Role of the Chemokine Receptor CXCR4 in Waldenstrom Macroglobulinemia.
Authors: Ghobrial Irene M. , Melhem Mona , Singha Ujjal , George Diane , Timm Michael , Francis Lanie , Alsayed Yazan , Huston Alissa , Roodman G. David , Greipp Philip , Gertz Morie , Witzig Thomas 
Published: April 2020

Abstract
Waldenstrom Macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow, and often in the lymph nodes. The mechanisms by which cells migrate/home to the bone marrow are poorly understood. The chemokine receptor CXCR4 and its ligand SDF-1 have been implicated in the migration and homing of lymphocytes. The objective of this study was to begin to identify the role of CXCR4 in WM. Paraffin embedded tissues were obtained after informed consent from 10 bone marrow samples of asymptomatic WM/monoclonal gammopathy of undetermined significance (MGUS) (n=5) and symptomatic WM (n=5). Four normal bone marrow samples were obtained for control. CXCR4 expression was determined using anti-CXCR4 antibody (Affinity Bioreagents, Golden, CO) 1:200 dilution for 16hrs, and a scale of 0–3+ was used to determine relative expression in the lymphocytes. Anti-CD20 antibody was used to determine the presence of lymphocytes in the samples. To determine whether the receptor is functional, migration was performed with the WM cell line (WSU-WM, kind gift from Dr. Al-Khatib, Wayne State University) and using a transwell migration assay (Costar, Corning, NY) with serial dilutions of SDF-1 (10-100nM). Comparison between the asymptomatic and symptomatic WM groups was performed by c2 test, and Mann-Whitney test, as appropriate. The baseline patient characteristics are displayed in table 1. CXCR4 expression was present on the surface of scattered lymphocytes in the normal bone marrow (median expression 1+). The expression of CXCR4 was increased in the asymptomatic/MGUS cases of WM (median expression 2+). In contrast, CXCR4 expression was downregulated in 4/5 cases of symptomatic WM specifically all the ones with extensive involvement of the bone marrow with lymphoplasmacytic cells (median expression 0). SDF-1 induced a bell- shaped response of migration in the WM cells with the maximal migration occurring at 1-10nM SDF-1. SDF-1 1-10nM induced 340% increase in migration as compared to untreated cells. However, high doses of SDF-1 100nM did not induce any migration as compared to untreated cells indicating inhibition of migration in response to high doses of SDF-1. In summary, we demonstrate that the CXCR4 receptor is functional and induces migration indicating that it plays a role in the migration of WM cells to the bone marrow. In addition, we demonstrate that CXCR4 expression is downregulated in WM with high infiltration of cells in the bone marrow as compared to those with minimal infiltration. These results may be due to the high level of SDF-1 in the bone marrow that induce downregulation of CXCR4. High levels of SDF-1 may downregulate the receptor and inhibit migration in order to confine the cells within the bone marrow. Supported in part by an ASH scholar award and the RFW.
Patient characteristics Asymptomatic WM/MGUS, (range), (N=5) Symptomatic WM, (range) (N=5) Median age at diagnosis 67.6 (55.8–78) 71.5 (61–77) Median IgM level (mg/dL) 997 (904–1490) 5410 (2000–7820) Median Hb (gm/dL) 12.6 (11.1–15.1) 10.8 (9.3–11.6) Organomegaly (liver or spleen) N 20% Lymphadenopathy N 40% Median involvement of tumor cells in the BM in % 5% 30(10–60)