Lymphoplasmacytic Cells In Bone Marrow Research Articles

Waldenström macroglobulinemia: 2023 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients and is found in most IgM MGUS patients. MYD88 is not required for the diagnosis. Age, hemoglobin level, platelet count, β2 microglobulin, LDH, and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or a BTK inhibitor. The preferred Mayo Clinic induction is either rituximab and bendamustine (without rituximab maintenance) or zanubrutinib. Bortezomib, cyclophosphamide, fludarabine, thalidomide, everolimus, Bruton Tyrosine Kinase inhibitors, carfilzomib, lenalidomide, bendamustine, and venetoclax have all been shown to have activity in relapsed WM. Given WM's natural history, the reduction of therapy toxicity is an important part of treatment selection.

Waldenström macroglobulinemia: 2021 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients and is found in the majority of IgM MGUS patients. Age, hemoglobin level, platelet count, β2 microglobulin, LDH and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or ibrutinib. Purine nucleoside analogues are active but usage is declining in favor of less toxic alternatives. The preferred Mayo Clinic induction is rituximab and bendamustine. Bortezomib, fludarabine, thalidomide, everolimus, Bruton Tyrosine Kinase inhibitors, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in relapsed WM. Given WM's natural history, reduction of therapy toxicity is an important part of treatment selection.

Waldenström macroglobulinemia: 2019 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in >90% of patients and is found in the majority of IgM monoclonal gammopathy of undetermined significance patients. Age, hemoglobin level, platelet count, β2 microglobulin, and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or ibrutinib. Purine nucleoside analogs are active but usage is declining for less toxic alternatives. The preferred Mayo Clinic induction is rituximab and bendamustine. Potential for stem cell transplantation should be considered in selected younger patients. Bortezomib, fludarabine, thalidomide, everolimus, ibrutinib, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in relapsed WM. Given WM's natural history, reduction of therapy toxicity is an important part of treatment selection.

Annual clinical updates in hematological malignancies: a continuing medical education series.

Disease overview: Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, and lymphadenopathy. Diagnosis: Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. Risk stratification: Age, hemoglobin level, platelet count, β 2 -microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Risk adapted therapy: Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all US patients with WM and can be combined with alkylating agent or purine nucleoside analogue, or both. The preferred Mayo Clinic nonstudy therapeutic induction is rituximab, cyclophosphamide, and dexamethasone. Future stem cell transplantation should be considered in induction therapy selection. Management of refractory disease: Bortezomib, thalidomide, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM's natural history, reduction of complications will be a priority for future treatment trials.

Waldenström macroglobulinemia: 2017 update on diagnosis, risk stratification, and management.

Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients. Risk Stratification: Age, hemoglobin level, platelet count, β2 microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Risk-Adapted Therapy: Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all U.S. patients with WM and can be combined with bendamustine, an alkylating agent, or a proteosome inhibitor. Purine nucleoside analogues are widely used in Europe. The preferred Mayo Clinic nonstudy therapeutic induction is rituximab and bendamustine. Potential for stem cell transplantation should be considered in induction therapy selection. Management of Refractory Disease: Bortezomib, fludarabine, thalidomide, everolimus, ibrutinib, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM's natural history, reduction of complications will be a priority for future treatment trials. Am. J. Hematol. 92:209-217, 2017. © 2016 Wiley Periodicals, Inc.

