Single Cell Network Profiling (SCNP) Identifies Altered Signaling Between Patient Risk Groups in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

B-CLL follows a variable clinical course, with a subset of patients progressing quickly. The reasons for this outcome disparity are not fully understood; however, evidence suggests that B-cell receptor (BCR) signaling is a driving event in disease onset and progression. B-CLL cells also receive survival signals through additional receptors. SCNP is a multiparametric flow cytometry-based assay that measures, quantitatively at the single cell level, changes in intracellular signaling proteins in response to extracellular modulators. This provides a functional measure of pathway activity and intraclonal signaling differences within the larger B-CLL cell population. In prior studies, we observed higher αIgM-induced (→) p-ERK signaling in B-CLL samples from patients who had a shorter time to first treatment (TTFT) (Cesano et al. ASH 2011 Abstract 2834). Herein we examined the feasibility for SCNP to further define patient risk stratification.

The current study was designed to 1) map signaling profiles in early-stage B-CLL and to 2) identify signaling associations with clinical subgroups defining B-CLL prognosis (IGHV mutational status, cytogenetic risk, CD38 / ZAP-70 expression).

Between 2006 and 2007, peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from a cohort of 39 untreated B-CLL patients, Rai stage 0 - II, at different time points during their clinical course. Of the 39 samples evaluated; 15 and 20 expressed CD38 (≥30% of cells) and ZAP-70 (≥20% of cells), respectively; 19 were IGHV unmutated (98% cutoff); and cytogenetic risk groups were evenly represented. SCNP analysis quantitatively measured 22 intracellular signaling proteins within CD19⁺CD5⁺ B-CLL cells, using a panel of 14 disease-relevant modulators. The Wilcoxon rank-sum test was used to identify signaling associations with CD38 and ZAP-70 expression, cytogenetic risk categories, del17p (includes p53 gene deletion) and IGHV mutational status.

Significant associations between patient risk categories and signaling are summarized in Table 1. IGHV unmutated and ZAP-70⁺ samples showed (1) elevated α-IgM or α-IgD-induced BCR signaling, either alone or in combination, (2) decreased CpGβ → p-ERK induction and (3) increased thapsigargin-induced (Ca²⁺ signaling) signaling. Of note, CD38⁺ samples did not show the same associations, but shared with unmutated IGHV samples a higher responsiveness to IFNα and weaker induction of p21 in response to bendamustine. The unfavorable cytogenetic group samples showed increased αIgM→p-ERK and had higher basal p-S6 that further increased with IgD crosslinking. Lack of p21 induction was also associated with unfavorable cytogenetics, which includes deletion of p53 (del17p), a regulator of p21 expression.

This is the second, independent SCNP analysis of B-CLL signaling showing decreased bendamustine→p21 and increased αIgM→p-ERK signaling in samples with unfavorable cytogenetics. Further associations with IGHV unmutated status included increased BCR signaling in multiple nodes, altered TLR9 responsiveness, and decreased drug-induced p21. These data support the potential utility of SCNP in: (1) identifying in one assay those patients with a more aggressive form of B-CLL, including both unmutated IGHV and p53 pathway alteration, and (2) identification of patients with signaling profiles that may be more likely to respond to targeted therapies.

Ptacek:Nodality, Inc.: Employment, Equity Ownership. Evensen:Nodality, Inc.: Employment, Equity Ownership. Friedland:Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership.