ALBERT

Description: Abstract 2949 Poster Board II-925 Background: The efficacy of rituximab is dependent on a number of host immune interactions, including binding through excitatory (FcγRIIA, FcγRIIIA) as well as inhibitory (FcγRIIB) Fcγ receptors. In previous studies, we showed that polymorphisms in FcγRIIIA-158 predicted outcome to single agent rituximab therapy. Patients displaying L/H or L/R at FcγRIIIA-48 or at least one valine (V/V or V/F) at FcγRIIIA-158 demonstrated greater responses to rituximab versus those patients who expressed FcγRIIIA-48-L/L or FcγRIIIA-158 F/F, respectively (JCO 23:474). The predictive role of FcγRIIIA polymorphisms in patients receiving combination therapy with rituximab has not been addressed to date in WM. We therefore investigated the predictive role of FcγRIIIA-48, -158, as well as other important polymorphisms implicated in modulating IgG antibody binding and activation: FcγRIIA-27, -131, and FcγRIIB-187 in 65 patients with WM who received combination rituximab therapy. Patients and Methods: Sixty-four WM patients with a median age of 61, prior therapies of 0, IgM of 3,540 mg/dL, Hct of 32.3%, B2M of 2.7 g/L, who participated on a clinical study and whose outcomes have previously been reported were included in this analysis. Treatment included rituximab in combination with cyclophosphamide (n=43), thalidomide (n=14), or lenalidomide (n=7). Categorical responses for all patients were as follows: CR/VGPR 7 (11%); PR (n=30; 46.2%); MR (n=18; 27.7%); Non-Responders (n=9; 14.1%) for an overall response rate of 86%. Twenty seven patients have progressed with a median follow-up of 19.4 months. Polymorphic variants at FcγRIIA-27, -131, FcγRIIB-187, and FcγRIIIA-48, -158 were determined by Taq Man real time PCR analysis and sequencing, and impact on overall response, categorical response rates, and progression free survival determined. Results: The expression of H/H at FcγRIIA-131, or at least one valine (V/V or V/F) at FcγRIIIA-158 was associated with improved categorical response, particularly the attainment of CR/VGPR. For FcγRIIA-131, H/H was expressed in 2/9 (22.22%) WM patients who were non-responders; 13/38 (34.2%) patients attaining a major (≥ PR) response, and 4/7 (57.14%) patients who attained a CR/VGPR. For FcγRIIIA-158, the expression of at least one valine was observed in 3/9 (33.3%) WM patients who were non-responders; 20/38 (52.62%) patients attaining a major (≥ PR) response, and 5/7 (71.42%) patients who attained a CR/VGPR. Polymorphisms at FcγRIIA-27, and FcγRIIB-187 showed no association with response. The expression of L/H or L/R at FcγRIIIA-48 was observed in 3/7 (42.86%) patients with CR/VGPR, whereas 2/9 (22.22%) of patients who were non-responders expressed this polymorphism. Subset analysis showed that among patients who received cyclophosphamide based therapy, no differences in polymorphic variation for FcγRIIA-131, FcγRIIIA-48, and -158 were observed between non-responders, major responders and those achieving CR/VGPR. Conclusions: Taken together, the results of this study support a role for the use of FcγRIIA-131, FcγRIII-158, and possibly FcγRIIA-48 as determinants of better categorical responses in WM patients receiving combination therapy with an immunomodulatory agent. The combined use of cyclophosphamide with rituximab therapy appears to negate the inferior outcomes predicted by polymorphic variants in FcγRIIA-131, FcγRIIIA-48, and -158 which have previously been shown to be associated with lower response rates to single agent rituximab therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4159 