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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 3942 Poster Board III-878 Waldenström's macroglobulinemia (WM) is characterized by bone marrow infiltration with lymphoplasmacytic cells (LPCs), and increased presence of mast cells. CD27 is a marker of memory B-cells, and is heterogeneously expressed on the cell surface of WM cells. In recent studies, we showed that the soluble CD27 (sCD27) is produced by WM LPC and is a faithful marker of disease burden in WM patients. Importantly, sCD27 triggers via CD70 the expression of CD40L on neighboring Mast Cells, which in turn supports the growth and survival of WM LPC. sCD27 therefore represents a promising target for the treatment of WM. The mechanism for sCD27 release in WM remains to be elucidated, but is likely cleaved from cell surface CD27. We therefore examined the effects of various matrix metalloproteinase (MMP) inhibitors on the production of sCD27. sCD27 levels were measured by ELISA from supernatants of cultured BCWM.1 and BL2126 B-cells transfected with a vector expressing either FLAG-tagged CD27 (pCD27-FLAG) or a control vector. PMA treatment of pCD27-FLAG transfected BCWM.1 WM and BL2126 cells led to sustained sCD27 release in cultured supernatants; in contrast, PMA treatment of control vector transfected cells showed minimal sCD27 release. Importantly, treatment of pCD27-FLAG transfected BCWM.1 WM and BL2126 cells with MMP 8 and MMP 3 inhibitors (Calbiochem) led to four-fold and two-fold decrease in sCD27 levels, respectively (Figure 1). In contrast, no significant change in sCD27 release was observed following treatment with MMP 2/9 or non-specific MMP inhibitors. These studies demonstrate that matrix metalloproteinases play an important role in sCD27 release, and provide the framework for efforts examining MMP 8 and 3 specific inhibitors in the treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 1947 Poster Board I-970 Recurrent infections are commonly observed among patients with WM, and may be related to the presence of IgA and IgG hypogammaglobulinemia. The etiology for this finding remains unclear, and has been speculated to be on the basis of tumor-induced suppression. We therefore evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 untreated WM patients and addressed the associated clinicopathological findings, and impact of therapy. The median age of these patients was 60, median IgM was 2,910, and median bone marrow (BM) infiltration was 40%. Of these patients, 131 (63.3%) and 120 (58.0%) patients demonstrated decreased serum IgA and IgG levels respectively, while 102 (49.3%) of these patients were abnormally low for both. BM infiltration, serum IgM levels, complete blood counts, absolute lymphocyte counts, b2-microglobulin, or the WM International Prognostic Scoring System score had no impact on the odds ratio of having IgA or IgG, or both IgA or IgG hypogammaglobulinemia by logistic regression analysis. The presence of adenopathy and/or splenomegaly was surprisingly associated with a lower incidence of hypogammaglobulinemia (p≤0.03). The presence of IgA, IgG or both IgA and IgG hypogammaglobulinemia did not predict for the occurrence of recurring infections, which were nearly all respiratory in nature and consisted of sinus (n=53; 25.85%), bronchial (n=16; 7.80%), unspecified upper respiratory tract (n=14; 6.83%), and pneumonic (n=7; 3.41%) infections. Lower IgA and IgG levels were however associated with disease progression in watch and wait patients. To understand the impact of WM directed therapeutic intervention on uninvolved immunoglobulin levels, we analyzed changes in IgA and IgG levels in a cohort of 93 patients who underwent treatment for WM. With a median follow-up of 12 months, no significant recovery in the median IgA and IgG levels was observed with any therapy during the course of follow-up, including in those patients who had follow-up in excess of 1 (n=46), 2 (n=25), and 3 (n=8) or more years post-therapy, and in those achieving a major remission including complete response. Lastly, we sequenced 8 genes (AICDA; BTK; CD40; CD154; NEMO, TACI, SH2D1A, UNG) implicated in immunoglobulin deficiency in 19 WM patients with IgA and/or IgG hypogammaglobulinemia. We observed an intronic variation at position c.1056-6T>C in 2 patients, and a hemizygous missense mutation at c.337G>A in another patient for NEMO, as well as a heterozygous missense mutation at c.425A>T in the highly conserved catalytic site of UNG for one patient. The results of these studies demonstrate that IgA and IgG hypogammaglobulinemia is common, and does not predict for recurrent infection risk in WM. Moreover, IgA and IgG hypogammaglobulinemia persists despite therapeutic intervention and response. These studies highlight the importance for further investigations into the IgA and IgG hypogammaglobulinemia of WM, as well as the signaling pathways involved in B-cell differentiation and immunoglobulin heavy chain class switching in the pathogenesis of WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3750 Poster Board III-686 Rituximab is an important mainstay of therapy in patients with Waldenstrom's macroglobulinemia, though the benefit of maintenance rituximab has not been established. As such, we examined the outcome of patients followed at our Institution who responded to a rituximab based therapy, and assessed the impact of maintenance rituximab. Two hundred forty eight patients were included in this analysis, of whom 86 (35%) received maintenance rituximab following response. No difference in baseline age, serum IgM, IgA, IgG, BM disease involvement, complete blood counts, B2M levels, as well as categorical response rates (CR, VGPR, PR and MR) following induction therapy were observed between cohorts. 181 (73%) patients were previously untreated, with a similar proportion in both cohorts. Induction therapy for all patients included rituximab alone (n=79), or in combination with bortezomib (n=40); cyclophosphamide (n=44); immunomodulatory agent (n=31); or a nucleoside analogue (n=54) containing regimen. The median number of rituximab infusions given as induction was 6 for both cohorts, while the median number of rituximab infusions given as maintenance was 8. Maintenance rituximab was administered as 1 infusion (at 375 mg/m2) every 3 months for 63 (73%) patients, and as 4 weekly infusions (at 375 mg/m2) every 6 months for 23 (27%) patients, with a median of 2 years of treatment. Both progression free (56.3 vs. 28.6 months; p=0.0001) and overall survival (>120 vs. 116 months; p=0.0095) were longer in those patients who received maintenance rituximab (Figs. A, B). Improved progression free survival was evident despite previous untreated or treated status, induction with rituximab alone or in combination therapy, as well as induction with a cyclophosphamide, nucleoside analogue or bortezomib containing regimen (p=0.0001). Among patients receiving maintenance rituximab, progression free survival was 53.3 versus 61.3 months (p=0.4931) for those patients receiving one infusion of rituximab every three months compared to those receiving 4 weekly infusions every 6 months, respectively. Best response serum IgM was 598 vs. 1380 mg/dL (p Disclosures: Treon: Genentech BioOncology Inc.: Consultancy, Honoraria, Research Funding; Biogen IDEC Inc.: Consultancy, Honoraria, Research Funding.

