Analysis of Chemokine and Adhesion Markers in Waldenstrom Macroglobulinemia.
Authors: Ngo Hai T. , Leleu Xavier , Moreau Anne-Sophie , Hatjiharissi Evdoxia , Jia Xiaoying , O’Sullivan Garrett , Runnels Judith , Villarreal Rose , Hunter Zachary , Treon Steven , Hideshima Teru , Anderson Kenneth , Ghobrial Irene 
Published: October 2019

Abstract
Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), indicating continuous entry and egression of malignant cells in and out of the BM. The process of homing and trafficking involves chemokines, cytokines, and adhesion molecules. In this study, we sought to determine the level of chemokines, cytokines and adhesion molecules in the BM and peripheral blood (PB) of patients with WM.
Method: We compared BM supernatant to serum samples from 10 patients with WM, including 5 matched samples from the same patients, and PB serum and BM supernatant from 2 normal healthy controls. The Luminex® xMAP® Multiplex (Upstate, VA) allows detection of human cytokines, chemokines, and growth factors using the Beadlite® products (Upstate, VA). We focused on chemokines that regulate migration: Eotaxin, GRO, IP10, MIP1a, MIP1b, MCP1, MCP3, MDC/CCL22, and RANTES; cytokines that regulate proliferation: Interleukin (IL)-2, -4, -6, -8, -10, and -15; and adhesion molecules: ICAM, VCAM. The means fluorescence intensity (MFI) was measured for all molecules. Chemokine and adhesion receptor expression on primary WM cells and WM cell line BCWM.1 was assessed using flow cytometry. Adhesion was measured using the in vitro adhesion assay (EMD Biosciences, CA), and the anti-VLA-4 antibody (10–100ug/ml, BD Pharmingen, CA).
Results: We first compared the BM supernatant of patients with WM to healthy donors. Adhesion molecules ICAM (mean MFI 5624 in WM vs 720 in control) and VCAM (mean MFI 9096 in WM vs 2135 in control) were significantly higher in the WM supernatant, p=0.03 and p=0.05 respectively. In addition, the chemokines IP-10 (Mean MFI 8186 in WM vs 1647 in control), MDC/CCL22 (mean MFI 13015 in WM vs 4221 in control), and GRO (mean MFI 7463 in WM vs 1337 in control), that regulate migration, were significantly upregulated in WM BM supernatant. We found higher expression of MIP1a in the PB of WM patients compared to patient BM supernatant (mean MFI 5715 in PB vs 1484 in BM), p=0.02. Similarly, RANTES and IL-10 were significantly higher in patients’ PB sera. There was no statistically significant difference between the PB sera of patients and healthy controls. The expression of CXCR3 (receptor of IP-10) and CCR4 (receptor of MDC/CCL22) were highly expressed on BCWM.1 (mean 90% expression). We then determined the expression of adhesion receptors on BM CD19+ cells from patient samples and BCWM.1. The WM cells and cell line expressed very high levels of surface adhesion receptors VLA4 and LFA1 (mean expression 95%). Anti-VLA-4 antibody 10ug/ml completely abrogated adhesion to fibronectin, 6% compared to control.
Conclusion: Adhesion molecules VCAM and ICAM and their receptors VLA-4 and LFA-1 were significantly elevated in the bone marrow of WM patients, indicating activation of adhesion receptors and localizing WM cells in the BM. The anti-VLA-4 antibody completely abrogated adhesion in vitro. In addition, the chemokines IP-10, MDC/CCL22 and GRO were upregulated in the BM of patients, which induce homing of cells into the BM. This study, therefore, systematically defines chemokines, cytokines and adhesion markers in WM, providing the preclinical framework for inhibition of adhesion molecules in clinical protocols to improve patient outcome in WM. * HN and XL are co-first authors.