Targeting IRAK1/IRAK4 Signaling in Waldenstrom's Macroglobulinemia

Background: MYD88 L265P somatic mutations are highly prevalent in Waldenström's macroglobulinemia (WM) (NEJM 367(9):826-33). MYD88 L265P activates multiple downstream signaling pathways including BTK and IRAK1/IRAK4 that support malignant cell growth and survival (Nature 470(7332):115-9; Blood 122(7):1222-32). Ibrutinib targets BTK, and shows high overall and major clinical response rates, though no complete responses are observed indicating alternative survival signaling.Methodology: Phospho-flow analysis of IRAK1, IRAK4, and BTK was performed in primary WM cells taken from untreated WM patients, and those on ibrutinib therapy. IRAK1 or IRAK4 knockdown experiments were performed using lentiviral shRNA transduction. Immunoprecipitation, western blot and phospho-flow studies were used to detect protein expression and phosphorylation in WM cells. Cell survival following IRAK4 or IRAK1 knockdown, ibrutinib and/or IRAK4/IRAK1 inhibitor (EMD Millipore) treatment was assessed by Annexin V staining, AlamarBlue® Cell Viability Assay or CellTiter-Glo® Luminescent Cell Viability Assays.Results: Phospho-flow analysis of bone marrow lymphoplasmacytic cells taken from WM patients following > 6 months of continued ibrutinib treatment demonstrated highly active IRAK1 and IRAK4, but not BTK. These findings prompted us to dissect the relative impact of IRAK1 and IRAK4 in supporting WM cell survival. Using lentiviral transduction, we identified shRNAs that produced similar levels of protein reduction by western blot analysis for both IRAK1 and IRAK4. Compared to scrambled control vector, knockdown of IRAK1 or IRAK4 both produced decreased tumor cell survival in MYD88 mutated BCWM.1 and MWCL-1 cells. More pronounced apoptosis, as well as sustained reduction in tumor cell growth occurred following knockdown of IRAK1 versus IRAK4. Treatment of primary WM cells taken from untreated patients, patients on ibrutinib therapy, as well as MYD88 mutated WM cells lines with ibrutinib and a toolbox IRAK4/IRAK1 inhibitor resulted in more robust reductions in NFkB signaling, and at least additive tumor cell killing versus either agent alone.Conclusions: MYD88 L265P mutated WM cells show greater dependence on IRAK1 versus IRAK4 directed signaling. IRAK1/IRAK4 signaling may contribute to persistent WM cell survival following ibrutinib treatment. Combined BTK and IRAK inhibition leads to augmented blockade of NFKB signaling and enhanced WM cell killing. The studies provide a framework for the development and investigation of IRAK inhibitors, alone and in combination with ibrutinib in WM patients. DisclosuresNo relevant conflicts of interest to declare.

Waldenström macroglobulinemia: 2015 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients. Age, hemoglobin level, platelet count, β2 microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all US patients with WM and can be combined with alkylating agent or purine nucleoside analog (or both). The preferred Mayo Clinic nonstudy therapeutic induction is rituximab, cyclophosphamide, and dexamethasone. Future stem cell transplantation should be considered in induction therapy selection. Management of Refractory Disease: Bortezomib, thalidomide, everolimus, ibrutinib, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM's natural history, reduction of complications will be a priority for future treatment trials.

As a Rare Presentation of Waldenstrom’s Macroglobulinemia; Fever

To the Editor, Waldenstrom’s macroglobulinemia (WM) is a B cell lymphoplasmacytoid lymphoma, which is characterized by a presence of serum monoclonal immunoglobulin M and clonal lymphoplasmacytic cells in bone marrow.1,2 Lymphadenopathy, splenomegaly, leucopenia, thrombocytopenia, anemia, hyperviscosity and peripheral neuropathy are commonly seen in WM.2 B-symptoms (weight loss, fever or night sweats) may also be detected in WM patients.2 Although fever is a B symptom, WM is not considered as a diagnosis in a febrile patient.3 In literature fever accompanied with WM were reported either with other B-symptoms or due to infection. Thus, at the time of diagnosis most of WM patients have no fever.4-6 Here we would like to present a patient, who has been diagnosed with WM during an evaluation of fever. A 51-year-old Caucasian woman has been admitted to our hospital with a 4-day-fever and fatigue. She has been treated with inhaler steroid and s-2 agonist because of asthma. She had no dysuria, cough, sputum, weight loss, night sweats or diarrhea. In physical examination liver was palpable as 2 cm at the right mid-

A Prospective Multicenter Study Of The Bruton's Tyrosine Kinase Inhibitor Ibrutinib In Patients With Relapsed Or Refractory Waldenstrom's Macroglobulinemia