Abstract 4993 The attainment of higher response rates, as well as better categorical responses has occurred with the introduction of novel biological agents, including rituximab in the treatment of WM. However, there is a paucity of knowledge as to whether the attainment of better categorical responses is associated with improved outcomes. We therefore analyzed the impact of categorical responses on progression free survival (PFS) in 159 WM patients naïve to rituximab therapy and who received a rituximab based regimen: cyclophosphamide (n=58), nucleoside analogue (n=43), immunomodulatory agent (n=35), or bortezomib (n=23). The median follow-up for all patients was 83.5 months. Baseline characteristics were as follows: median age 62 years; serum IgM 3670 mg/dL; hematocrit 31.7%. Eighty-two patients had a B2M ≥3.0 g/L, and the IPSS score was I (n=64); II (n=57) and III (n=38). One hundred eight (68%) patients were previously untreated. Categorical responses were as follows: Complete Response (CR) defined as absence of clinically evident bone marrow or extramedullary disease, normal range serum IgM and negative immunofixation study (n=14); Very Good Partial Response (VGPR) defined as ≥90% reduction in serum IgM (n=21); Partial Response (PR) defined as ≥ 50% to Disclosures No relevant conflicts of interest to declare.

Abstract 2950 Poster Board II-926 Background: Multiple studies in closely related diseases such as Chronic Lymphocytic Leukemia (CLL) have revealed distinct miRNA profiles. This increasing appreciation for the role of miRNA expression in disease pathogenesis and homeostasis within cancer biology lead us to profile the miRNA expression of CD19+ bone marrow cells from 11 patients with Waldenstrom's Macroglobulinemia (MR) and 5 healthy donors. The median age for patients was 72 years (range 49-81), WM ISS Prognostic Score was 1 (range 0-4), bone marrow disease involvement was 40% (range 5-80%), and serum IgM was 3,330 (range 202-6,110 mg/dL). Five patients (45.5%) were previously treated and 4 (36.4%) had extramedullary disease. Patients and Methods: CD19+ cells were selected using auto-MACs cell sorting (Miltayni Biotec) and total RNA was extracted with Trizol (Invitrogen). Micro-RNA profiling was conducted using TaqMan low density arrays (Applied Biosystems) allowing for stem-loop based qPCR detection of 670 miRNAs per sample. Results were validated using RT2 miRNA SYBR green based qPCR (SABiosciences). Relative quantification was calculated by ddCT using U6 endogenous controls and normalized to the first healthy donor sample. Results were analyzed using custom perl scripts running bootstrap calculated 95% CIs and approximate permutation testing from 10,000 resampling groups for both means and medians resulting in a robust and distribution independent characterization of each population. Additional Mann-Whitney-Wilcoxon, general linear modeling (GLM), ANOVA, and Spearman correlation testing was conducted using R (R Project for Statistical Computing). Results: We identified miR-29c (+3.2 fold), miR-339-5p (+2.0 fold), and miR-21 (+3.2 fold) as significantly up-regulated in WM patients (p Conclusions: The above findings demonstrate a distinct miRNA profile in WM, and implicate several miRNAs in the pathogenesis of WM, including genes involved in the regulation of tumor cell senescence and IL-12A. These findings provide a framework for the exploration of novel signaling pathways and therapeutic approaches in WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 4789 Patients with WM commonly display low cholesterol levels, particularly LDL and VLDL levels. Given these observations, we examined their levels in 110 patients with WM, and observed decreased total cholesterol ( Patients and Methods Eighteen patients, 8 of whom were previously untreated, and who were asymptomatic with slowly progressing disease were enrolled in this study. The median age for these patients was 67 (range 42-88 years), median IgM was 2,610 (571-5,880 mg/dL), and median hematocrit was 35.4 (range 30.2-41.4%). Patients received Simvastatin at 20 mg daily for the first week, then dose escalated weekly by 20 mg a day to a maximum of 80 mg daily by week 4. Patients were maintained on therapy until progression. Results With a median follow-up of 6 months (range 3-18 months), 7 patients have demonstrated objective disease progression. No objective responses have been observed in any treated patients. Among non-progressing patients, serum IgM levels (2,610 vs. 2545 mg/dL) and hematocrit (35.4% vs. 34.5%) remain stable (p=NS). Grade 2 toxicities included elevation in CPK (n=1), and musculoskeletal pain (n=3). No grade 3 toxicities were observed. Conclusions Simvastatin does not produce objective responses in patients with WM, though may have potential for suppression of disease progression in some patients with WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 761 Introduction: Rituximab is an important treatment option in Waldenstrom's Macroglobulinemia (WM). We and others have previously reported a paradoxical flare in serum IgM levels following rituximab administration which can occur within hours of its administration, and can affect 40-50% of WM patients leading often to symptomatic hyperviscosity, and or aggravation of other IgM mediated morbidities. We have observed a similar flare phenomenon in 3 WM patients receiving IVIG therapy, suggesting that the IgM flare may result from Fcg receptor signaling. We therefore sought to clarify the mechanism for the IgM flare, in hopes that knowledge of its mechanistic attributes might facilitate development of pre-emptive treatments. Patients and methods: CD19+ sorted BM lymphoplasmacytic cells (LPC) from WM patients were incubated in the presence or absence of rituximab or IVIG for 24-48 hours, alone and in the presence of co-cultured monocytes. IgM levels in supernatant were then determined at 24-48 hours by ELISA. Sera and genomic DNA from 68 WM patients treated with rituximab, of whom 27 (40%) experienced an IgM flare, were also evaluated. Serum IgM and cytokine levels were determined for these patients before and following rituximab. Polymorphisms in FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 were also determined for all 68 WM patients, including both patients who did and did not demonstrate an IgM flare. Results: We first observed that the direct incubation of WM LPC with either rituximab or IVIG did not induce significant IgM release. We therefore sought to delineate if other bystander immune cells contributed to the IgM flare phenomenon either directly or by cytokine release. We observed that rituximab and IVIG could stimulate both healthy donor and WM patient monocytes to produce IL-6, which was assessed by both RT-PCR and ELISA. IL-6 transcription was increased as early as 3 hours, and was associated with an increase in IL-6 secretion. Since monocytes express various Fcg receptors, we next sought to clarify which receptor was potentially responsible for the induction of IL-6 release. We therefore stimulated monocytes with activating F(ab')2 fragments directed at FcgRI, FcgRIIA, FcgRIIB, and FcgRIIIA. Only treatment with an FcgRIIA F(ab')2 activating fragment resulted in robust IL-6 transcription and secretion by monocytes. Since IL-6 plays an important role in stimulating IgM secretion by WM cells, we next examined IL-6 levels in patients who received rituximab and compared their levels over the course of therapy in patients with and without an IgM flare. A spike in IL-6 levels was observed in those patients who experienced an IgM flare, and correlated with the time course of the IgM flare. Given these findings, we co-cultured primary BM WM cells as well as BCWM.1 cells in the presence or absence of monocytes using a Tran swell co-culture system, and observed that overnight incubation with rituximab or IVIG resulted in increased IgM production. This effect could be blocked by simultaneous incubation with an IL-6 blocking antibody. Similar results were obtained by culturing WM LPC with supernatants obtained from rituximab or IVIG stimulated monocytes incubated with and without an IL-6 blocking antibody. Lastly, the frequency of polymorphisms for all 68 WM patients for FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 did not defer from published data bases, and were similar between rituximab treated patients who either experienced or not the IgM flare. Conclusion: Taken together, the above data suggest that the IgM flare observed in WM patients following rituximab and IVIG administration is likely triggered by IL-6 in response to stimulation of bystander immune cells including monocytes, possibly through FcgRIIA binding. Efforts aimed at blocking IL-6 release should be explored as a pre-emptive means of preventing the IgM flare in WM patients being considered for rituximab and IVIG therapy. 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 762 Background: The existence of a familial form of Waldenstrom's Macroglobulinemia (WM) has previously been described by us and others. Up to 20% of WM patients have a first degree relative with either WM, or a closely related B-cell disorder. The genetic basis for these findings has so far remained elusive. Patients and Methods: As part of these efforts, we enrolled 482 WM patients and their first and second degree family members, for a total of 148 families. Families were then classified as follows: Sporadic (Only 1 case of WM present); Familial WM Only (if ≥ 2 cases of WM were present); Familial Mixed B-cell (if only the proband had WM, and other B-cell disorders were present in other family members). Detailed medical as well as familial history for cancer and autoimmune disorders was collected, along with complete blood counts, quantitative immunoglobulins, and serum protein electrophoresis for all participants. Buccal and peripheral blood DNA samples were also collected. Genome wide association studies were run on peripheral blood DNA samples using SNP 6.0 platform from Affymetrix, and analyzed using R (R project for statistical computing) and the Affymetrix Genotyping Console. Data from 99 individuals was available for this analysis. Results: Among the 148 families enrolled, 89 (60.1%), 17 (11.5%), and 42 (28.4%) were classified as Sporadic, Familial WM Only, and Familial Mixed B-cell Cohorts. Analysis of the SNP 6.0 data revealed that copy number polymorphism (CNP) 88 was strongly associated with Familial Mixed B-cell presentation. A copy number of 0 for CNP88 was found in 2/5 (40%) probands (WM patients) from the Sporadic Cohort; 1/6 (16.7%) patients from the Familial WM Only Cohort; and 8/9 (88.9%) patients from the Familial Mixed B-cell Cohort (p= 0.011 and 0.09 vs. Sporadic and Familial WM Only Cohorts, respectively). Among proband family members, a copy number of 0 for CNP88 was found in 7/15 (46.7%) individuals from the Sporadic Cohort; 9/26 (34.6%) individuals from the Familial WM Only Cohort; and 34/37 (91.9%) individuals from the Familial Mixed B-cell Cohort (p Conclusions: The results of these studies implicate the homozygous deletion of GSTM1 in the Familial Mixed B-cell presentation for Waldenstrom's Macroglobulinemia. These findings are invariably germain not only to WM predisposition, but also to other closely related B-cell disorders which cluster with Familial 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.

