PB1879 MULTIPARAMETRIC FLOW‐CYTOMETRY ANALYSIS OF SURFACE MARKERS EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: PATHOGENIC AND PROGNOSTIC IMPLICATIONS.
Authors: Guastafierro S. , Spada A. , Ferrara M.G. , Scalia G. , Raia M. , Falcone U. , Parascandola R.R. , Miranda G. , Sica A. , Chiurazzi F. , Lucivero G. 
Published: June 2019

Background:Chronic lymphocytic leukaemia (CLL) is a highly heterogeneous disease with many possible clinical courses. CLL clinical variability is related to the immunobiology of CLL clones in each patient and to differences in leukemic‐cell survival, activation and proliferation, interactions with the microenvironment.Aims:This study aimed to analyze the expression of surface molecules in monoclonal CD19+CD5+CD23+ clones from CLL patients and to evaluate direct or reverse correlations within their expression in order to define their pathogenic relevance.Methods:Peripheral blood (PB) samples from 81 subjects with untreated CLL were analyzed by a FACSCanto II System (Becton Dickinson) equipped with 3 lasers for simultaneous analysis of 6 surface molecules detected with monoclonal antibodies conjugated to different fluorochromes and specific for the following membrane molecules: CD45, CD3, CD4, CD8, CD56, CD19, CD23, CD22, CD5, CD20, CD43, CD38, CD10, CD103, CD25, CD11c, CD31, CD40, CD27, CD49d, CD80, CD86, CD69,CD95, CD71, CD72, CD41, CD42b, kappa and lambda light chains. Statistical analysis included determination of means and standard deviations of percentages of CD19+ cells and of CD19+ cells co‐expressing the membrane molecules previously indicated. Within the molecules co‐expressed by CD19+ lymphocytes, we calculated the correlation coefficient by Pearson's test.Results:PB CD19+ B lymphocytes represented approximately 70% of circulating lymphocytes. According to the phenotypic pattern of CLL cells, more than 85% of CD19+ cells, that showed a dim expression of kappa or lambda chains, co‐expressed CD5 (90.7 ± 14.5%), CD23 (87.6 ± 12.2%). Furthermore, CD19+ cells co‐expressed at high levels CD22 (94.2 ± 5.3%), CD43 (85.7 ± 14.1%), CD31 (84.3 ± 15.6%) and CD72 (72.6 ± 23.3%). The phenotypic heterogeneity of the CLL clones was more pronounced in the co‐expression of CD27 (62.1 ± 20.5%), CD40 (58.0 ± 26.8%), CD20 (60.4 ± 24.6%), CD25 (42.2 ± 30.1%), CD49d (26.3 ± 32.7%), CD95 (20.9 ± 14.0%), CD38 (21.8 ± 27.7%), CD69 (20.2 ± 18.1%), CD41 (10.5 ± 7.7%), CD42b (10.4 ± 6.2%), CD11c (8.6 ± 9.9%). Low percentages of co‐expression (<6.0%) were observed for CD10, CD103, CD80 and CD86.Calculation of Pearson's correlation coefficient showed the strongest direct correlation between CD23 and CD43 expression (r = 0.60); moderate correlations between CD40 and CD95 (r = 0.54), CD31 and CD72 (r = 0.49), CD38 and CD95 (r = 0.40), CD41 and CD42b (r = 0.53), CD25 and CD95 (r = 0.50). Weak reverse correlations were observed between CD20 and CD43 (r = ‐0.45), CD23 and CD40 (r = ‐0.41), CD49d and CD23 (r = ‐0.39) or CD43 (r = ‐0.45).Summary/Conclusion:In our study, the strongest direct correlation was observed between CD23 and CD43; these molecules, expressed by high percentages of CLL B‐cells, regulate the trans‐endothelial migration and have anti‐adhesion function. Both CD23 and CD43 present a reverse correlation with CD49d, a well known poor prognosis marker. CD23 and CD43 show a reverse correlation with CD40 and CD20, respectively. This findings suggest an opposite regulatory role in CLL‐clone survival and proliferation. A second cluster of correlations is related to CD95 expression. CD95/Fas expression correlated directly with CD38, a poor prognosis marker, but also with CD40 and CD25, molecules involved in T/B cooperation and cell activation, respectively. The observed correlations in the expression of the examined surface molecules might have relevance in the biology of CLL individual clones and contribute to regulate trafficking, homing, and survival and/or proliferation of CLL cells.