Different Pattern of Expression of CD52, CD20 and CXCR-4 in Peripheral Blood, Bone Marrow and Lymph Nodes in B-Cell Chronic Lymphocytic Leukemia (B-CLL),
Authors: Jaksic Ozren , Gizdic Branimir , Veic Tajana Stoos , Jaksic Vlatka Pandzic , Kusec Rajko , Pejsa Vlatko , Jaksic Branimir 
Published: October 2019

Abstract
Abstract 3877
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by variable clinical presentation with different involvement of various lymphoid compartments i.e. peripheral blood, bone marrow and lymphoid organs such as lymph nodes and spleen. Also there is a well documented intraclonal and interclonal variability of B-CLL cells in different microenvironments regarding a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variable distribution of tumor mass has strong association with prognosis and a well documented influence on response to antibodies like rituximab and alemtuzumab. There is a well documented efficacy of rituximab in cases with with 11q deletion (associated with significant lymphadenopathy), and known resistance to alemtuzumab in pateints with bulky lymphadenopaty (>5cm).
Aim of this study was to evaluate level of expression of CD20 and CD52 along with key chemokine receptor CXCR-4 and proliferation marker Ki-67 on B-CLL lymphocytes and intra and interclonal differences dependent on different microenvironment, ie peripheral blood (PB), bone marrow (BM) and lymph nodes (LN).
PB, BM and LN samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression levels of CD20, CD52, CXCR-4 and Ki-67 molecules on CD19+CD5+ B-CLL cells were analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests.
We have analyzed samples taken from 25 typical B-CLL patients with median age of 72 years. There were 14 males and 11 females. Mean beta-2 microglobulin was 4.3mg/l, mean TTM size was 9.8 and mean TD was 0.74. There were 13, 5 and 7 patients in Binet stage A, B and C, respectively. There were 4 previously treated patients (patients were not treated 3 months before sampling) of whom one patient was previously treated with both rituximab and alemtuzumab. Among included patients there were patients with 11q deletion and with 17p deletion. Median expression level (MFI) of CD52 on B-CLL cells was 171, 193, and 352 for PB, BM and LN respectively (p<0.05) and on T cells was 224, 171 and 137 (p<0.05). Median expression level (MFI) of CD20 on B-CLL cells was 7.5, 5.7 and 4.7 for PB, BM and LN respectively (p<0.05). These results were very consistent in this clinically and cytogenetically heterogenous group of B-CLL patients showing the same pattern in almost all patients. Median expression level (MFI) of CXCR-4 was 9.6, 5.9 and 2.6 (p<0.05) and Ki-67 was 1.08, 1.27 and 1.64 (p<0.05) for PB, BM and LN respectively. There was no correlation of CXCR-4 with CD20 and C52 expression in any compartment and there is positive correlation of Ki-67 with both CD20 and CD52 in PB but not in other compartments.
Relatively unexpected results demonstrating the lowest level of expression of CD20 on B-CLL cells in lymph nodes compared to PB and BM and the highest expression of CD52 on B-CLL cells in LN compared to PB and BM (but with the opposite pattern on T cells) is inversely related to known efficacy of agents (ie rituximab and alemtuzumab) targeting these molecules in these lymphoid compartments. These results indicate that other factors in selected microenvironment (beside number of molecules on cell surface) regulate sensitivity of B-CLL cells on rituximab and alemtuzumab in vivo. These may include other cells (like T cells) and soluble factors. Also in this study levels of expression were not clearly related to molecules involved in recirculation (CXCR-4) and proliferation (Ki-67) indicating that other factors in microenvironment may be important for the observed expression levels. These results warrant further studies to indentify these factors which may eventually uncover novel therapeutic targets.

Disclosures:
No relevant conflicts of interest to declare.