Background: Hypogammaglobulinemia of the “uninvolved” immunoglobulins is commonly observed in Waldenstrom’s macroglobulinemia (WM), and has often been attributed to disease-related suppression. However, there is a paucity of information related to the pathogenesis of hypogammaglobulinemia in these disorders. Methods: We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 patients with WM, and addressed the impact of therapy and response on IgA and IgG levels for 93 of these patients who required subsequent treatment. We also performed extensive sequence analysis of the promoter, all exonic, and flanking intronic regions from peripheral blood of 19 untreated WM patients who demonstrated IgA and/or IgG hypogammaglobulinemia for 8 genes often observed in common variable immunodeficiency disorders (CVID) and B cell deficiency i.e. AICDA, BTK, CD40, CD154, NEMO(IkBkG), TACI, SH2D1A, UNG. Results: At baseline, 120/207 (58.0%), 131/207 (63.3%), and 102/196 (49.3%) patients had abnormally low levels of serum IgG (C (n=2) and a hemizygous missense mutation at codon 113 for NEMO (n=1), and a heterozygous missense mutation at codon 142 in UNG (n=1). Conclusions: The results of these studies demonstrate that IgA and IgG hypogammaglobulinemia is a constitutive feature of patients with WM, which neither correlates with, nor is impacted by disease burden, despite therapeutic intervention and response. The results also suggest that patients with WM may harbor sequence mutations, which may be a contributor to the pathogenesis and/or morbidity of WM.

Since the adoption of rituximab as a mainstay of therapy in non-Hodgkin’s lymphoma, there has been growing debate on the importance of adriamycin and vincristine as treatment components used in the therapy of indolent non-Hodgkin’s lymphoma, including Waldenstrom’s macroglobulinemia (WM). We therefore examined the outcome of symptomatic WM patients who required therapy based on consensus guidelines and received treatment at our Institution with CP-R (n=20), CVP-R (n=17), or CHOP-R (n=23). Baseline characteristics for all 3 cohorts were as follows: Median Age Median Prior Therapies Bone Marrow Involvement sIgM (mg/dL) Hct PLT B2M CP-R 65 (range 42–74) 0 (range 0–2) 45% (range 5–95%) 2620 (range 551–6750) 33.4 270 2.3 CVP-R 60 (range 32–81) 1 (range 0–2) 50% (range 20–90%) 2220 (range 185–8430) 30.0 169 3.3 CHOP-R 54 (range 42–72) 0 (range 0–2) 50% (range 5–90%) 5150 (range 241–12400) 31.0 239 3.6 Responses to therapy, including median decrease in serum IgM and best response for IgM, Hct, and PLT counts were as follows: ORR(CR+PR+MR) ≥PR CR/nCR % decrease sIgM Post-sIgM Post-Hct Post-PLT p=N.S. for all treatment cohorts. CP-R 90% 80% 0% −54% 1150 38.0 300 CVP-R 88% 71% 12% −67% 790 36.1 219 CHOP-R 83% 70% 17% −63% 794 38.3 230 Adverse events attributed to therapy, including rituximab related IgM flare were as follows: Neutropenic fever Hospitalizations Treatment related neuropathy IgM flare IgM flare requiring plasmapheresis P=N.S. except as follows: (a) p=0.02; (b) p=0.00006; (c) p=.0.004 versus CPR. CP-R 0% 0% 0% 25% 10% CVP-R 18% 12% 59%b 29% 11% CHOP-R 26%a 17% 35%c 23% 17% The results of this study demonstrate comparable response characteristics among WM patients treated with CP-R, CVP-R, or CHOP-R though a trend for attainment of more CR/nCR was observed among those patients receiving CVP-R and CHOP-R. Importantly, significantly more toxicity was observed, particularly neutropenic fever and treatment related neuropathy among patients treated with CVP-R and CHOP-R versus CP-R. The results of this study suggest that in WM, the use of CPR may provide analogous treatment responses to more intense cyclophosphamide based regimens, while minimizing treatment related complications.

