Angiogenic Activity in IgV_H Mutated and Unmutated Chronic Lymphocytic Leukemia (CLL): Indications for the Therapeutic Use of VEGF-Signaling Inhibitors.

Background: CLL is characterized by an heterogeneous clinical course, with some patients living many years without therapy and other patients presenting an aggressive disease. Different biologic properties of the leukemic cells have important prognostic implications. In particular, the IgV_H gene mutational status, chromosomal abnormalities, expression of CD38 and ZAP-70 have been correlated with the clinical course of the disease. There is also growing evidence that angiogenesis may be linked to the pathogenesis and outcome of CLL. In particular, vascular endothelial growth factor A (VEGF-A) has been detected in the serum and bone marrow biopsy cells analyzed by immunostaining: both events have been correlated with a poor prognostic likelihood.

Aims: The goal of this study was to obtain a more complete characterization of the angiogenic potential of CLL cells, to correlate the angiogenic capacity with other prognostic parameters and to investigate the possibility of overcoming this property by specific VEGF-signaling inhibitors.

Methods: Eighteen patients with a diagnosis of CLL, based on morphologic and immunophenotypic criteria, have been evaluated for the expression of CD38 and ZAP-70, and for the IgV_H gene mutational status. An Angiokit test (TCS Cell Works) was utilized to evaluate the capacity of purified CD19+ CLL cells to stimulate the growth and tubule formation of human endothelial cells and the relative roles of an anti-VEGF specific antibody and of PTK787/ZK 222584 (PTK/ZK, Schering, Novartis), a novel, oral angiogenesis and lymphangiogenesis inhibitor that blocks tyrosine kinase signaling from all known VEGFRs. The quantification of endothelial tubule formation was carried out using a specific software. After stimulation with a biotinylated goat anti-human IgM F(ab)₂, leukemia cells were evaluated for gene expression profiling using the Affymetrix HGU133 Plus 2.0 gene chip.

Results: In 9 patients, the leukemic cells were CD38+, ZAP-70+ and showed an unmutated IgV_H status; in the remaining 9, the leukemic cells were CD38−, ZAP-70− and with a mutated IgV_H status. In CLL patients with poor prognostic features - CD38+, ZAP-70+ and unmutated IgV_H - purified leukemic cells showed a significantly greater capacity to stimulate the growth and tubule formation (3407 ± 968 tubules) of human endothelial cells compared to leukemic cells obtained from CLL patients with good prognostic factors, i.e. CD38−, ZAP-70− and mutated IgV_H, (1550 ± 624 tubules). This property was amplified following sIgM cross-linking, that resulted in the upregulation of angiogenetic genes, including: EGR-1, SDF2L1, TNFAIP3, PTGER4, FIBP, NR4A3. A reduction of VEGF-A-induced tubule number was observed when leukemic cells were cultured with the anti-VEGF antibody (43–82% of inhibition) and, to a further extent, with the PTK/ZK inhibitor (90–96% of inhibition) compared to purified CLL cells alone.

Conclusions: In CLL patients with poor prognostic factors, leukemic cells display an angiogenic capability significantly higher than that of cells from patients with favorable prognostic factors. The current findings provide the rationale for investigating the potential clinical role of anti-angiogenic agents, such as PTK/ZK, in the management of CLL patients.