Single Cell Network Profiling (SCNP) assay reveals B Cell Receptor Signaling heterogeneity in the Context of ZAP-70 Expression in patients with Chronic Lymphocytic Leukemia (CLL)

CLL is a disease with considerably variable clinical course. Among early stage CLL patients that are provided with the “watch and wait” standard of care, a significant subset of these patients undergo rapid disease progression during which many of them are refractory to currently available therapies. Although several biological indicators including expression level of ZAP-70 have been associated with CLL patient risk stratification, technical challenges related to assay consistency have limited their broad use in the clinical setting. It has been observed that ZAP-70 expression is sufficient to transduce B cell receptor (BCR) signaling and that BCR signaling activation by anti-IgM was enhanced in ZAP-70 positive CLL cells including spleen tyrosine kinase (Syk), a cytosolic protein kinase that is recruited by the activated BCR and forms a protein complex with ZAP-70 that in turn initiates downstream signaling resulting in CLL cell survival. Based on the BCR biology and established prognostic data, we undertook a study designed to interrogate critical signaling pathways associated with BCR while simultaneously assessing the quantity of the ZAP-70 protein in each individual cell. If successful, this approach may glean a better prognostic prediction than existing techniques while building an assay system suitable for translational studies in the varied clinical groups. We used SCNP, a new technology platform which uses multi-parametric flow cytometry to measure biological pathways focusing on intra-cellular signaling and post-translational changes in response to modulators, to examine the relationship between BCR signaling and ZAP-70 expression in patients with CLL. The goal of this investigation is to generate hypotheses that guide development of sensitive and accurate prognostic tests and support translational therapeutic development targeting the BCR pathway for CLL patient management. Methods: SCNP was performed to measure basal and modulated BCR signaling in cryopreserved PBMCs from 10 CLL patients (5 ZAP-70 negative and 5 ZAP-70 positive) who were not currently receiving treatment. Anti-IgM or hydrogen peroxide was used to evoke BCR signaling, which was measured by SCNP using phospho-specific antibodies against signaling proteins including p-Syk, p-ERK, p-p38, p-Lck, p-NF-kB and p-SAPK/JNK. ZAP-70 protein expression was measured simultaneously with the signaling assessment by staining with a ZAP-70 (clone 136F12) antibody. Analysis was performed using WinList™ to gate the CD5+, CD19+ CLL cells separately from the CD5+, CD3+ T cells. Results: The biological heterogeneity among CLL patients was clearly evident when the basal and evoked levels of BCR signaling protein phosphorylation were assessed using SCNP. In comparison to normal B cells, elevated basal levels of p-Syk, p-NF-kB and p-SAPK/JNK were observed in 7 of 10 CLL samples (MFI two fold greater than unstained cells). Following BCR stimulation with anti-IgM, a trend of positive correlation between the nodes (i.e. proteomic readouts in the presence of modulator) p-Syk, p-Lck, p-ERK and p-p38 and increasing amounts of ZAP-70 was observed. Interestingly, p-Syk was found to correlate well with ZAP-70 expression level (Spearman=0.76). However, following hydrogen peroxide modulation, there was no positive correlation observed between ZAP-70 expression and p-Syk, p-ERK, p-p38, and p-Lck, whereas there appeared to be a trend toward elevated p-SAPK/JNK and p-NF-kB in the context of diminished ZAP-70 expression. An SCNP study in a large set of clinical CLL samples is ongoing to further investigate the relationships between BCR signaling and ZAP-70 expression observed in this study in an attempt to understand the impact of ZAP-70 expression on signaling and the relation of both measures to patient outcome. Conclusions: The findings reported here suggest that measurement of BCR signaling pathways using SCNP reveal biological heterogeneity within the known prognostic groups based on the expression level of the ZAP-70 protein. These findings, when validated, may permit identification of high risk individuals at an early stage of disease, as well as guiding selection of suitable treatment strategies in the two differing prognostic groups.