Micro-Array and Protein Analyses Reveal a Preferential Autocrine VEGF Survival Loop in CD38⁺ Sub-Clones When Compared with CD38⁻ Sub-Clones Derived from the Same CLL Patient.

CD38 expression is a marker of poor prognosis in chronic lymphocytic leukemia (CLL) but to-date no clear rationale for this has been elucidated. Using high speed cell sorting and subsequent gene expression profiling, we investigated the transcriptional characteristics of purified CD38⁺ and CD38⁻ sub-clones derived from the same patient (n = 12). Our analysis revealed a distinct, differential gene expression profile between CD38⁺ and CD38⁻ subsets with 35 genes found to be consistently over expressed in the CD38⁺ cells (of which CD38 was one; 9.1 fold). Of particular interest were four genes, CXCL-2 (9.4 fold), vascular endothelial growth factor (VEGF) (5.8 fold), MIP1α /CCL3 (5.4 fold) and Interleukin 1β (IL-1β ) (4.1 fold). In a number of different tumor models IL-1β has been shown to induce the expression of CXCL-2, VEGF, and MIP1α resulting in angiogenesis, survival signaling and/or tumor proliferation. Protein analysis revealed 2–3 fold elevated levels of IL-1β and VEGF in CD38⁺ cells confirming that the elevated transcription was translated. Furthermore, the differential transcription of these genes corroborates our previous findings; CD38⁺ cells have a higher proliferative index and higher levels of the anti-apoptotic protein Mcl-1. Our data strongly suggests that the CD38⁺ clones preferentially utilise an autocrine VEGF loop and given that the CD38⁻ clones also express VEGF receptor the potential for bystander paracrine effects of secreted VEGF seem likely in bimodal patients. Therefore, the relative over expression of IL-1β and VEGF provide a rationale for the disease progression and poor clinical prognosis of CD38⁺ CLL patients.