specific lymphocytes were immunomagnetically selected, analyzed for purity by flow cytometry [FACS] and compared to commercial BM. RESULTS: Hips produced a median 593×106 MNC (SD=54×106), knees yielded 387×106 MNC (SD=54×106); viability was > 93% by Trypan Blue. The median for commercial marrow was 354×106 MNC (SD=24×106) [n=12,25mL]. Selection of CD34+, CD19+, CD138+ and CD3+ cells from processed TJA BM resulted in substantial populations of enriched cells; the purity of each subset was determined to be > 90% by FACS. Age-matching to hematologic malignancies [median age at diagnosis: 67 - NCI's SEER database] was possible with TJA controls, the median age was 66.6, whereas for commercial donors, it was 27.2 [n=37]. CONCLUSIONS: Using our novel methodology, the intramedullary aspirates routinely discarded during primary, elective TJA represent a viable, and underutilized, reservoir of NHC, and are superior to purchased marrow specimens in terms of cost, yield and appropriateness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3991.

B-cell chronic lymphocytic leukemia (B-CLL) results from the clonal expansion of CD5+/CD23+ B-lymphocytes. The malignant transformation is assumed to occur at the level of mature B lymphocytes. Newer studies, however, have questioned this assumption. Estrov et al were able to generate CLL B-lymphoid

