Intra-Clonal Diversity Identified by CD19-Mediated Signaling Correlates with Disease Progression In Chronic Lymphocytic Leukemia (CLL)
Authors: Herishanu Yair , Kay Sigi , Dezorella Nili , Hazan-Hallevi Inbal , Trestman Svetlana , Perry Chava , Deutsch Varda Deutsch , Polliack Aaron , Naparstek Elizabeth , Katz Ben-Zion 
Published: October 2019

Abstract
Abstract 3577
Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with CLL. Subsets of the CLL clone that respond more robustly to external stimuli may gain a growth and survival advantage which may possibly promote clonal evolution. Here we report an intra-clonal diversity in response to CD19 engagement in CLL cells identified by using a non-conventional flow cytometric analysis. Monoclonal anti-CD19 antibody rapidly induced cellular aggregation of CLL cells identified as a high side scatters lymphoid population (HSLP). The percentage of the HSLP within the whole CLL population was highly reproducible in individual patients, but was extremely variable between patients (range 0–39.4%, median-19.95%). This inter-individual heterogeneity was attributed to the intrinsic ratio between CD19 responsive and unresponsive CLL subsets and was seen to correlate with time to treatment (TTT), but not with ZAP-70 expression. During a median follow-up period of 45.6 months (n=46), CLL patients with more than 20% of HSLP had a median TTT of 50.7 months from the time of diagnosis while this median has yet not been reached in patients with a lower proportion of HSLP (p=0.019). The rapid induction of cellular aggregation via CD19 was completely abrogated by disruption of the cholesterol-rich plasma membrane rafts by methyl-cyclodextrin, but minimally affected by protein kinase and actin cytoskeleton inhibitors. The relatively ineffective blocking of cellular aggregation by cytoplasmatic signaling inhibitors suggests that this response is mainly attributed to CD19 association with other trans-membrane components, which possibly occurs within the lipid rafts. The CLL sub-population which responded to CD19 clustering expressed higher mRNA levels of c-Myc, and exhibited different morphological features from the non-responsive CLL cells subset. The CD19-responsive subset also correlated with higher CD20 expression and a marked decrease in HSLP was noted in samples taken 24 h after rituximab administration (n=6). As expected, rituximab resulted in a significant decrease in the total CLL counts, with the specific elimination of the HSLP. Comparison of HSLP induction by CD19 in normal B-cells and in leukemic cells from B-type ALL revealed very low levels of HSLP in B-cells from healthy individuals and B-ALL (n=5, range 0.5–2.3%, median-0.9%). Leukemic cells derived from other indolent B-cell lymphoproliferative disorders (non-Hodgkin lymphomas and hairy cell leukemia) had the highest proportion of HSLP of all samples screened (n=8, range 34–60%, median-38.3%).

Conclusions:
we have identified intra-clonal diversity for CD19-mediated cellular aggregation in CLL which correlates with time to treatment initiation. This phenomenon was completely lipid raft dependent and restricted to a distinct CLL cell subset. The heterogeneous CD19 response may reflect basic differences in the juxta-membrane signaling patterns within the CLL clone.

Disclosures:
No relevant conflicts of interest to declare.