IntroductionWhole genome sequencing has revealed highly prevalent somatic mutations in WM (Hunter et al, ICML-12, 2013). MYD88 L265P is present in >90% of patients with Waldenstrom's Macroglobulinemia (WM), and supports malignant growth via signaling involving Bruton's Tyrosine Kinase (BTK). Ibrutinib inhibits BTK, and in vitro induces apoptosis of WM cells bearing MYD88 L265P (Yang et al, Blood 2013). WHIM-like mutations in CXCR4 are present in 1/3 of patients with WM, and their expression induces BTK activity and confers decreased sensitivity to ibrutinib mediated growth suppression in WM cells (Cao et al, ASH 2013, submitted). We therefore evaluated the efficacy and tolerability of ibrutinib in relapsed or refractory WM, and examined the impact of MYD88 L265P and WHIM-like CXCR4 mutations on ibrutinib response given our laboratory findings. Patients and MethodsSymptomatic WM patients who received at least 1 prior treatment were enrolled on this prospective clinical trial. Intended therapy consisted of 420 mg of oral ibrutinib daily for 2 years or until progression, or unacceptable toxicity. Sanger sequencing was used to determine MYD88 and CXCR4 mutations in sorted bone marrow lymphoplasmacytic cells (BM LPC) from 43 and 40 patients, respectively. Forty of 43 (93%) and 10/40 (25%) patients had MYD88 L265P and WHIM-like CXCR4 mutations, respectively. Results63 patients including 17 with refractory disease were enrolled; all 63 are evaluable for response and toxicity. Median baseline characteristics: Age 63 (range 44-86) ; Prior therapies 2 (range 1-6); Hematocrit 30.8% (range 24.5-41.5); Hemoglobin 10.5 g/dL (8.2-13.8 g/dL); serum IgM 3,610 mg/dL (range 735-8390 mg/dL); serum M-protein 2.14 g/dL (range 0.5-5.4 g/dL); B2M 3.9 mg/L (range 1.3-14.2 mg/L); BM disease involvement 65% (range 3.2-95%). At best response, median serum IgM levels and M-protein declined to 1,340 mg/dL and 0.84 g/dL, respectively (p<0.00001). Median hematocrit and hemoglobin rose to 38.1% and 12.6 g/dL, respectively (p<0.00001). Post-treatment bone marrow assessment at 6 months is available for 34 patients at this time, and showed a reduction from 70% to 45% in WM disease involvement for these patients (p=0.0006). With a median follow-up of 6 (range 2-15 cycles), the best overall response rate i.e. minor response (MR) or better using consensus criteria adapted from the 3rd International Workshop on WM is 81% (4 VGPR; 32 PR, 15 MR), with a major response rate (PR or better) of 57.1% and a median time to response of 4 weeks. 11 patients have stable disease, and 1 patient was a non-responder. Independent disease and response assessments are planned. Grade >2 treatment related toxicities include thrombocytopenia (n=9; 14.3%); neutropenia (n=12; 19.1%); stomatitis (n=1; 1.6%); atrial fibrillation (n=1; 1.6%); diarrhea (n=1; 1.6%); herpes zoster (n=1; 1.6%); hematoma (n=1; 1.6%); hypertension (n=1; 1.6%) and epistaxis (n=1; 1.6%). 59 patients remain on study with 7 on reduced doses of ibrutinib. Reasons for discontinuation include non-response (n=1) in a patient with wild type MYD88; MDS/RAEB (n=1) in a heavily pre-treated patient who attained PR, but who had 5q deletions pre-dating protocol therapy; thrombocytopenia (n=1) in a patient with splenic entrapment; and patient decision (n=1). In patients who underwent tumor sequencing, attainment of major responses was impacted by mutations in CXCR4, but not MYD88 L265P. The major response rate was 77% for patients with wild-type CXCR4 vs. 30% in those with WHIM-like CXCR4 mutations (p=0.018). Decreases in serum IgM (p=0.047) and IgM M-spike (p=0.012), as well as improvements in hemoglobin (p=0.058) were greater in patients with wild-type CXCR4. Patients with wild-type CXCR4 also had increased peripheral lymphocytosis following ibrutinib treatment versus those with WHIM-like CXCR4 mutations (p=0.001). ConclusionsIbrutinib is highly active, and well-tolerated in patients with relapsed or refractory WM. Rapid reductions in serum IgM and improved hematocrit occur in most patients receiving ibrutinib. The presence of WHIM-like CXCR4 mutations impacts responses and peripheral lymphocytosis in WM patients undergoing ibrutinib treatment. Disclosures:Off Label Use: Ibrutinib is a Bruton's Tyrosine Kinase (BTK)inhibitor that, in vitro, induces apoptosis of Waldenstrom's macroglobulinemia cells bearing somatic mutations of MYD88 L265P.

Waldenström macroglobulinemia: 2013 update on diagnosis, risk stratification, and management

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, and lymphadenopathy. The presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. Age, hemoglobin level, platelet count, β2 microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all US patients with WM and can be combined with alkylating agent or purine nucleoside analog (or both). The preferred Mayo Clinic nonstudy therapeutic induction is rituximab, cyclophosphamide, and dexamethasone. Future stem cell transplantation should be considered in induction therapy selection. Bortezomib, thalidomide, everolimus, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM's natural history, reduction of complications will be a priority for future treatment trials.