Abstract 2954 Poster Board II-930 The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with potential tumor-suppressor activity. Hypermethylation in the DLC-1 promoter is an aberrant epigenetic modification associated with transcriptional silencing of DLC-1 in various types of human cancers. To explore the epigenetic alteration of DLC-1 in Waldenström's macroglobulinemia (WM), we investigated the methylation status of the DLC-1 promoter and its correlation with DLC-1 mRNA expression in cell lines and primary WM patient samples. Initial analysis using methylation-specific PCR (MSP) showed that DLC-1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1. Using quantitative RT-PCR, DLC-1 mRNA expression was detectable in BCWM.1, but not WM-WSU. These results suggested a correlation between the DLC-1 methylation status and mRNA expression. Similarly, among multiple myeloma (MM) cell lines, we showed that RPMI and U266 exhibited complete methylation, whereas INA6 showed partial methylation, and no methylation was detectable in MM1S and MM1R cells. Similar in WM cells, DLC-1 methylation status was highly correlated with the mRNA expression in these MM cell lines. Of 37 WM patient samples examined, 24 (65%) exhibited methylation in the DLC-1 promoter. In contrast, no methylation of DLC-1 was observed in 4 healthy volunteers. The methylation status was further confirmed using bisulfite DNA sequencing in a subset of WM patients. Quantitative RT-PCR analysis showed that DLC-1 mRNA expression was significantly lower in WM patients compared to healthy volunteers (p=0.001). Treatment with demethylation agents azacytidine or 5-aza-deoxycytidine resulted in significant reactivation of DLC-1 transcription in the WM cell lines WM-WSU and BCWM.1. In addition, a synergistic induction of DLC-1 transcription was observed in the presence of azacytidine and the HDAC inhibitor Vorinostat in BCWM.1 and primary WM patient cells. Moreover, functional studies showed that overexpression of DLC-1 induced cell growth arrest and apoptosis in BCWM.1. DLC-1 methylation status was also correlated with serum sCD27 levels in WM patients (p=0.004), which is secreted by WM cells, serves as a marker of disease burden and facilitates CD40L directed paracrine stimulation by mast cells. Taken together, these results suggest that the down-regulation of DLC-1 via aberrant DNA methylation plays a role in the pathogenesis of WM, and represents a novel therapeutic target in the treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 4785 Waldenstrom's Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration of CD19+ cells and production of a monoclonal IgM protein. Despite advances in treatment, WM remains incurable. As part of these efforts we sought to define the role of HDAC-inhibitors in WM. Gene expression profiling of bone marrow CD19+ cells from 30 WM patients and 10 healthy donors showed over-expression of HDAC4, HDAC9, and Sirt5 in WM patients. Evaluation of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA or Vorinostat), Trichostatin A (TSA), LBH-589 (Panobinostat), and sirtinol demonstrated dose dependent killing of BCWM.1 cells with IC50 of 3.5 uM, 70 nM, 0.8 uM, and 30 uM, respectively, whilst the combination of these agents with bortezomib resulted in at least additive tumor cell killing. TSA is more potent than bortezomib in inducing apoptosis in primary WM tumor cells in patients with prior treatment. TSA and bortezomib showed synergistic effect in 25% of the patients samples tested. We also observed that TSA and bortezomib-induced apoptosis of BCWM.1 cells depended on the activation of a similar set of caspases. Conversely, changes in cell cycle regulators were distinctly different between TSA and bortezomib treated BCWM.1 cells. The results of these studies demonstrate over-expression of distinct members of HDAC in WM cells, and provide a framework for the examination of HDAC-inhibitors as monotherapy, as well as combination therapy with bortezomib in the treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3930 Poster Board III-866 Background The use of gene expression profiling (GEP) was used to dissect the molecular profile of Waldenstrom's macroglobulinemia. Bone marrow CD19+ cells from 22 WM patients and 8 healthy donor (HD) were used in these studies, with application of analytics geared toward non-normally distributed data. Patient characteristics were as follows: median age 64 years; bone marrow disease involvement 35%; serum IgM 3,295 mg/dl; beta-2 microglobulin (B2M) 2.7 mg/L; WM ISS Prognostic Score 2. Four patients (18%) previously received rituximab, and 4 (18%) patients had a family history of WM and/or related B-cell disorders. Materials and Methods GEP was performed using the Affymetrix U133 plus 2 platform on CD19+ selected, CD138 depleted bone marrow cells. Array quality checks, normalization, and unsupervised hierarchical clustering were conducted using dChip (Li and Wong 2001 PNAS). These results were then used for further analysis via custom perl scripts that used 10,000 resampled groups to calculate bootstrap percentile based 95% confidence intervals (CI) for both mean and median values. Comparisons between groups were evaluated using approximate permutation testing. To help identify potential biomarkers, absence/presence calls from DCHIP based on the perfect match vs. mismatch comparisons were tabulated for each group and the contingency table resulting from group comparisons were analyzed using a Fisher's exact test. A gene was considered significant if 50% of its probes displayed at least a 2-fold change, mutual exclusion of means/median values and respective 95% CI, and p < 0.01 for both mean and median comparisons. This data was then compared with dChip clustering results and analyzed using Ingenuity Pathway Analysis (Ingenuity Systems). Results Significantly down regulated genes included DLL1 (-13.5 fold, expressed 0% WM vs. 88% HD, P Conclusions The results of these studies demonstrate differential expression of several novel genes in WM including g protein coupled receptors and genes involved in interferon signaling. Importantly, these studies demonstrate for the first time differential expression of several gene candidates involved in B-cell differentiation that distinguish sporadic versus familial WM. Moreover, GEP revealed a unique profile for patients presenting with poor prognostic disease. Lastly, these studies reveal the up-regulation of 2 tumor suppressor genes, and the anti-apoptotic gene MCL-2 in WM patients treated with rituximab. The findings of these studies therefore have important implications in the pathogenesis, prognostication and treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3731 Poster Board III-667 Azacytidine is a potent demethylation agent and has been used for the treatment of myelodysplastic syndrome. Despite a role in reactivating epigenetically silenced tumor-suppressor genes, the mechanisms of action for azacytidine are not well understood. The relationships between the reactivation of tumor-suppressor genes and the significance for clinical responses also remain to be established. Azacytidine can incorporate into both RNA and DNA strands and may therefore act through multiple pathways. We therefore investigated the therapeutic potential of azacytidine in Waldenstrom's Macroglobulinemia (WM), and characterized certain molecular changes associated with azacytidine. We found that azacytidine exhibited significant dose-dependent cytotoxicity against WM cell lines, and primary WM cells. Treatment of BCWM.1 WM cells with 2 uM of azacytidine rapidly induced cell cycle arrest at G1, and also induced apoptosis at 48 hours. Cleavage of caspase 3, 7, 8, 9 and PARP-1 suggested an involvement of mitochondrial and death receptor pathways in azacytidine-induced apoptosis. The BH3 proteins Puma and Bim, and the pro-apoptotic protein Bax were up-regulated while the anti-apoptotic proteins Bcl-2 and Bcl-xl remained unchanged after treatment with azacytidine. In addition, azacytidine did not induce Akt and NFκB pathways. To further elucidate molecular changes associated with azacytidine treatment, we performed genome-wide microarray expression in BCWM.1 WM cells treated with azacytidine. We observed that the gene encoding fatty acid synthase (FASN) was among the most down-regulated of targets. FASN has been linked to tumor cell proliferation, and its expression was observed by us to be upregulated by microarray studies in WM patients, and further validated as a highly over-expressed target by quantitative RT-PCR analysis in WM patients versus healthy donors. Moreover, by these studies we show that knockdown of FASN by small interfering RNA resulted in growth arrest, and also induced apoptosis in BCWM.1 WM cells. Cerulenin, a specific inhibitor of FASN derived from a natural product, induced strong cytotoxicity against BCWM.1 and primary WM cells at 20 uM in 24 hours. In addition, a synergistic induction of apoptosis in BCWM.1 WM cells was observed in the presence of both azacytidine and cerulenin. The results of this study support an important role for FASN in azacytidine-induced cytotoxicity in WM cells, and highlight a novel mechanistic pathway for this agent, as well as a novel target for drug therapy in WM. A clinical study investigating the activity of azacytidine in WM is planned. Disclosures: No relevant conflicts of interest to declare.

