Genetic Basis of WM. A familial basis for WM has been suggested by previous reports showing clustering of the disease among relatives of patients with WM. Recently, we reported the results of a study among 257 consecutively diagnosed patients with WM, wherein almost 20% of patients had a first degree relative with either WM or a closely related malignancy including non-Hodgkin’s and Hodgkin’s lymphoma, multiple myeloma, and chronic lymphocytic leukemia. Importantly in this study, we found that patients with “familial WM” presented at an earlier age, and with higher serum IgM and bone marrow involvement.

In an effort to delineate the genetic basis of WM, we have focused our efforts on genes with a role in differentiating B-cells into plasma cells. These studies have identified anomalies in the expression of several genes including BLIMP-1, PAX-5, XBP-1, and IRE1, and suggest that WM may be a genetically heterogeneous disease. At the same time, our studies have sought to identify the predisposition to WM, particularly among familial WM patients. Ongoing research to clarify several genes is underway.

Mast Cells Support the Growth and Survival of WM Cells. Given the propensity for WM to be located in the bone marrow, part of our effort to delineate the influences that permit growth and survival of WM cells has focused on bone marrow microenvironmental support. Interestingly, we, and others, have observed excess mast cells (MC) in bone marrow biopsies of WM patients. These mast cells are commonly found admixed with tumor aggregates. We therefore sought to clarify the role of MC in WM. Co-culture of 0.5% PFA fixed, or sublethally irradiated cell lines - HMC-1, LAD2 or KU812 - and sorted bone marrow lymphoplasmacytic cells (LPC) from 10 WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies.

CD40 Ligand is constitutively expressed by MC in WM patients and supports WM cell growth. We identified a particular growth signal expressed by MC in WM patients, CD40 ligand (CD40L), a member of the tumor necrosis family (TNF) family of ligands. CD40L serves as a potent inducer of both normal as well as malignant B-cell proliferation through interaction with CD40. By immunohistochemical, multicolor flow cytometric and RT-PCR analysis, we found CD40L widely expressed by bone marrow MC of WM patients, but in none of 5 normal donors. Moreover, by multicolor flow cytometry, CD40 was expressed on bone marrow tumor cells from 14 of 17 (83%) patients; we confirmed functionality by the G28.5 CD40 agonistic antibody, which induced dose dependent proliferation of WM cells from 4 of 4 patients. Importantly, expansion of tumor cells from 3 of 4 patients in mixed cultures with paraformaldehyde fixed MC was blocked in a dose dependent manner by use of a CD40L blocking protein (CD40:Fc). These studies, therefore, demonstrate that CD40L is constitutively expressed on bone marrow MC in WM and supports the growth of WM tumor cells.

B-Lymphocyte Stimulator Protein (B-LYS), A Proliferation Inducing Ligand (APRIL), CD70 and CD30 Ligand (CD30L) are expressed by MC and tumor cells in WM. In addition to CD40L, we have also looked at other TNF family members that may have a role in growth and survival of B-cells and IgM production, an important mediator of morbidity and potential precursor to symptomatic hyperviscosity and auto-immune consequences, including neuropathies, rheumatoid athritides, cold agglutininemia, and cryoglobulinemia, in up to half of WM patients. These studies led to the identification of four TNF family members (B-LYS, APRIL, CD70, and CD30 Ligand) as widely expressed antigens on WM MC. Interestingly, B-LYS, APRIL, CD70 and CD30L were also widely expressed on WM tumor cells. Importantly, the receptors for B-LYS (BCMA, TACI and BAFF-R), APRIL (BCMA, TACI) and CD70 (CD27) were identified on WM tumor cells by both flow cytometric analysis and RT-PCR. In contrast, the receptor for CD30 Ligand was found on WM cells in only 1 of 21 cases by flow cytometric analysis. We also recently found that a soluble version of CD27 is highly expressed in the serum of patients with WM, and induces the expression of CD40L and APRIL on MC from WM patients and in mast cell lines. Notably, an antibody to CD70 (SGN-70) blocked soluble CD27 induction of CD40L and APRIL on MC, and is currently being developed as a therapeutic agent for WM and myeloma in cooperation with Seattle Genetics.

Studies to address the functional role of these ligands are underway or under consideration, and will focus on the role of these ligands in normal immunoglobulin (IgA, IgG) production. This is of particular interest given the presence of universal IgA and IgG hypogammaglobulinemia in WM patients and previous reports suggesting a role for BLYS, APRIL and CD40L in IgM->IgA and IgM->IgG heavy chain class switching.

Targeting Mast Cells in WM. Campath-1H is a monoclonal antibody used in the treatment of CD52 expressing B-cell malignancies. Recently, we and others have observed that Campath-1H shows unusually high activity in patients with relapsed/refractory WM. We therefore examined bone marrow MC from patients with WM and other MC disorders for cell surface expression of CD52 to determine if MC might be an apt target for Campath-1H in pre-clinical trials. Multicolor flow cytometric analysis demonstrated CD52 expression on bone MC (FceRI+, CD117+) from 8 of 10 WM and 2 of 2 systemic mastocytosis (SM) patients patients; 3 of 3 healthy donors; as well as on the LAD and HMC-1 MC lines. Moreover, RT-PCR analysis confirmed CD52 expression in sorted bone marrow MC from 6 of 7 WM and 2 of 2 SM patients, and 6 of 6 healthy donors. Importantly, Campath-1H induced high levels of antibody dependent cell mediated cytotoxicity (ADCC) activity against LAD2 mast cells using activated NK effector cells. No direct cytotoxicity or antiproliferative activity by Campath-1H was observed on LAD2 cells. These studies demonstrate that CD52 is widely expressed on human MC and provide support for the use of Campath-1H in the treatment of WM and other systemic mast cell disorders.

Imatinib mesylate (Gleevec) has been widely used in myeloid disorders with both excellent results and good tolerance. Besides its well known activity as an inhibitor of the bcr-abl protein, imatinib has also been shown to target other tyrosine kinase proteins such as the c-kit receptor (stem cell factor receptor, CD117), a receptor universally expressed by WM patient mast cells which demonstrate growth in response to stem cell factor. Importantly, we and others have shown that imatinib inhibits proliferation of mast cell lines, as well as ex vivo expanded bone marrow MC from normal donors. Importantly, in co-culture experiments conducted in stem cell factor media enriched with LAD2 MC and sorted WM cells, imatinib inhibited growth of both tumor and WM cells with a pharmacologically achievable IC50 of 1-10 mmoles/L.