Waldenstrom's macroglobulinemia (WM) is a B-cell malignancy characterized as an IgM secreting lymphoplasmacytic lymphoma. Familial predisposition is common in WM. Studies to date by us and others have revealed three identifiable clinical subtypes for WM predisposition: * Sporadic; proband has WM, but there is an absence of WM or other B-cell disorders in other family members; * Familial, Mixed B-cell Disorders Subtype; proband has WM, and various B-cell disorders are manifested by other family members. * Familial, WM Only Subtype; proband has WM, and only WM is present in other family members; While these studies suggest a separate genetic predisposition for WM, the correlation of additional cancer risk among all patients with WM and their kin, and well as those sub typed by familial WM predisposition may herald important information for common genetic risks to cancer. We therefore examined the incidence of additional malignancies in 923 consecutive WM patients seen at our Institution, and characterized the frequencies of additional malignancies based on familial subtype and against SEER data. In addition, we also characterized the incidence of solid cancers in kin of WM patients, and sub typed these cancers based on familial WM presentation. Of the 923 patients, 221 (23.9%) patients had at least one additional malignancy to WM. Among these patients, 32 had 2, and 4 (0.43%) had 3 additional malignancies. For 167/221 (75.5%), the associated cancers were diagnosed before WM. The associated malignancies for all patients were as follows: Prostate (n=53; 9.2% of all males); Breast (n=27; 7.7% of all females); Skin (Basal and Squamous; n=61; 6.6%); Skin (Melanoma; n=16; 1.7%); Lung (n=12; 1.3%); Thyroid (n=10; 1.1%); Colorectal (n=7; 0.8%); Bladder (n=8; 0.9%) Other B-cell Malignancies (n=18; 2.0%); Renal (n=6; 0.7%); MDS (n=6; 0.65%); Other (n=11; 1.2%). The incidence of Lung (p=0.002) and Prostate (p=0.07) were higher among WM patients with Familial, Mixed B-cell Disorders Subtype. To avoid potential treatment related impact on additional cancer development, we next adjusted the observed versus expected frequencies based on SEER-17 data. The age adjusted incidence for development of any malignancy among WM patients was 7.6 fold higher when the development of another cancer antedated the diagnosis of WM. Among all WM patients, the incidence of solid cancers among first degree kin were as follows: Prostate (n=98; 10.6%); Breast (n=133; 14.4%); Skin (Basal and Squamous; n=21; 2.3%); Skin (Melanoma; n=21; 2.3%); Lung (n=116; 12.5%); Thyroid (n=9; 1.0%), Colorectal (n=79; 8.6%); Renal (n=8; 0.9%); and Gastric (n=20; 2.2%). The incidence of Breast (p=0.0098), and Skin (Melanoma) (p=0.037) cancers were higher among first degree kin of patients with the Sporadic versus Familial, Mixed B-cell Disorders Subtype. In summary, the above data suggest an increased risk for additional cancers among all WM patients, as well as specific risks for lung and prostate cancer among patients with Familial, Mixed B-cell Disorders Subtype. Moreover, these data also show the association of specific types of solid cancers in first degree kin of WM patients, particularly for WM patients with the Sporadic Subtype. n= Age (Yrs) Gender % Treated Additional Cancers Sporadic 666 60 (29–91) 64% M; 36% F 515 (77%) 163 (24.4%) Familial, Mixed B–cell Disorders 212 58 (36–85) 57% M; 43% F 156 (73%) 50 (23.5%) Familial, WM Only 45 61 (35–89) 56% M; 44% F 35 (77%) 8 (17.7%) Total 923 59 (29–91) 62% M; 38% F 706 (76.4%) 221 (23.9%) Disclosures: No relevant conflicts of interest to declare.

Description: 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

Description: 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.

Description: 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

Description: 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.

Description: Abstract 3736 Poster Board III-672 Introduction Waldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by overproduction of a monoclonal IgM paraprotein which can produce morbidity including hyperviscosity, as well as autoimmune related neuropathy, hemolysis, and thrombocytopenia. Therefore approaches aimed at both suppressing IgM production, as well as selectively inducing apoptosis of WM cells represent an ideal treatment strategy for WM. FcgRIIB is an inhibitory receptor that is expressed on B-cells, and whose expression we recently identified as highly over-expressed in WM. Importantly, FcgRIIB possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM), and which becomes phosphorylated at Tyr 292 upon activation, and is then followed by inhibition of BCR signaling and induction of apoptosis. We therefore validated the expression of this receptor in WM, and examined the impact of its ligation on tumor cell killing, IgM secretion and downstream signaling events in WM cells. Patients and Methods Bone marrow lymphoplasmacytic cells (LPC) from 12 WM patients which were sorted for CD19+ and CD138+, and BCWM.1 WM cells were subjected to real-time PCR and flow cytometric analysis. Cells were then subjected to co-culture with anti- FcgRIIB (AT10, 7.3) or control antibodies for 24-48 hours, and their effects on survival, IgM production and downstream signaling were assessed. Results Real-time PCR and flow-cytometric analysis demonstrated strong expression of FcgRIIB in WM patient bone marrow CD19+ and CD138+ cells, thus confirming our recent microarray results. Importantly, the expression of FcgRIIB in WM LPC correlated with the memory B-cell marker CD27. Anti-FcgRIIB antibody treatment dramatically reduced constitutive, and/or IL-6 induced IgM production in CD19+ and CD138+ sorted primary WM LPC, as well as CD32hiCD138hi BCWM.1 cells. This effect was observed in some experiments at an early time point that had not effected survival. Among primary CD138+ WM LPC and CD32hiCD138hi expressing BCWM.1 cells, treatment with anti-FcgRIIB antibodies for 48 hours led to increased apoptosis in 10 of 12 patients, as assessed by Annexin V and PI staining which occurred despite blockade with a pan-caspase inhibitor, and was even more pronounced when anti-FcgRIIB antibodies were cross-linked. Western blot analysis revealed that treatment of CD32hiCD138hi expressing BCWM.1 cells with cross-linked anti- FcgRIIB antibodies led to phosphorylation of Tyr 292 of the FcgRIIB ITIM which was not observed in the absence of cross-linking. Binding of FcgRIIB by both the AT10 and 7.3 antibodies resulted in dephosphorylation of Akt, which was further reduced in the presence of cross-linking. Coincident with the above, the pro-apoptotic molecule JNK also underwent phosphorylation in the presence of anti-FcgRIIB binding. Conclusion Taken together, these studies validate our previous microarray data by showing that the inhibitory receptor FcgRIIB is strongly expressed on LPC from WM patients, and ligation thereof leads to suppression of IgM production and induction of apoptosis thereby identifying FcgRIIB as a novel therapeutic target in WM. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: 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.

Description: Abstract 1912 Background: Waldenstrom's Macroglobulinemia (WM) is a rare low grade non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in (LPC) the bone marrow, an elevated serum IgM, and frequently accompanied with hyperviscosity syndrome. The insulin receptor substrates (IRS) are important mediators of the insulin like receptor family and PI3K signaling leading to PKC and AKT activation. Methods: TaqMan low density arrays were used to evaluate the relative levels of 667 miRNAs in 11 WM patient and 5 age matched healthy donor (HD) CD19+ bone marrow cells. The results of this screen were validated using individual stem loop RT-PCR assays. Additional mRNA targets were identified using an existing gene expression profiling (GEP) data set of 22 WM patients and 8 HD using the Affymetrix U133 plus 2 platform. GEP findings were validated using an independent cohort of 18 WM patients and 7 HD. Results: Aberrant miRNAs identified were miR-21 (+3.27 fold p=0.035), miR-29c (+3.17 fold; p=0.003), miR-155 (+5.53 fold; p=0.082), miR-9* (-3.94 fold; p=0.001), miR-27b (-4.94 fold; p=0.001), miR-126 (-21.52 fold; p=0.006), miR-126* (-25.55 fold; p=0.039), miR-145 (-34.27 fold; p