Waldenström’s macroglobulinemia (WM) is an incurable B cell malignancy characterized by the accumulation of IgM secreting clonally related bone marrow lymphoplasmacytic cells (LPC) including CD19+ B-cells and CD138+ plasma cells. Despite intense research efforts, the pathogenetic basis for this disease remains to be clarified. MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes by inducing translational inhibition and through cleavage of targeted transcripts by partial or complete base pairing. We therefore evaluated the expression of 384 microRNAs in CD19+ and CD138+ sorted bone marrow lymphoplasmacytic from 13 untreated WM patients, and compared their expression profiling to analogous lymphoplasmacytic cells taken from the bone marrows of 13 healthy donors. Data obtained from microRNA arrays was analyzed using SDS, RQ manager, R and dChip softwares. Relative expression was calculated using the comparative Ct method through RQ manager and dChip softwares. Of the 384 microRNAs evaluated in CD19+ patient cells, miR-192, -125b, -21, -155 demonstrated significant upregulation, whereas miR-181c, -572, and -650 were significantly down regulated compared to healthy donor CD19+ bone marrow cells (p

Cognitive impairment is age-related, becoming increasingly prevalent after 65 years of age. In community settings, its most common cause is dementing illness, usually Alzheimer’s disease, for which there is no cure. Other causes of cognitive impairment exist, including some for which specific treatment may be available. Even if the impairment is due to a dementing disorder that will progress inexorably to mental incompetence, detection is justifiable for purposes of prognosis and life planning.

Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

Waldenstrom’s macroglobulinemia is a B-cell disorder characterized by the infiltration of lymphoplasmacytic cells (LPC) in the bone marrow (BM), along with an IgM monoclonal gammopathy. Characteristic of WM is an increased number of mast cells (MC) which are found in association with LPC, and stimulate LPC growth through several TNF-family members including CD40L, APRIL and BLYS (Ann Oncol17:1275; Blood104:917A). As such, we have targeted MC in WM. One important growth and survival factor for MC is stem cell factor (SCF), which signals through CD117. Imatinib mesylate blocks SCF signaling through CD117, and induces apoptosis of WM BM MC and LPC, both of which highly express CD117 (Blood2004; 104:314b). As such, we performed this Phase II study of imatinib mesylate in patients with relapsed and refractory WM. Intended therapy consisted of imatinib mesylate which was initiated at 400 mg po qD over the first month, and subsequently dose escalated to 600 mg po qD for up to 2 years. Dose de-escalation to 300 mg po qD was permitted for toxicity. Twenty-eight patients were enrolled, 27 of whom are eligible for evaluation at final analysis. Median age was 61 (range 33–80 years), and median prior therapies was 2 (range 1–5). Twenty-four and four patients had relapsed and refractory disease, respectively. With a median follow-up of 6.3 months for all evaluable patients, median serum IgM levels declined from 3,110 to 2,530 mg/dL at best response (p=0.002). On an evaluable basis, 7/27 (26%) and 2/27 (7%) of patients attained a ≥25% and ≥50% decrease in serum IgM, respectively. Responses were prompt, and occurred at a median of 2.1 months. The median time to progression for responding patients was 8.4 (range 2–15 months). Hematological toxicities were the most common feature and ≥grade 2 toxicities included anemia (n=12), leucopenia (n=5), edema (n=3), thrombocytopenia (n=2) and neutropenia (n=1). Tryptase levels, which measure mast cell burden, declined in all patients for whom pre- and post-serum was available from 6.7 (range 2.2–10 ng/ml) to 3 (range 1–5.5 ng/ml) (p

Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

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.

8017 Background: Rituximab is active in Waldenstrom's macroglobulinemia (WM), producing response rates of 40–50%. Lower response rates are observed among patients with the FcγRIIIA-158 FF polymorphism; high B2M (≥3.0 mg/dL), and high serum IgM levels (≥6,000 mg/dL). Thalidomide enhances rituximab-mediated ADCC of lymphoplasmacytic cells and produces response rates of 25% in WM. As such, we conducted this phase II study using thalidomide and rituximab in patients naïve to either agent. Methods: Intended therapy was: Weeks 1–52: Thalidomide (200 mg po qHS for 2 wks, then 400 mg po qHS) Weeks 2–5, 13- 16: Rituximab (375 mg/m2/wk) Twenty-five patients were enrolled, 20 of whom were previously untreated, with a baseline median age of 62 (range 44–86 yrs), BM involvement of 40 (range 5–80%), serum IgM of 3,670 (range 924–8,610 mg/dL), B2M of 2.6 (range 1.4–8.3 mg/L), Hct of 34.1 (range 23.6–42.6%). Results: Grade ≥2 toxicities to thalidomide included neuroparesthesias (n=11); somnolence (n=3); confusion (n=3); rash (n=2); tremors (n=2); bradycardia (n=2) and palpitations (n=1). Among patients experiencing neuroparesthesias, 10 demonstrated resolution to grade 1 (n=3) or complete resolution (n=7) at a median of 6.7 (range 0.4- 22.5 months). Dose reduction of thalidomide occurred in all patients and led to discontinuation in 11 patients. Twenty-three patients were evaluable. Responses among evaluable patients: CR (n=1); PR (n=15); MR (n=2); SD (n=1) for an overall and a major response rate of 78% and 70%, respectively. Median serum IgM decreased from 3,670 (924–8,610 mg/dL) to 1,590 (36–5,230 mg/dL) (p2M levels (71% vs. 89% for No significant financial relationships to disclose.

8018 Background: WM is an indolent B-cell lymphoma. NA are widely used in the treatment of WM, and are considered as appropriate first line agents for the treatment of WM (Gertz et al, Semin Oncol 2003; Treon et al, Blood 2006). Increased incidences of disease transformation and development of MDS/AML have been observed among patients with indolent B-cell malignancies receiving NA. We therefore sought to delineate the incidence for these events in a large population of WM patients treated at our institution. Methods: 326 previously treated patients with the consensus panel definition of WM, who received treatment with (n=173) or without (n=153) a NA were included in this analysis. Baseline characteristics between NA and non-NA treated patients were not significantly different and were as follows: median age 59 years; male/female ratio 1.4; median B2M 2.9 mg/L; serum IgM 3,000 mg/dL; BM involvement 40%; Hct 34%; WBC 5,100/ul, and PLT count 243,000/ul. For patients receiving NA, treatment consisted of either fludarabine (n=117; 68%), cladribine (n=48; 27%) or both (n=8; 5%). For non-NA treated patients, therapy included chlorambucil, rituximab, CVP, CHOP, thalidomide, and cyclophosphamide alone or in combination with rituximab, and alemtuzumab. Median follow-up of patients was 64 (range 10–270) months. Results: Among NA treated patients, 10 (5.7%) patients had transformation to an aggressive NHL (to DLBCL) (n=7; 4%) or developed MDS/AML (n=3; 1.7%). Disease transformation and development of MDS/AML occurred at a median time of 48 (range 7–114), and 48 (range 38–52) months following NA treatment, respectively. In contrast, among non-NA treated patients, only 1 patient demonstrated disease transformation (to DLBCL) at 10 months and no patients developed AML/MDS (p=0.025). Conclusions: These data demonstrate an increased incidence of disease transformation and development of MDS/AML among WM patients treated with NA. No significant financial relationships to disclose.

