Role of Microenvironment-Associated Chemokines and Cytokines for Binet Stage A CLL Patients Included in a Prospective Trial (CLL1 trial) of the German CLL Study Group (GCLLSG): sIl2Ralpha Is An Independent Predictor of Progression-Free Survival (PFS),

In CLL, proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T cells and CD68⁺ nurselike cells (NLCs) and display signs of B cell receptor (BCR) activation, suggesting that CLL proliferation is T cell- and BCR-driven. In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined, especially in the light of distinct prognostic and biological subgroups of patients (pts).

In order to address this issue, we measured serum levels of chemokines and cytokines in a prospective cohort of 157 previously untreated Binet stage A pts., a small subgroup of the risk-stratified CLL1 trial of the GCLLSG. Median follow-up time of that subgroup was 50.3 months. Median time to progression was 61.3 months. Median time from diagnosis to study entry was 1 year. Serum samples had been centrally collected at study entry and stored at −80°C. Sera were analyzed on a luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. In a pilot phase, sera of 21 pts were pre-analytically screened for a total of 62 different chemokines. Median serum levels of 27 different chemokines and cytokines were found to differ from healthy controls in a significant manner. Those 27 chemokines and cytokines were subsequently analyzed in 157 pts. For all parameters univariate and multivariate analyses were performed for progression-free survival.

Serum levels of CCL3 and CCL4, chemokines known to be secreted by CLL cells upon BCR engagement, were elevated compared to healthy controls. High CCL3 levels correlated strongly with high CCL4 levels (p<0.001) and tended to be associated with unmutated IgHV status (p=0.06), supporting the role of BCR triggering in biologically selected CLL pts. Concerning CCL3 and CCL4, no significant difference in PFS was detected. Serum levels of CCL2 and CCL17, both binding chemokine receptor CCR4, were concordantly elevated compared to healthy controls (p<0.001), suggesting a possible role of chemoattraction of CCR4+ T cells towards these chemokines in CLL. CXCL12, CCL21, and CXCL10, chemokines associated with chemotaxis of CLL cells, were concordantly elevated compared to healthy controls. Pts with serum levels of CCL21 beyond the median also had higher levels of CXCL10 (p<0.001) and CXCL12 (p=0.02). CLL pts have been found to have abnormal neovascularization in the bone marrow and lymph nodes. Elevated VEGF levels were strongly associated with elevated EGF level (p<0.001). High VEGF levels correlated with high white blood cell counts (p=0.008) and showed a trend towards association with del(11q) (p=0.07) and lymphadenopathy (p=0.165). Concerning VEGF, no significant difference in PFS was detected. In contrast, pts with high levels of sIl2R alpha showed a significant shorter PFS. When confirmed by conventional ELISA, median PFS in pts within the highest quartile of sIl2Ralpha levels were 22 months compared to 72 months in the lower three quartiles (p<0.001). When we analyzed sIl2R alpha together with genetic abnormalities like del(11q), trisomy 12, del(13q), del(17p), the risk stratification model used in CLL1 (high risk versus low risk for disease progression), the hierarchical model as published by Döhner et al., and IgHV status in a multivariate Cox regression, we found that sIl2R alpha (OR 2.5, 95% CI 1.4–4.8, p=0.004) and IgHV mutational status (OR 4.0, 95% CI 2.2–7.3, p<0.001) were both independent prognostic variables.

The assessment of sera in a small subgroup of the CLL1 study cohort of the GCLLSG revealed that the levels of several chemokines and cytokines were elevated when compared to healthy controls. Median serum levels of different chemokines correlated with distinct biological characteristics of CLL pts, like genetic abnormalities or clinical parameters. In addition, sIl2R alpha could be identified as an independent prognostic variable for PFS in early CLL.

Eichhorst:Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Hallek:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bergmann:Celgene: Honoraria.