Divergence in CD19-Mediated Signaling Unfolds Intraclonal Diversity in Chronic Lymphocytic Leukemia, Which Correlates with Disease Progression

Abstract

Emerging data on intraclonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. In this study, we report intraclonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into CD19-responsive (CD19-R) and -nonresponive subpopulations. Engagement of CD19 by anti-CD19 Ab rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA, exhibited distinct morphological features, and were preferentially abolished in rituximab-treated patients. Both subpopulations reacted to sIgM stimulation in a similar manner and exhibited similar levels of Akt and Erk phosphorylation, pointing to functional signaling divergence within the BCR. CD19 unresponsiveness was partially reversible, where nonresponding CD19 cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19–signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow–derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with >15.25% of the CD19-R cell fraction had a shorter median time to treatment compared with patients with <15.25% of CD19-R cell fraction. In conclusion, divergence in CD19-mediated signaling unfolds both interpatient and intraclonal diversity in CLL. This signaling diversity is associated with physiological implications, including the location of the cells, their responses to anti-CLL therapeutics, and disease progression.

Blinatumomab for the Treatment of Philadelphia Chromosome–Negative, Precursor B-cell Acute Lymphoblastic Leukemia

“…There is evidence to suggest a role for CD19 in enhancing malignant pathways (10). For example, high levels of CD19 expression were shown to correlate with Akt activation and overexpression of c-Myc (12,13). Targeting CD19 in vitro seems to confer antitumor activity by induction of cell-cycle arrest (14), apoptosis (9), and sensitization of neoplastic cells to chemotherapy (15,16).…”

HLA-G is a component of the chronic lymphocytic leukemia escape repertoire to generate immune suppression: impact of the HLA-G 14 base pair (rs66554220) polymorphism

“…This is also in line with previous studies, which found that sHLA-G levels in plasma were higher in CLL patients than in healthy controls. 17,18 A second issue favoring the view that quantification of soluble rather than membrane HLA-G is an informative and dependable assay derives from old and new facts linked to the unique lipid structure of the CLL cell surface, 33,34 potentially increasing the instability of the membrane form. In agreement with this, in myeloma cells mHLA-G is also released from the cell membrane in microparticles.…”

Measurement of lymphocyte aggregation by flow cytometry–physiological implications in chronic lymphocytic leukemia

“…In a previous study, we found that in some cases aggregation of chronic lymphocytic leukemia (CLL) cells is a physiological outcome of CD19 engagement, and can be measured by flow cytometry . Moreover, we have shown that the extent of CLL cell aggregation in a given patient is associated with disease progression . We show herein that aberrant CLLs exhibit elevated levels of CD19‐induced cellular aggregation, in a similar manner to circulating non‐Hodgkin's lymphoma (NHL) cells.…”

“…Acquisition of an aggregate as a single event is considered as coincidence, and common flow cytometry protocols recommend the exclusion of such events, pending on their occurrence as random events . In a previous study, we found that in some cases aggregation of chronic lymphocytic leukemia (CLL) cells is a physiological outcome of CD19 engagement, and can be measured by flow cytometry . Moreover, we have shown that the extent of CLL cell aggregation in a given patient is associated with disease progression .…”

“…Anti‐CD37 antibodies, or type II anti‐CD20 antibodies, induce B‐cell aggregation which is associated with the induction of apoptosis in lymphoma and chronic lymphocytic leukemia (CLL) cells . CD19 engagement results in CLL cells aggregation associated with anti‐β 2 integrin mediated adhesion . However, lymphocyte aggregation is often estimated by observation of bright field and/or fluorescent microscope images, due to the lack of methodology to measure lymphocytes aggregates in a robust and accurate manner .…”