IgA and IgG hypogammaglobulinemia are a common finding in patients with WM which may contribute to recurrent infections seen in up to half of WM patients. The etiology for this finding remains unclear. As part of these studies, we investigated the prevalence and genetic basis for IgA and IgG hypogammaglobulinemia among 89 previously untreated patients with the clinicopathological diagnosis of WM. Their median IgM was 2,980 (180–12,400) mg/dl, median BM involvement was 35% (5%–95%) and median B2M was 2.7 (1.4–13.7 mg/dL). Fifty-six (63%) and 66 (74%) of these patients demonstrated IgG (

WM is an incurable B-cell disorder characterized by the presence of lymphoplasmacytic cells in the bone marrow and an IgM monoclonal protein. The presence of hypocholesterolemia has often been observed in WM patients, though the incidence and significance has not been investigated. As part of these studies, we evaluated lipoprotein levels in 110 patients with WM. The median age for this cohort was 62, and median number of prior therapies was 1. Twenty-one (19.1%) patients were on a lipid-lowering agent (lovastatin, simvastatin, pravastatin). We observed decreased total cholesterol (

In previous studies, we demonstrated that Rituximab and Bortezomib are active agents as monotherapy in patients with Waldenstrom’s macroglobulinemia (WM), producing response rates of 40–80%. Moreover, we and others have demonstrated synergistic activity for these agents together, and with steroids in preclinical studies. As such, we examined the activity of combined bortezomib, dexamethasone and rituximab in patients with the consensus panel diagnosis of WM. Intended therapy consisted of IV bortezomib at 1.3 mg/m2 and IV dexamethasone 40 mg on days 1,4,8, and 11, and Rituximab at 375 mg/m2 on day 11 which constituted one cycle of therapy. Patients received four consecutive cycles, followed by a three month pause, and then 4 more cycles, each given three months apart. Ten patients are eligible for evaluation at interim analysis. Following a median of 4 cycles of therapy, median serum IgM levels for all evaluable patients declined from 5,095 (range 700–8030 mg/dL) to 2,850 (range 280–3660 mg/dL) (p=0.001). The overall response rate was 100%, with 5 and 5 patients achieving a minor (≥25% decrease in IgM) and major (≥50% decrease in IgM) response, respectively. Responses were prompt, and occurred at a median of 1.1 months. Therapy was well tolerated with III/IV toxicities consisting of sepsis (n=1), pneumonia (n=1), and thrombocytopenia (n=1). Four patients developed herpes zoster while on treatment, resulting in the implementation of prophylaxis for all patients with valcyclovir. At a median of 4 cycles, no sensory or motor neuropathies have occurred. The interim results of this study demonstrate that BDR is a highly active, and well-tolerated regimen in the primary therapy of WM.

Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

Introduction: WM is a B-cell disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells (LPC), along with excess mast cells (MC) which support the growth and survival of BM LPC through multiple TNF-family ligands including CD40L, APRIL and BLyS/BAFF. Importantly, BM LPC stimulate cell surface expression of TNF-family ligands through release of sCD27 which induces CD70 on MC. We therefore have sought the development of agents which could target CD27-CD70 interactions. As such, we examined the therapeutic potential of directly targeting CD70 using the fully humanized monoclonal antibody SGN-70 (Seattle Genetics, Inc., Bothell WA). Methods-Results: As part of these studies, we used flow cytometric analysis to evaluate the expression of CD70 on primary WM patient BM LPC and MC, as well as 2 WM cell lines (BCWM.1 and WM-WSU). These studies demonstrated cell surface expression of CD70 on BM LPC and MC from 20/26 (77%) and 10/11 (90%) WM patients, respectively. We next assessed the ability of the SGN-70 antibody to eradicate primary WM LPC (n=5) and WM cell lines by assessing for direct induction of apoptosis, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) as well as induction of TNF family ligands on primary WM MC and the LAD2 MC line. Following incubation of WM LPC with SGN-70 (0.01–20 μg/ml), no direct induction of apoptosis or CDC activity was observed. However, SGN-70 mediated significant dose-dependent ADCC against WM LPC and MC at concentrations of 0.1–20 ug/ml. Importantly, SGN-70 blocked sCD27-induced expression of CD40L and APRIL on primary WM MC and LAD2 MC. To further evaluate the therapeutic potential of SGN-70 in an in vivo model, SCID-hu mice bearing BCWM.1 WM cells were treated with SGN-70 (1 mg/kg, i.p., qOD) Serum human IgM and sCD27 levels were measured by ELISA to monitor for tumor engraftment and disease progression. SGN-70 initiated 6 weeks following tumor engraftment blocked tumor growth in 12/12 treated mice, whereas all 5 untreated mice demonstrated disease progression. The results of these studies provide the framework for clinical trials to examine the therapeutic potential of the SGN-70 monoclonal antibody in WM.

Waldenstrom’s macroglobulinemia is a B-cell disorder characterized by the infiltration of lymphoplasmacytic cells (LPC) in the bone marrow (BM), along with an IgM monoclonal gammopathy. Characteristic of WM is an increased number of mast cells (MC) which are found in association with LPC, and stimulate LPC growth through several TNF-family members including CD40L, APRIL and BLYS (Ann Oncol17:1275; Blood104:917A). As such, we have targeted MC in WM. One important growth and survival factor for MC is stem cell factor (SCF), which signals through CD117. Imatinib mesylate blocks SCF signaling through CD117, and induces apoptosis of WM BM MC and LPC, both of which highly express CD117 (Blood2004; 104:314b). As such, we performed this Phase II study of imatinib mesylate in patients with relapsed and refractory WM. Intended therapy consisted of imatinib mesylate which was initiated at 400 mg po qD over the first month, and subsequently dose escalated to 600 mg po qD for up to 2 years. Dose de-escalation to 300 mg po qD was permitted for toxicity. Thirteen patients were enrolled and are eligible for evaluation at interim analysis. Median age was 64 (range 53–80 years), and median prior therapies was 2 (range 1–5). Nine and four patients had relapsed and refractory disease, respectively. Following a median of 3 months of therapy, median serum IgM levels for all evaluable patients declined from 3190 (range 763–8130 mg/dL) to 2095 (range 695–7340 mg/dL) at best response (p=0.009). On an intent to treat analysis, 6/13 (46.2%) of patients attained a ≥25% decrease in serum IgM. Responses were prompt, and occurred at a median of 2.5 months. Overall, therapy was well tolerated with ≥grade 2 toxicities as follows: anemia (n=4); edema (n=3); hyperglycemia (n=2); leukopenia (n=2); thrombocytopenia (n=1); neutropenia (n=1); and resulted for 4 patients in dose modification (n=4) or treatment cessation (n=3). The interim results of this study demonstrate that imatinib mesylate is an active and well-tolerated salvage therapy for WM.

