Osteopontin - A New Angiogenic Factor Which Correlates with Circulating Endothelial Cells in Acute Myeloid Leukemia.

There is evidence that acute myeloid leukemia (AML) development and progression could be determined by the expression profiling of specific angiogenic proteins. Previously we have found that the number of circulating endothelial cells (CEC) and endothelial progenitors (CEP) in peripheral blood of AML patients is a good noninvasive marker of angiogenesis and correlates with tumor mass and response to treatment. Osteopontin (OPN) is hematopoietic stem cell niche component which controls tumor growth and angiogenesis. It promotes VEGF dependent tumor progression, induces endothelial cell survival and migration and modulates expression of angiopoietins. The role of OPN in AML is unknown. We evaluated the level of circulating OPN, angiopoietin-1 (ANG-1) and ANG-2 in AML patients. In addition we correlated the levels of angiogenic cytokines with CEC number, known prognostic factors and response to treatment.

OPN, ANG-1 and ANG-2 were measured in plasma samples in 69 newly diagnosed AML patients with median age 53 (range 18-78 years) and 16 controls with median age 36 (range 22-73 years) using enzyme-linked immunosorbent assay (ELISA). According to median level of cytokines patients were subdivided into “low-expressers” and “high”-expressers group. CEC were evaluated by the four colour flow cytometry using a panel of previously described monoclonal antibodies. CEC deriving from angiogenic microvessels were characterized by CD36 expression.

The median levels of circulating OPN (162,5 pg/ml) and ANG-2 (5184 pg/ml) were significantly higher in AML patients as compared to healthy controls (70 pg/ml; p<0.0001 and 2624,3 pg/ml; p<0.001 respectively). In contrast, the level of ANG-1 in AML pts (1038 pg/ml) was lower than in control group (1710 pg/ml; p<0.002). OPN plasma level was significantly higher in AML patients with more than 50% of blasts in the bone marrow (197 pg/ml) and elevated LDH activity (170 pg/ml) compared to group with BM blasts <50% (152 pg/ml; p<0.05) and normal LDH (137,5 pg/ml; p<0.03). In analogy, we found the significant positive correlation between ANG-2, but not ANG-1, with WBC count (p=0.05), circulating blast count (CBC) (p<0.005) and LDH activity (p=0.05). There was no statistical association between the levels of OPN, ANG-1, and ANG-2, and patients' age, sex, AML subtype according FAB classification and cytogenetic risk group. Interestingly, in AML patients OPN level correlated positively with the number of CEC deriving from angiogenic microvessels (p<0.02). Moreover, we observed tendency to positive correlation between ANG-2 level and CD36+ CEC count (p=0.09). In addition, in AML we have found significant positive correlation between OPN and ANG-2 level (p<0.03). From the 49 patients receiving intensive induction chemotherapy, 28 (56%) achieved complete remission (CR). We observed that the CR rate in “low-expressers” of OPN (67%) was higher than in the “high-expressers” (33%), however a difference did not reached statistical significance. This may be due to not very large number of evaluated patients.

The levels of circulating OPN and ANG-2, but not ANG-1, are significantly higher in AML patients than in healthy subjects and correlate with tumor burden as well as the number of “angiogenic” circulating endothelial cells. This data clearly demonstrate an important role of circulating OPN as well as ANG-2 in angiogenesis and AML biology. A better understanding of precise functions of OPN and angiopoietins may create new options for therapeutic interventions in AML.

Robak:Celgene: Consultancy; Roche: Honoraria, Research Funding; Genmab: Research Funding; Cambridge Antibody Technology: Research Funding; GlaxoSmithKline: Honoraria.