CLL is the most common leukemia in the Western world. It is characterized by the clonal expansion of CD5+/CD19+ B cells, expressing surface immunoglobulins (sIg), most often of the IgM subset. Clinically, it can either manifest as an indolent disease with little impact on the lifespan of a subset of patients or as an aggressive, highly drug-resistant disease with lethal outcome but all possible scenarios in between these extremes are routinely observed. The degree of somatic mutations of the sIg has been shown to affect survival; patients expressing highly mutated sIg have a better prognosis than patients whose sIg are less than 2% different from germline sequences. Therefore, it is intuitive that signaling through the sIg, or B-cell receptor (BCR), must have a role in determining the fate of the CLL cells, veering the intracellular machinery towards proliferation or apoptosis.
We have systematically investigated the downstream events caused by BCR stimulation via crosslinking of the sIg in a series of about 60 heterogeneous CLL patients and 15 normal donor peripheral blood mononuclear cells (PBMCs). Using multiplexed phosphoflow cytometry we were able to assess the intensity of multiple signaling events at specific stages of the BCR signaling pathway. By combining phosphospecific staining and classic immunophenotyping we were also able to evaluate signaling intensity at the at the single cell level as well as correlating the signal intensity of CLL-specific surface markers to that of the phosphoproteins analyzed. We used principal component analysis to combine phosphospecific staining within individual cells and generate a variable that discriminates efficiently between CLL and PBMCs B-cells.
We found that signaling properties of CLL B-cells were impaired when compared to normal B cells from healthy donors, both at the level of upstream members of the BCR signaling pathway (pBLNK, pSYK, pZap70, pBTK, pPLCg2) and downstream components such as pp38 and pERK. However, within the cohort of CLL samples we identified a small subpopulation in which signaling proceeded as well or more efficiently than in normal B cells. We also used our principal component variable to quantify the heterogeneity of signaling responses within populations of CLL B-cells and correlate them with clinical parameters that are known prognostic indicators in CLL.
In conclusion, multiplexed phosphoflow cytometry of individual CLL cells provides a novel and highly specific method to determine the use of signaling pathways (such as BCR-mediated) in comparison to normal B cells. Our studies indicate a significant decrease in BCR signaling of CLL B cells, which involves both upstream and downstream components.
van den Brink:Cytheris: Research Funding.
Asterisk with author names denotes non-ASH members.