7556 Background: Responses to sildenafil citrate (Viagra), a phosphodiesterase-5 inhibitor used to treat erectile dysfunction, have been observed in patients with Waldenstrom’s Macroglobulinemia (WM). Moreover, sildenafil citrate induces apoptosis of WM lymphoplasmacytic cells (Clin Lymphoma 5:205, 2004). We therefore conducted a prospective phase II study of sildenafil citrate in patients with slowly progressing WM who did not meet consensus eligibility for active therapy (Semin Oncol 2003; 30:116). Intended therapy was as follows: Week 1 25 mg po qD Week 2 50 mg po qD Week 3 75 mg po qD Week 4, then Months 2–24 100mg po qD or Maximum Tolerated Dose Methods: Thirty patients were enrolled, 18 of whom were previously untreated. All patients demonstrated progressing disease prior to enrollment. Median age was 66 (range 43–85 yrs), baseline BM involvement was 30% (range 5–90%), serum IgM was 3,640 (range 790–6,720 mg/dL), hematocrit was 37.1% (range 32.7–58.5%), and B2M was 2.3 (range 1.5–8.9 mg/dL). Patients were evaluable for response after 3 months of therapy. Results: At a median of 3 months, serum IgM levels declined in 19/30 (63%) patients from a median of 3,640 (range 790–6,720 mg/dL) to 2,965 (range 1,170–6,110 mg/dL). 5/30 patients (17%) demonstrated at least a minor response (≥25% IgM decrease). Two patients were taken off study for non-response to therapy. Toxicities were mild and included headaches, sinus congestion, facial flushing, dyspepsia, and generally resolved on their own with prolonged sildenafil citrate usage. Only two patients required dose modification to 25 mg and 75 mg po qD, respectively. Corollary studies to determine putative mechanisms of action for sildenafil citrate in WM were also performed and will be updated at the meeting. Conclusions: This prospective clinical trial provides preliminary evidence for activity of sildenafil citrate in patients with advancing WM. With minimal toxicity, sildenafil citrate appears to have suppressed disease progression in more than half of patients and has resulted in objective responses, thus warranting further investigation in WM and possibly other B-cell disorders. No significant financial relationships to disclose.

INTRODUCTION: Xbp-1 is a ubiquitously expressed basic leucine zipper protein which serves as key transcription factor for plasma cell differentiation and immunoglobulin production. XBP-1 mRNA undergoes splicing at an IRE-1a specific cleavage site to produce a spliced form (Xbp-1s) which due to a frame shift results in a more stable and potent transcription factor than the unspliced form (Xbp-1us). Previous observations demonstrated that Xbp-1 was highly expressed in multiple myeloma (MM), though its expression in other B-cell disorders including Waldenstrom’s macroglobulinemia (WM) remains to be clarified. METHODOLOGY: In this study, we investigated the different forms of Xbp-1 in CD19+ selected bone marrow (BM) cells from 61 patients with the consensus panel definition of WM using semi-quantitative PCR analysis. CD19+ selected BM cells from 6 healthy donors, and CD138+ selected BM cells from 4 MM patients were used as controls. All RT-PCR results were normalized to b-actin levels. Sequencing of Xbp-1 in CD19+ selected LPC was also performed for 20 WM patients. RESULTS: In 9/61 (15%) WM patients, Xbp-1 transcripts were undetectable. Among WM patients expressing Xbp-1, as well as all MM patients evaluated, higher levels of XBP-1us were observed in comparison to normal healthy donors (p=0.001). Decreased XBP-1s transcript levels were also observed among WM and MM patients versus healthy donors, but did not reach statistical significance. Sequence analysis of Xbp1 in CD19+ selected BM LPC demonstrated variants 10/20 WM patients in exon 1 that are currently under investigation. CONCLUSIONS: Abnormal expression of Xbp-1 is common in patients with WM, and includes loss of expression, and aberrations in splice patterns. The genetic basis for these findings is under investigation.

INTRODUCTION: Rituximab is active in Waldenstrom’s macroglobulinemia (WM), producing major (CR + PR) response rates of 40–50% using an extended dose schedule. Lower response rates though are observed among patients with the FcgRIIIA-158 F/F polymorphism (JCO 23:474) and higher serum IgM levels (>6,000 mg/dL) (Ann Oncol 16:132). Thalidomide enhances rituximab mediated ADCC killing of lymphoplasmacytic cells and produces response rates of 25% in WM. As such, we conducted this phase II study using combination thalidomide and rituximab in patients naïve to either agent. METHODS: Intended therapy was as follows: Weeks 1–52 Thalidomide (200 mg po qHS for 2 weeks, then 400 mg po qHS);Weeks 2–5, 13–16 Rituximab (375 mg/m2/week). Twenty-five patients were enrolled, 20 of who were previously untreated, with a baseline median age of 62 (range 44–86 yrs), BM involvement of 40 (range 5–90%), serum IgM of 3670 (range 924–8610 mg/dL), B2M of 2.6 (range 1.4–7.8 mg/L), Hct of 33.0 (range 24.5–37.7%). RESULTS: Grade 3/4 toxicities to thalidomide included paresthesia (n=10); somnolence (n=8); rash (n=7); neuropathic pain (n=6); confusion (n=4); tremors (n=2); and bradycardia (n=2). Dose reduction occurred in all patients and led to premature discontinuation in 11 patients. All evaluable patients received the intended rituximab therapy. Paradoxical IgM spikes following rituximab occurred during both courses of therapy and were observed in 12 patients. Two patients succumbed, unrelated to therapy, and were unevaluable. Responses were as follows: CR (n=1); PR (n=15); MR (n=2); SD (n=1) for an overall and a major response rate of 78% and 70%, respectively, among evaluable patients. Four patients had no response to therapy. With a median follow-up of 19 months, 6 patients have progressed. The median duration of response was 19.6+ months (range 7 to 26+ months) Overall response was associated with median cumulative dosed thalidomide: CR/PR/MR (29,275 mg) vs. SD/NR (7,400 mg); p=0.004. Importantly, overall responses were unaffected by FcgRIIIA-158 polymorphism status (75% vs. 71% for VV/FV vs. FF); serum IgM (83% vs. 80% for 6,000 mg/dL); and B2M levels (86% vs. 78% for CONCLUSION: These studies demonstrate that thalidomide in combination with rituximab is highly active in WM, though additional studies are warranted to determine the ideal dose and schedule for thalidomide in view of toxicities. Moreover, thalidomide in combination with rituximab may overcome unfavorable prognostic determinants previously reported with rituximab monotherapy in WM.

