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

Abstract

AbstractAbstract 2876 Background: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. Objectives: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). Methods: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. Results: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.Table 1Signaling Associations to Patient Risk Groups (Wilcoxon p-value)Signaling NodeCD38 ≥ 30%IGHV UnmutatedZAP-70 ≥ 20%Unfavorable CGX (del11q22.3 and/or del17p13)Favorable CGX (13q14.3)↑ αIgM → p-ERK0.0130.0059↑ αIgM → p-LYN0.0030.0060↑ αIgM → p-PLCγ20.0140.0244↑ αIgM → p-SYK0.024↑ αIgM → p-ERK*0.0180.04850.013↑ αIgM+αIgD → p-AKT0.043↑ αIgM+αIgD → p-ERK0.00390.0018↑ αIgM+SDF1α → p-ERK0.00500.0074↑ αIgD → p-S60.01190.034↓ CpGβ → IkB0.030↓ CpGβ → p-ERK0.0170.0355↑ R848 → IkB0.0055↓ R848 → IkB0.031↑ IFNα → p-STAT10.0026↑ IFNα → p-STAT30.0159↑ IFNα → p-STAT50.00240.0047↑ IL2 → p-STAT50.008↑ Thapsigargin → p-AKT0.0470.0102↑ Thapsigargin → p-ERK0.00920.0018↓ Thapsigargin → p-ERK0.0448↓ Bendamustine → p210.00210.0250.0003↑ Fludarabine → p-H2AX0.0073*Measured at 60 minutes; all other αIgM modulations measured at 10 minutes. Conclusions: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. Disclosures: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.