CD27-CD70 interactions in the pathogenesis of Waldenstrom macroglobulinemia.

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

Genetic linkage of Fc gamma RIIa and Fc gamma RIIIa and implications for their use in predicting clinical responses to CD20-directed monoclonal antibody therapy.

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

CD52 is expressed on human mast cells and is a potential therapeutic target in Waldenstrom's Macroglobulinemia and mast cell disorders.

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

CD5, CD10, and CD23 expression in Waldenstrom's macroglobulinemia.

CD5, CD10, and CD23 are cell surface antigens used to distinguish B-cell disorders. The expression of these antigens and their clinical significance in Waldenstrom's macroglobulinemia (WM), an uncommon B-cell disorder, remains to be clarified. We therefore determined expression of CD5, CD10, and CD23 by flow cytometric analysis on bone marrow lymphoplasmacytic cells (CD19+ k/l light chain restricted) for 171 serially biopsied patients with findings of the consensus panel definition of WM. Importantly, we also correlated laboratory and clinical data, as well as existence of a familial history of a B-cell disorder in view of reports suggesting familial predisposition in WM. These studies demonstrated tumor cell expression of CD5, CD10, and CD23 in 15 of 171 patients (9%), 11 of 161 patients (7%), and 37 of 105 patients (35%), respectively. Coexpression of CD23 with CD5 or CD10 was common. Tumor Lymphoplasmacytic from 10 of 15 (66%) and 3 of 11 (27%) patients with WM that expressed CD5 and CD10, respectively, also showed expression of CD23 (P = 0.01 and P = 0.08, respectively). Among patients with CD23 expression, increased serum immunoglobulin (Ig) M levels were observed compared with patients without CD23 expression (P = 0.05). No differences in age at diagnosis; presence of adenopathy and/or splenomegaly; bone marrow involvement; serum IgA, IgB, and b2 macroglobulin levels; hematocrit; platelet count; or familial history of WM or a related B-cell disorder were observed among patients with and without CD5, CD10, and CD23 expression. These studies demonstrate that CD5, CD10, and CD23 are commonly found in WM and that their expression should not exclude the diagnosis of WM. Moreover, expression of CD23 may define a clinically distinct subset of patients with WM.

Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism.

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

A SCID-hu in vivo model of human Waldenström macroglobulinemia.

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