Waldenstrom’s Macroglobulinemia (WM) is incurable B-cell malignancy characterized by the infiltration of the bone marrow infiltration with lymphoplasmacytic cells (LPC) and the presence of an IgM monoclonal gammopathy. A significant impairment in understanding the biology and advancing therapeutics for WM has been the lack of a representative cell line and animal model. We therefore report on work accomplished in our laboratories in establishing the BWMC.1 cell line, which was derived from long term culture of CD19+ selected lymphoplasmacytic cells isolated from the bone marrow (BM) aspirate of a previously untreated 55-year-old female patient with IgMk secreting WM. BCWM.1 cells morphologically resembled LPC and had prominent nucleoli and ample cytoplasm by Wright-Giemsa staining, and were propagated in RPMI 1640 medium supplemented with 10% FBS. 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−, ck+, cl−. BCWM.1 cells also expressed the survival proteins APRIL and B-Lymphocyte Stimulator (B-LYS), and their receptors TACI, BCMA and BAFF-R. ELISA studies demonstrated secretion of IgMk in culture. Karyotypic and M-FISH studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements through FR1c/JHc amplification of genomic DNA. BCWM.1 cells were positive for LMP1 expression by histochemical staining and PCR analysis. Importantly, inoculation of BCWM.1 cells directly into the human BM milieu of SCID-hu mice resulted in engraftment of tumor cells and serum detection of human IgMk in four of five SCID-hu mice within two weeks. In contrast, subcutaneous injection of BCWM.1 cells in SCID mice did not lead to engraftment, suggesting a role for the human BM microenvironment in supporting the expansion of BCWM.1 cells. These studies therefore support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the SCID-hu mouse model may be used as a convenient model for studies focused on both the pathogenesis and development of targeted therapies for WM.

The unfolded protein response (UPR) maintains the quality of newly synthesized secretory and transmembrane proteins such as immunoglobulins in the endoplasmic reticulum (ER) of eukaryotic cells. The most proximal sensors of the UPR are the ATF6, IRE1a-Xbp1, and PERK proteins. Bip, an ER stress-induced pro-survival molecular chaperone, controls the trafficking of Atf6 and is considered as an indicator of the onset of the UPR. EDEM, downstream of Xbp1 splicing, regulates the extraction of misfolded proteins into the cytosol for proteasome-mediated destruction. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected. We studied the UPR stress system in patients with Waldenstrom’s Macroglobulinemia (WM), a B-cell disorder characterized by excess secretion of IgM. As part of these efforts, we examined the UPR genes Bip-Atf6, Ire1a-Xbp1, Perk, as well as genes downstream of the UPR pathway including Xbp1 spliced-Edem, eIF2a and Gadd34 using semi-quantitative PCR analysis of CD19+ selected bone marrow lymphoplasmacytic cells (LPC) from 46 patients with the consensus panel diagnosis of WM. We found that UPR gene expression was highly heterogeneous with 16 (35%) patients expressing all genes, whilst 11 patients (24%) expressed only Xbp1 and Edem. Among patients expressing all UPR genes, median serum IgM (p=0.025) and B2M (p=0.05) levels, as well as bone marrow involvement (p=0.06) were higher, with levels of Bip showing greatest correlation to serum IgM and B2M levels, and BM involvement (p=0.01). These results confirm that UPR gene expression is related to disease activity in WM, and suggest a particular role for Bip as a prognostic factor in this disease.

The homozygous expression of phenylalanine (F/F) at codon 158 of FcγRIIIa (CD16) is associated with inferior clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with indolent non-Hodgkin’s lymphoma, in contrast to higher response rates observed for Waldenstrom’s macroglobulinemia patients expressing at least one valine (V/F, V/V) and follicular NHL patients who are homozygous for valine (V/V). We attempted elucidate the potential basic mechanism(s) behind these clinical observations by analyzing all the potential implications of this polymorphism among the 3 polymorphic subgroups at FcγRIIIA-158 (F/F, V/F, and V/V). We therefore used peripheral blood isolated natural killer (NK) cells from a pool of 52 unrelated healthy donors who were genotyped for FcγRIIIA-158. We evaluated allele-specific differences for FcγRIIIa gene expression by quantitative RT-PCR and demonstrated higher transcript levels among V/V (23.2 ng/mL) versus V/F (6.7 ng/mL) and F/F (6.2 ng/mL) (p

INTRODUCTION: In previous studies, we observed that CC-5013 enhanced rituximab mediated ADCC killing of lymphoplasmacytic cells (BJH2005; 128:192). As such, we conducted a phase II study of CC-5013 with rituximab in WM patients naïve to either agent. Intended therapy was as follows: Weeks 1-48 CC-5013 (25 mg po qD for 3 weeks, then 1 week off); Weeks 2–5, 13–16 Rituximab (375 mg/m2/week). METHODS: Twelve patients were enrolled, 10 of whom were previously untreated with a median age of 65 (range 53–76 yrs), along with baseline BM involvement of 50 (range 5–90%), serum IgM of 4175 (range 1180–7130 mg/dL), hematocrit of 31.9% (range 24–36.6%), and B2M of 3.5 (range 1.8–6.0 mg/L). RESULTS: Four patients were taken off study due to intolerance to therapy (3 to CC-5013, 1 to CC-5013 and Rituximab) and were non-evaluable. Unexpected acute decreases in hematocrit occurred in 10/12 (85%) patients within the first 2 weeks of therapy, resulting in hospitalization for 4 patients. The median decrease in hematocrit, which coincided with CC-5013 administration was 4.2% (range 1.7-7.5%), and was not associated with blood loss, hemolysis, or generalized myelosuppression. Other grade 3/4 toxicities attributed to CC-5013 therapy were expected and included neutropenia (n=4); tinnitus (n=2); thrombocytopenia (1); myositis (n=1); and rash (n=1). Five patients had a spike in serum IgM levels following rituximab, accompanied with epistaxis (n=1), headaches and blurry vision (n=2) requiring plasmapheresis. Toxicities led to the discontinuation and dose reduction of CC-5013 for 8 and 4 patients, respectively, and discontinuation of rituximab in 1 patient due to recurring allergic reactions. Discontinuation of CC-5013 within the first 28 days of therapy occurred in 6 out of 8 patients. Responses among the 8 evaluable patients: PR (n=3); MR (n=4); SD (n=1); ORR 88% among evaluable patients. In two patients with bulky adenopathy and splenomegaly, a significant reduction in the node/spleen size was noted by week 12. Both of these pts have subsequently achieved a PR. With a median follow-up of 7+ months, no patient who received at least 2 months of CC-5013 have progressed. CONCLUSION: The response data achieved with CC-5013 and rituximab in this study is encouraging, though the dose and schedule of CC-5013 administration, and mechanism for the unexpected acute drops in hematocrit observed in WM patients warrants further investigation. Such studies are in progress in our laboratory.