Use of whole genome sequencing to identify highly recurrent somatic mutations in Waldenström’s macroglobulinemia.

8107 Background: Waldenstrom’s Macroglobulinemia (WM) is an IgM secreting lymphoplasmacytic lymphoma. The genetic basis for this disease remains to be clarified. Methods: We performed whole genome sequencing (WGS) using CD19+ selected bone marrow lymphoplasmacytic cells (LPC) from 30 WM patients. For 10 of these patients, paired CD19+ depleted peripheral blood samples were used for WGS as normal controls. Results: The most common somatic variants identified and validated by Sanger sequencing included MYD88 L265P, an activating mutation for IRAK/TRAF6/NFKB and MAPK signaling, which was observed in 27/30 (90%) patients; the N-terminal domain of CXCR4, which included mutations associated with WHIM syndrome and confer constitutive CXCR4 signaling resulting from dysfunctional receptor endocytosis, a finding observed in 8/30 (27%) patients, and ARID1A (5/30; 17%), a tumor suppressor gene. Less common somatic variants were also identified in MUC16 (4/30; 13%), TRAF2 (3/30; 10%), TRRAP (3/30; 10%) and MYBBP1A (2/30; 7%). Conclusions: Using WGS and confirmatory Sanger sequencing, we have identified several somatic variants with oncogenic function, the most common of which include MYD88 L265P, the N-terminal domain of CXCR4, and ARID1A.

Waldenström Macroglobulinemia: 2012 update on diagnosis, risk stratification, and management

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, and lymphadenopathy. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. Age, hemoglobin level, platelet count, β(2) microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all US patients with WM and can be combined with alkylating agent or purine nucleoside analog (or both). The preferred Mayo Clinic nonstudy therapeutic induction is rituximab, cyclophosphamide, and dexamethasone. Future stem-cell transplantation should be considered in induction therapy selection. Bortezomib, thalidomide, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM's natural history, reduction of complications will be a priority for future treatment trials.

MicroRNA-21 and −155 Are Induced by a Widely Expressed Mutation in MyD88 (L265P) in Waldenstrom’s Macroglobulinemia,

Abstract 3957 Introduction:MicroRNA dysregulation has been described in Waldenstrom’s Macroglobulinemia (WM), an indolent non-Hodgkin’s lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells (LPC). Whether MicroRNA dysregulation represents a primary oncogenic event, is reactive to an oncogenic event, or is related to the stage of B-cell differentiation remains to be clarified. As part of these efforts, we sought to delineate aberrant MicroRNAs which emerge by direct comparison of WM LPC with memory B-cells, from wherein the WM clone is believed to have emerged. Further to these efforts, we also sought to clarify whether MicroRNA dysregulation is related to the presence of a somatic mutation in MYD88 (L265P), a widely expressed mutation in WM patients, which also induces NF-kB and STAT3 activation via IRAK 1/4 (Nature 2011, 470:115–119). Patients and Methods:CD19+ selected bone marrow cells from WM patients were isolated by immunomagnetic sorting. Healthy donor B-cells (CD19+) were isolated from peripheral blood, and further sorted using a memory B-cell (CD19+ CD27+) isolation kit (Miltenyi, Auburn CA). MicroRNA profiling was performed using Taqman low-density arrays. Taqman stem-loop Real-Time PCR was used for validation of MicroRNA changes. MYD88 L265P expressing BCWM.1 and MWCL-1 cells were used for lentiviral transduction experiments, and inhibitory studies using an IRAK 1/4 kinase inhibitor (407601, EMD, Gibbstown, USA) in order to assess changes in MicroRNA expression. STAT3 and IKKα phosphorylation were detected by Western blot using phospho-specific antibodies. Results:MicroRNA profiling was performed using bone marrow LPC from 11 WM patients, and compared to total B-cells, and selected memory B-cells from 7 healthy donors. Real-time PCR was also performed in 27 additional patients for a total of 38 patients which showed that MicroRNA-21, −155 and −29c were increased, and MicroRNA-9*, −27b, −126, and −145 were decreased when WM LPC were directly compared with total healthy donor CD19+ cells. When WM LPC were directly compared to healthy donor memory B-cells, only MicroRNA-21 and −155 remained significantly higher in WM LPC. To investigate whether MicroRNA-21 and −155 are impacted by the MYD88 L265P mutation, we next performed lentiviral knock-down experiments in BCWM.1 and MWCL-1 cells. Levels of MicroRNA-21 and −155 decreased following knockdown of MYD88, whereas over-expression of MYD88 L265P led to enhanced Micro-RNA 21 expression. In addition, MicroRNA-21 but not −155 decreased following treatment of BCWM.1 and MWCL-1 cells with an IRAK 1/4 kinase inhibitor potentially signifying differences in downstream activation of MicroRNA-21 and −155 by MYD88 L265P. Conclusion:Differences in MicroRNA expression vary based on which population of healthy donor B-cells are compared to WM LPC. Direct comparison of WM LPC to healthy donor memory B-cells reveals differences in MicroRNA-21, and −155. Importantly, MicroRNA-21 and −155 are induced by the MYD88 L265P mutation through distinct signaling pathways. The data suggest a reactive role for MicroRNA-21 and −155 in response to the MYD88 L265P mutation in WM. Disclosures:No relevant conflicts of interest to declare.