e14582 Background: HDAC inhibitors (HDAC-I) have emerged as a promising class of therapeutics for the treatment of lymphoplasmacytic malignancies, particularly in combination strategies. The basic mechanisms by which these agents modulate malignant cell growth remain to be clarified. In recent studies, we showed that SAHA, TSA, and Sirtinol have significant preclinical activity in Waldenstrom's macroglobulinemia (WM). As part of our effort to identify potential molecular targets for these agents, we performed gene expression profiling (GEP) using WM cells treated with these agents. Methods: We implemented the Bootstrap re-sampling technique on five BCWM1 GEP datasets to characterize the range of gene expression observed. Individual BCWM1 cells were treated with SAHA, TSA and Sirtinol at their established IC50 dosages for 6 and 24 hours prior to gene expression profiling. The genes which changed by two-fold, and fell outside of the 90% range of expression after drug treatment were identified. Results: SAHA upregulated TNF-α and downstream proteins including HSP70, TNFSF9, SERPINB2, as well as STAT4- dependent genes. Sirtinol upregulated metalloproteinase (MMP) and phosphodiesterase (PDE) family members through retinoic acid signaling. We also observed that SAHA, TSA and Sirtinol differentially upregulate elements of the MAP kinase pathways, including signaling through Erk, JNK, and p38 MAPK, indicating a global stress response to HDAC inhibition. Conclusions: The above studies suggest that HDAC inhibitors differentially effect growth of WM cells through both global as well as pathway specific alterations in signaling. Importantly, this data provides a platform for studying the mechanistic role of HDACs in WM, and provide a framework for the use of HDAC-I as single and combination. No significant financial relationships to disclose.

PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

19505 Background: The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with tumor-suppressor activity. Hypermethylation of the DLC-1 promoter leads to transcriptional silencing of DLC-1, and its re-expression blocks proliferation and/or induces apoptosis. We therefore sought to determine the methylation status of the DLC-1 promoter and its impact on DLC-1 transcription and tumor growth in Waldenstrom’s macroglobulinemia (WM). Materials and Methods: The methylation status of the DLC-1 promoter was analyzed in WM (BCWM.1 and WM-WSU), multiple myeloma (INA6, MM1S, MM1R, RPMI 8226 and U266), and bone marrow CD19+ cells from 19 WM patients and 4 healthy individuals using methylation-specific PCR. The methylation pattern was subsequently determined by cloning and bisulfite DNA sequencing. DLC-1 mRNA expression was measured by quantitative RT-PCR. Results: The DLC- 1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1 WM cells. In myeloma cells, ...

8585 Backgound: Waldenstrom’s macroglobulinemia (WM) is a B-cell, IgM-secreting lymphoplasmacytic lymphoma. In this disease, serum IgM levels are used to assess both progression and response to tre...

8617 Background: Waldenstrom’s macroglobulinemia (WM) is a B-cell malignancy characterized by the presence of a serum monoclonal IgM protein, which is used to monitor both disease burden and respon...

8540 Background: Waldenstrom’s macroglobulinemia (WM) is a B-cell malignancy characterized by IgM secreting lymphoplasmacytic lymphoma. Recurrent sinus infections are commonly observed in WM patien...