γWe and others have previously described polymorphisms in codon 158 and 48 for FcγRIIIα as predictors of clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with low-grade non-Hodgkin’s lymphoma, including Waldenström’s Macroglobulinemia. The expression of at least one valine (V) at FcγRIIIα-158 and Leucine/Histidine (L/H) at FcγRIIIα-48 is associated with higher response rates to rituximab, though primacy for FcγRIIIα-158 over 48 polymorphisms for determining rituximab response is attributed to genetic linkage. As part of these studies, we sought to determine if polymorphisms in FCγRIIA were linked to FCγRIIIA since higher response rates have been observed in patients who express Histidine/Histidine (H/H) in FcγRIIA-131. We therefore performed sequence analysis of the whole FcγRIIa gene and exons 3 and 4 of the FcγRIIIa gene in 52 healthy donors. The genetic association between polymorphisms was calculated using the statistical program GOLD and expressed as linkage disequilibrium (D’). Single nucleotide polymorphisms were commonly found in FcγRIIIa at codons 158, 48 and in FcγRIIa at codons 131, 27. The genotype frequencies for polymorphisms are as follows: FcγRIIIa-158: 25% F/F; 63% F/V; 12% V/V; FcγRIIIa-48: 67% L/L; 10% L/R; 17% L/H; FcγRIIa-131: 29% H/H; 46% H/R; 25% R/R; FcγRIIa-27: 71% Q/Q; 27% Q/W; 2% W/W. Strong linkage was found between the FcγRIIIa-158 and −48 (D’=1, p=0.001), as well as between FcγRIIa-131 and -27 (D’=1, p=0.00082). Specifically, the expression of F at FcγRIIIa-158 was found to be associated with expression of L at FcγRIIIa-48, whereas the expression of H at FcγRIIa-131 was associated with the expression of Q at FcγRIIa-27. Importantly, we observed links among polymorphisms at FcγRIIa and FcγRIIIa. Specifically, FcγRIIIa-48 was linked to FcγRIIa-27 (D’=0.765, p

In this multi-center phase II study, 27 patients with the consensus panel definition of Waldenstrom’s macroglobulinemia received bortezomib as a single agent. All but one patient had relapsed/or refractory disease, and the median number of prior therapies was 2 (range 0–3). Intended therapy consisted of 8 cycles of bortezomib at 1.3 mg/m2 on days 1,4,8,11. Twenty-six patients have completed therapy and are eligible for evaluation. The median age for this group was 62 (44–79 years) and baseline laboratories were as follows: serum IgM: 4,565 (1,067–9,330 mg/dl); bone marrow (BM) involvement 30% (1–95%); and hematocrit 34.9% (25.6–45.3%). Following a median of 6 cycles of therapy, serum IgM levels decreased in 25/26 patients to a median of 2,685 (125–5,650 mg/dl), while hematocrit rose in 20/26 patients to a median of 37.2% (30.9–46.3%). The overall response rate was 84.6%, with 11 and 11 patients achieving a minor (

The tumor necrosis factor (TNF) receptor family member, CD27, is a transmembrane co-stimulatory molecule present on primed T and B lymphocytes that also secrete a soluble form (sCD27). Recent evidence has suggested that interactions between CD27 and its TNF-like ligand, CD70, play a critical role in regulating B-cell activation and survival, though the detailed mechanism(s) by which this occurs remain unclear. Waldenstrom’s Macroglobulinemia (WM) represents a lymphoplasmacytic lymphoma characterized by a monoclonal IgM gammopathy and possesses a mast cell component that may contribute to its pathogenesis (Blood 104; 646a). Using ELISA assays, we observed that WM patients displayed significantly higher levels of sCD27 in their sera (median 7.45, range 0–19.42 U/ml) versus healthy donors (median 0, range 0–2.78 U/ml; p=2.5 x 10−7). CD27 was expressed in 7/7 patients using RT-PCR analysis, but was expressed on the cell surface of tumor cells in 5/12 patients using flow cytometric analysis. Conversely, CD70 expression was widely expressed on both tumor cells (6/6 patients) and mast cells (10/11 patients) using flow cytometric analysis. In order to define the functional role of sCD27 in WM, we cultured BCWM.1 (CD27−CD70+) WM cells, and LAD1 (CD27−CD70+) mast cells with sCD27 (0.1–50 ug/mL), and observed no effect on proliferation or induction of apoptosis. Culture of LAD1 cells with sCD27 did, however, result in marked upregulation of the TNF family ligands CD40L (CD154) and a proliferation induction ligand (APRIL), which previous work in our laboratory had implicated as mast cell proliferation and survival factors in WM. Taken together, these studies suggest a novel functional role for sCD27, and imply a pivotal role in the pathogenesis of WM.

INTRODUCTION: In previous studies, we demonstrated that mast cells (MCs) may support the growth and survival of lymphoplasmacytic cells (LPC) in WM patients through expression of several ligands including CD40L, B-LYS, PDGFa and VEGF. While increased numbers of MC, as well as their association with LPC in the bone marrow (BM) of patients with WM has previously been reported, their enumeration before and following therapeutic intervention remains to be clarified. METHODOLOGY: We therefore enumerated MC using tryptase staining in whole sections of BM trephine biopsies before and after therapy for 19 patients (median age 59; range 49–85 years) with the consensus panel diagnosis of WM who received rituximab (n=4), rituximab plus fludarabine (n=14) or bortezomib (n=1). Biopsies from 7 non-WM, age matched patients with normal trilineage myelopoiesis and who demonstrated no evidence of disease were used as controls. RESULTS: At baseline, an increased number of BMMC was observed for WM patients (median 49/mm2; range 0.4–149.5/mm2) versus control patients (median 14/mm2, range 3.8–31.4/mm2); p=0.003. Among non-responding patients, the median number of BMMC rose from 42.76/mm2 (range 23.3–134.6/mm2) to 72.59/mm2 (range 59.1–77.9/mm2, n=4), while it remained stable for minor responders 14.9/mm2 (range 0.4–26.6/mm2) to 15.8/mm2 (range 11.9–44.8/mm2, n=3), and decreased for major responders (CR and PR) from 56.6/mm2 (range 7.1–149.5/mm2) to 34.13/mm2 (range 3.3–110.9/mm2, n=12). CONCLUSION: These preliminary studies demonstrate that BMMC are significantly increased in patients with WM, and their number following therapeutic intervention is dependent on extent of response.