CCL3 (MIP-1α) plasma levels and the risk for disease progression in chronic lymphocytic leukemia

Abstract

B-cell receptor (BCR) signaling has been inferred as an important mechanism for disease progression in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. In response to BCR activation, CLL cells secrete the chemokine CCL3, which fosters interactions between CLL cells and the leukemia microenvironment. CCL3 secretion correlates with expression of the 70-kDa ζ-associated protein (ZAP-70) and responsiveness of the CLL clone to BCR stimulation. Here, we measured CCL3 plasma levels by enzyme-linked immunosorbent assay (ELISA) in 351 CLL patients and examined CCL3 levels for associations with established prognostic markers and time from diagnosis to initial therapy. We found that CCL3 plasma concentrations were strongly associated with established prognostic markers. In a Cox proportional hazards regression model, CCL3 as well as established prognostic markers (immunoglobulin heavy chain variable-region mutation status, CD38 or ZAP-70 cytogenetics, clinical stage) were significantly associated with time to treatment. Multivariable analysis revealed that CCL3 (hazard ratio [HR] = 2.33, P < .0001), advanced clinical stage (HR = 2.75, P = .0025), poor risk cytogenetics (del 17p, HR = 2.38; del11q, HR = 2.36, P = .001), and CD38 expression (HR = 1.43, P = .023) were independent prognostic markers. Collectively, CCL3 is a novel, robust, and independent prognostic marker in CLL that can easily and reliably be measured by ELISA. CCL3 therefore should become useful for risk assessment in patients with CLL.

Figure 1

Relationship between CCL3, CCL4, and the time from diagnosis to initial therapy. (A) Kaplan-Meier curves show the probability of treatment-free survival according to the time since diagnosis. CLL patients are divided into groups of patients with low or high CCL3 (left-hand curve) or CCL4 (right-hand curve) plasma levels. These categories are based on CCL3 and CCL4 cutoff levels that correspond to the median CCL3 and CCL4 concentrations in our population (Table 2); the respective cutoff levels are displayed next to each of the curves. (B) Kaplan-Meier curves that display the proportion of untreated patients in 4 CLL subgroups, based on their CCL3 and CCL4 levels. Patients with high CCL3 (≥ 10 pg/mL) and low or high CCL4 show lower probabilities of treatment-free survival than patients with low levels of CCL3 (< 10 pg/mL) and high or low CCL4 levels.

Figure 2

Relationship between established prognostic markers and the time from diagnosis to initial therapy. Five Kaplan-Meier plots indicate the probability of treatment-free survival in our cohort of CLL patients, based on the presence or absence of established prognostics markers (mutation status, CD38, ZAP-70, β₂-microglobulin, and cytogenetics).

Figure 3

CCL3 plasma levels in CLL: distribution and sequential samples, and CCL3 immunohistochemistry. (A) The distribution of the plasma levels of CCL3 among 351 patients with CLL. (B) Sequential plasma levels of CCL3 detected in serial samples of CLL patients. The lines connect the symbols of individual patients representing the CCL3 concentrations (y-axis) in any one patient over time (x-axis). (C) Conventional (left) and immunofluorescence (right) immunodetection of CCL3 in CLL lymph node sections in a representative case (original magnification ×400). Images were captured with the use of an Olympus BX50 microscope and a ColorView digital camera, and processed with cellSens imaging software (all Olympus). CCL3⁺ cells (brown stain on the left side) tended to accumulate in areas of proliferation centers (left). Immunofluorescence double staining with CD79a (red) and CCL3 (green) revealed colocalization of CD79a⁺ and CCL3⁺ CLL cells, as indicated by the arrows. (Inset) Coexpression of CCL3 and CD79a in another case. More immunohistochemistry data are available in supplemental Figure 2.