Disruption of MYD88 Pathway Signaling Leads to Loss of Constitutive IRAK1, NF-κβ and JAK/STAT Signaling and Induces Apoptosis of Cells Expressing the MYD88 L265P Mutation in Waldenstrom’s Macroglobulinemia

Abstract 597 Introduction:Through whole genome sequencing of bone marrow lymphoplasmacytic cells (LPCs) from WM patients, we recently identified a broadly expressed somatic variant (MYD88 L265P) which was present in 90% of WM patients, as well as in BCWM.1 and MWCL-1 WM cells. The presence of this variant has been reported to confer oncogenic activity in cell lines derived from patients with DLBCL ABC subtype lymphomas by induction of IRAK 1/4 kinase activity, and downstream signaling via NF-κβ (Ngo et al, Nature 2011, 470:115-119). We therefore sought to delineate the activity of agents which either disrupt MYD88 homodimerization, a prerequisite to IRAK 1/4 auto-phosphorylation, or by direct inhibition of IRAK 1/4 kinase activity in WM. Patients and Methods:Primary WM LPCs were isolated from bone marrow biopsy specimens from WM patients, and CD19+ B-cells were isolated from the peripheral blood of healthy donors. The mutational status of MYD88 was identified by Sanger sequencing, which confirmed the presence of the L265P mutation in BCWM.1 and MWCL-1 cells, and absent in Ramos and MM1S cells. Western blot and phospho-flow studies using phospho-specific antibodies were performed for signaling proteins downstream of MYD88, cell killing evaluated by Annexin V/PI staining and AlamarBlue, and IgM and IL-6 release determined after treatment with and without inhibitors of MYD88 homodimerization (IMG-2005-5, IMGENEX, San Diego CA), and IRAK 1/4 kinase function (407601, EMD, Gibbstown, USA). Results:Phosphorylation of the MYD88 downstream signaling proteins IRAK1, IκBα, NFκβ-p65 and STAT3 was greater in MYD88 L265P expressing BCWM.1 and MWCL-1 cells, and primary WM patient LPC, versus MYD88 wild type expressing Ramos, MM1S, and healthy donor CD19+ B-cells. Inhibition of MYD88 homodimerization and IRAK 1/4 kinase activity led to decreased phosphorylation of IRAK1, IκBα, NFκβ-p65 and STAT3 in BCWM.1 and MWCL-1 cells. Importantly, both inhibitors induced robust apoptosis of BCWM.1 and MWCL-1 cells, and primary WM LPCs bearing the MYD88 L265P mutation in comparison to Ramos, MM1S, or healthy donor CD19+ B-cells. Activation of caspase-3 and PARP, decreased BCL-2 expression, and decreased release of IL-6 and IgM accompanied treatment of WM cells following treatment with inhibitors of MYD88 homodimerization and/or IRAK 1/4 kinase activity. Conclusion:Disruption of MYD88 pathway signaling leads to loss of constitutive IRAK1, NF-κβ and JAK/STAT activation and induces apoptosis of cells expressing the MYD88 L265P mutation in WM. The results of this study provide novel insights into the pathogenesis of WM, and provide a framework for the clinical investigation of inhibitors of the MYD88 pathway in WM. Disclosures:No relevant conflicts of interest to declare.