Waldenstrom’s Macroglobulinemia (WM) is an incurable B-cell malignancy characterized by bone marrow (BM) infiltration with a spectrum of clonally related cells, including small lymphocytes and lymphoplasmacytic cells (CD19+) as well as mature plasma cells (CD138+). The molecular pathogenesis of the disease remains to be defined. We therefore analyzed the gene expression profiles of CD19+ and CD138+ BM mononuclear cells from 30 untreated patients with WM and compared their gene expression profile to their normal counterparts from 10 healthy donors using Affymetrix microarrays (U133 plus 2.0). Since the microenvironment plays an important role in the pathogenesis of WM, we also profiled and compared gene expression profiling for CD19 and CD138 depleted BM mononuclear cells from the same patients and healthy donors. Gene expression analysis was performed using dChip software. Unsupervised hierarchical cluster analysis demonstrated distinct gene expression patterns between WM cells versus their normal counterparts. In supervised hierarchical cluster analysis selecting for genes with > 2 fold change in expression and a False discovery Rate (FDR) < 2%, a set of 1171, 582 and 360 genes were found to be differentially expressed between WM patient and healthy donor CD19+, CD138+, as well as CD19/CD138 depleted (microenvironmental) cells, respectively. Among the most significantly over-expressed genes in the CD19+ compartment in WM patients were: BCL2, TNFRSF13B, TNFRSF17, IGLL1, CCR2, CLLU1, whilst the AP1 family genes JUND and FOSB were among the most significantly down-regulated genes in both CD19+ and CD138+ cells in WM patients. Other interesting transcripts which were over-expressed in CD138+ cells from WM patients included those from genes involved in transcription regulation (ZKSCAN1, ZMYM1, ZNF189, ZNF19, and ZNF559) and interferon response (IFI16 and IFIH1). Of considerable interest was our observation that microenvironmental cells in WM patients demonstrated an overactive transcriptional profile composed of genes which are associated with immune and inflammatory responses including the Toll like receptors (TLR 1,5,7,8), interferon and cytokines (IFI16, IFNAR1, IL-10R, IL-8R), genes encoding extracellular matrix components (Fibronectin and Hepatocyte Growth Factor) as well as genes involved in apoptotic signaling (TNFSF10, TRAF4). These studies provide the first comprehensive molecular characterization of WM, dissecting the molecular features of the two immunophenotypically distinct populations of malignant cells, and providing for the first time evidence for a distinct molecular profile in BM microenviromental cells.

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma. Bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. We therefore investigated the in vitro effect of the new proteasome inhibitor NPI-0052 (N) alone and in combination with Bortezomib (B). Methods: WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake. Determination of the synergistic effect [combination index (CI)] of combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. We also tested the effect of N on WM cells in the co-culture with BMSCs. Activity of the 20S proteasome was determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity. Results: N induced cytotoxicity and inhibition of DNA synthesis (IC50 15nM) in BCWM.1 (48 h). Similar effects were demonstrated in IgM secreting cell lines and primary CD19+ WM cells (IC50 18–30nM). No cytotoxicity was observed on peripheral blood mononuclear cells. The combination of N+B significantly inhibited BCWM.1 proliferation compared to each agent alone: B (5nM) induced cytotoxicity in 8.5%, which increased to 26%, 40% and 53% in the presence of N 2.5nM (CI:0.83), 5nM (CI:0.72) and 10nM (CI:0.7) respectively, indicating synergism. To determine the mechanism of synergy, we investigated the effect of the two agents and their combination on proteasome activity and on signaling pathways, specficially the Akt pathway. Both N and B inhibited the three proteasome activities: the combination of N+B was increased compared to the effect of each agent alone on the caspase-like (C-l) activity of the proteasome. B and N used as single agents induced 29% and 34% inhibition of the C-l activity, respectively, compared to 60% when B and N were used in combination. The C-l activity preferentially cleaves substrates with an aspartic residue in P1 position, like the substrates of the caspases. N induced caspase-8, PARP cleavage and increase of Smac as well as down-modulation of the anti-apoptotic proteins c-IAP, XIAP, survivin, Bcl-2, Mcl-1. The combination of N+B induced a stronger and more significant induction of caspase-8, -PARP cleavage, as well as caspase-3 and -9, which were not affected by using N alone. Similarly, Smac modulation resulted in a more significant induction when cells were exposed to both proteasome inhibitors. N inhibited Akt phosphorylation in BCWM.1 cells (6h) in a dose-dependent manner. GSK3 phosphorylation and ribosomal protein-S6, Akt-downstream target proteins, were also markedly inhibited. Importantly, N inhibited Akt phosphorylation and Akt activity in BCWM.1, even when combined with B, which induced increase of Akt phosphorylation. Lastly, neither exposure to IL-6 nor adherence to BMSCs conferred protection to WM cells against NPI-induced cytotoxicity. Conclusion: NPI-0052 has significant antitumor activity in WM in vitro especially in combination with Bortezomib. These results provide the framework for clinical trials in WM.