CLL is a complex disease characterized by a progressive accumulation of CD5+ B-lymphocytes in the peripheral blood, bone marrow, lymph nodes, and other sites. Another important host system for the survival of the malignant population is the vasculature of the bone marrow and other involved sites. We report the results of a series of experiments performed on bone marrow trephine biopsies and bone marrow/peripheral blood samples from patients with CLL that support earlier observations that dysregulated angiogenesis is a common phenomenon in this disease. The proangiogenic factors VEGF and HIF-1α are expressed by the malignant cells and are associated with increased microvessel production in the bone marrow milieu. The potent antiangiogenic factor TSP-1 is not produced by the malignant cells in the marrow. In addition to endothelial surfaces, VEGFR-1 and VEGFR-2 are also expressed on the CLL cells, implying that VEGF may act both on the normal endothelial cells and on the malignant population.
Micovessel density is increased throughout. Several observations suggest a relatively localized paracrine effect of proangiogenic factors in CLL bone marrows and the possible importance of angiogenesis in the metabolically active edges of CLL infiltrates. Although most cases had a diffuse growth pattern, in cases with a nodular pattern of growth, the edges of the nodules had a relatively higher microvessel density than the centers or uninvolved areas of marrow.
Observations such as these are also noted in other malignancies, including both hematologic neoplasms and solid tumors. The explanation for this lies in the production of proangiogenic growth factors and hormones by tumor cells and/or the nontumoral matrix [24–26]. In addition, the production of growth factors in the tumor cells is often controlled by transcriptional regulators, the most prominent of which appears to be HIF-1α.
A major proangiogenic factor is VEGF, a homodimeric glycoprotein (molecular weight ~45 kD) encoded by a gene on chromosome 6p21-p12 [27, 28] which stimulates angiogenesis and vascular permeability by interacting with the tyrosine kinase receptor-2 (VEGFR-2 or KDR/Flk-1) and -1 (VEGFR-1 or Flt-1) . VEGF expression was apparent in both the granulocytes and lymphocytes of the CLL cases. Although there may be a slight increase in microvessel density in areas of involvement, there is an overall increase in microvessel density thoughout. The mere production of proangiogenic factors is not sufficient to explain the differential MVD noted in our patients. Another possible explanation for the localized microvessel production in CLL is the differential expression of other pro- and antiangiogenic factors such as HIF-1α and TSP-1 that may act in concert with VEGF to induce neovascularization.
VEGF is a mitogen that presumably could have activity in any cells expressing the VEGF receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1), and co-expression of at least one of its receptors supports the theory of an autocrine role for the cytokine in addition to the paracrine role associated with angiogenesis [30–32]. Knowledge of the extent of VEGF receptor expression in normal tissues is currently limited, but VEGFR-2 appears to demonstrate lineage restriction that is limited to endothelial cells in normal conditions , and is expressed in certain leukemias . In CLL, we have noted that both receptor classes are expressed on the malignant cells, and Kay et al have shown that mRNA encoding the VEGF receptors is upregulated in CLL cells . In the model proposed by Kay et al VEGF acts autocrinously, since two different classes of VEGF receptors are expressed on CLL cells. We provide further evidence of this by demonstrating the presence of VEGFR-1 and VEGFR-2 immunohistochemically in the malignant cells. In this regard, VEGF could act like the receptors for the growth factors bFGF and platelet-derived growth factor (PDGF), which are expressed in a wide variety of cell types and have a broad spectrum of mitogenic activity . Interestingly, there was a broad range of VEGF receptor expression in the lymphocytes. In some cases, large numbers of cells expressed VEGF receptors and in others receptor expression was virtually absent. We did not find a clear association of this finding with stage or MVD; however this may represent evidence of heterogeneous acquisition of an autocrine phenotype by CLL cells.
In normal systems, expression of the various angiogenesis-related factors is regulated, resulting in a dynamic equilibrium in which pro-and antiangiogenic factors are balanced. In conditions of localized tissue hypoxia, VEGF expression is enhanced by the transcription regulator HIF-1α. Increased expression of HIF-1α mRNA was noted by Kay et al in CLL cells grown in vitro . In our cases, a speckled pattern of staining with antibody directed against HIF-1 α was consistently present in CLL cells. Other cells were negative, with the exception of mast cells, which have a proven role in angiogenesis [35, 36], although production of HIF-1α by mast cells has not previously been reported. Because granulocyte staining for HIF-1α in our specimens was not observed, it seems unlikely that the cytoplasmic mast cell staining observed in our patients is the result of nonspecific (antibody-independent) interactions. The pattern of lymphocyte staining with this antibody suggests a tight clustering of antigens within the CLL cytoplasm, the significance of which is currently unknown.
In the smaller number of cases we have tested for the FISH detectable abnormalities, immunoglobulin mutational status, and immunophenotypic parameters predictive of poor prognosis, however, we found no difference in MVD between those patients with one or more of these poor prognostic indicators and their angiogenic status as determined by MVD.
In contrast to the apparent lack of correlation of these prognostic indicators with angiogenic status, Kini et al have identified that degree of angiogenesis, using MVD as an index, correlated with stage , and Molica et al have demonstrated that MVD at diagnosis correlates with upstaging and progression-free survival . These findings suggest that dysregulation of angiogensis is common in CLL, and as such may represent an early event in leukemogenesis. Since the clinical course of CLL may last many years, the acquisition of other genetic mutations may augment the earlier dysregulation of angiogenesis, accounting for the increased MVD seen in higher stage individuals. Patients with a shorter duration of disease may have more modest changes in angiogenic status.