Association between B-cell receptor responsiveness and disease progression in B-cell chronic lymphocytic leukemia: results from single cell network profiling studies

Abstract

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.

Figure 1.

BCR signaling profiles in B cells from B-CLL patients – exploratory study. (A) Representative flow cytometry histograms of BCR signaling phosphoproteins in basal conditions or following stimulation with anti-IgM in CD5⁺/CD19⁺ cells from B-CLL patients. (B) BCR signaling profiles in basal condition or following stimulation with anti-IgM across B-CLL samples. Data are expressed as log₂Fold difference in phosphoprotein median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI) and represented as a pseudo-color map. (C) Response to BCR modulation in unmutated (UM) and mutated (M) IGHV samples. Data are expressed as log₂Fold difference in RMFI of anti-IgM-modulated divided by unmodulated cells (log₂Fold change). The line in the middle represents the median value. The B-CLL subsets were confirmed by the two Wilcoxon's rank sum test. *P<0.05; **P<0.01; ns = not significant. (D) Correlation between pairwise combinations of phosphoprotein response to anti-IgM, measured as log₂Fold change, in unmutated and mutated B-CLL subsets.

Figure 2.

Binet stage A B-CLL patients grouped on the basis of responsiveness to BCR modulation were associated with biological prognostic parameters – exploratory study. Response to BCR engagement was expressed as log₂Fold difference in relative median fluorescence intensity (RMFI) of anti-IgM-modulated cells divided by unmodulated cells (log₂Fold change). Patients were then grouped based on BCR log₂Fold changes using the k-medoids clustering algorithm. (A) Scatter plot in the first two principal components of BCR response to anti-IgM. Each point represents a patient, the pattern or filled quadrants denotes cluster membership: empty indicates cluster 2, filled and red indicates cluster 1. (B) B-CLL patient clusters are represented as a pseudo-color map. The standard biological parameters of prognosis are aligned below the heat map.

Figure 3.

Kaplan-Meier curves of TTFT for subgroups of Binet stage A or Rai I/0 patients defined by p-ERK response to anti-IgM in the exploratory study (A); test study 1 using the anti-IgM→p-ERK | log2Fold (B) or the anti-IgM→p-ERK | Uu metric (C); test study 2 (D). High and low p-ERK values were referred to the median signal values in (A) and (B) and to the 0.66 cut-point for the Uu metric (see the Online Supplementary Design and Methods section). P values are from the log-rank test.