Waldenström macroglobulinemia: 2011 update on diagnosis, risk stratification, and management

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, and lymphadenopathy. Presence of IgM monoclonal protein associated with 10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. Age, hemoglobin level, platelet count, b2-microglobulin, and monoclonal IgM concentrations are characteristics required for prognosis. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-based therapy is used in virtually all US patients with WM and can be combined with alkylating agent or purine nucleoside analogue, or both. The preferred Mayo Clinic nonstudy therapeutic induction is rituximab, cyclophosphamide, and dexamethasone. Future stem cell transplantation should be considered in induction therapy selection. Bortezomib, thalidomide, lenalidomide, and bendamustine have all been shown to have activity in WM. Given WM’s natural history, reduction of complications will be a priority for future treatment trials.

Successful treatment with rituximab and thalidomide of POEMS syndrome associated with Waldenstrom macroglobulinemia

A POEMS syndrome is a rare disorder characterized by p olyneuropathy, o rganomegaly, e ndocrinopathy, m onoclonal gammopathy and s kin abnormalities including hyperpigmentation and hypertrichosis. Here we report a 55-year-old female case of a POEMS syndrome associated with Waldenstrom macroglobulinemia. The patient had bed-bound polyneuropathy, splenomegaly, IgM-λ type monoclonal (M) protein, elevated λ-type free light chain (FLC), infiltration of CD20-positive lymphoplasmacytic cells in bone marrow, edema and hypertrichosis, and was diagnosed to have an ‘atypical’ POEMS syndrome associated with macroglobulinemia. Nerve conduction studies and a sural nerve biopsy confirmed a demyelination and axonal degeneration without IgM deposition on myelin sheathes. None of neuron-related auto-antibodies characteristic of IgM paraproteinemic neuropathies was detected in her serum and cerebrospinal fluid. Weekly administration of rituximab (375 mg/m 2) combined with thalidomide (50 mg/day) was initiated. By eight weeks of the treatment, the ambulation activity of the patient was restored and her polyneuropathy completely disappeared as determined by clinical symptoms and electrophysiological examinations. This is the first case report presenting a POEMS syndrome associated with WM treated with rituximab and thalidomide. The further examinations of the present case should shed light on the pathogenesis of the ‘atypical’ POEMS syndrome.

High-Throughput Microrna Profiling in Patients with Waldenstrom's Macroglobulinemia.

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<0.05). Analysis of bone marrow derived CD138+ cells from WM patients demonstrated significant upregulation in miR-192, -193b, -17-3p, -585, -148b, whereas miR-29c, -155, -126, -148a, -125a, -181d, -30a-3p, let-7b, let-7c were downregulated in comparison to healthy donor CD138+ bone marrow cells (p<0.05). Importantly, characterization of the modulated microRNAs found in these studies demonstrated a critical role in growth and survival pathways through modulation of several genes including HOX, BCL-2 and c-myc. Taken together, these studies demonstrate significant differences in microRNA expression between comparable WM and healthy donor lymphoplasmacytic cell populations, and identify aberrancies in microRNAs with a pivotal role in the growth and survival of B-cells.

Establishment of BCWM.1 cell line for Waldenström's macroglobulinemia with productive in vivo engraftment in SCID-hu mice

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

Preclinical In Vitro and In Vivo Evidence Support a Therapeutic Role for the CD70 Directed Monoclonal Antibody (SGN-70) in Waldenström's Macroglobulinemia (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.

Mast cells in Waldenstrom's macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling

Bone marrow (BM) mast cells (MC) are commonly found in association with lymphoplasmacytic cells (LPC) in patients with Waldenström's macroglobulinemia (WM). We therefore sought to clarify the role of MC in WM. Co-culture of sublethally irradiated HMC-1 MC, KU812 basophilic cells, or autologous BM MC along with BM LPC from WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. Furthermore, by immunohistochemistry, multicolor flow cytometry and/or RT-PCR analysis, CD40 ligand (CD154), a potent inducer of B-cell expansion, was expressed on BM MC from 32 of 34 (94%), 11 of 13 (85%), and 7 of 9 (78%) patients, respectively. In contrast, MC from five healthy donors did not express CD154. By multicolor flow cytometry, CD154 was expressed on BM LPC from 35 of 38 (92%) patients and functionality was confirmed by CD154 and CD40 agonistic antibody stimulation, which induced proliferation, support survival and/or pERK phosphorylation of LPC. Moreover, MC induced expansion of LPC from 3 of 5 patients was blocked in a dose dependent manner by use of a CD154 blocking protein. These studies demonstrate that in WM, MC may support tumor cell expansion through constitutive CD154-CD40 signaling and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.