Discrimination between circulating endothelial cells and blood cell populations with overlapping phenotype reveals distinct regulation and predictive potential in cancer therapy.

BACKGROUND: Circulating endothelial cells (CECs) have been proposed to predict patient response to antiangiogenic cancer therapy. However, contradictory reports and inconsistency in the phenotypic identification of CECs have led us to compare three cell populations with partially overlapping phenotype in cancer patients receiving chemotherapy and the antiangiogenic agent bevacizumab. METHODS: Patients (n = 20) with locally advanced pancreatic cancer were monitored during 16 weeks of neoadjuvant treatment with gemcitabine and bevacizumab. Detection of circulating cell populations was based on the marker combination CD45, CD31, and CD146; levels of viable and dead (7-aminoactinomycin D-positive) cells were evaluated by flow cytometry in 2-week intervals. RESULTS: We were able to discriminate and concomitantly monitor three cell populations elevated in cancer patients. Whereas CECs were defined as CD45(-) CD31(+) CD146(+), the distinct populations of CD45(-) CD31(-) CD146(+) and CD45(-) CD31(high) CD146(-) cells were partly positive for CD3 and CD41, respectively. CECs and CD45(-) CD31(-) CD146(+) cells increased during therapy; the rise in dead cells was positively correlated with patient response or survival. Conversely, CD45(-) CD31(high) CD146(-) cells decreased in neoadjuvant treatment. A highly significant correlation was established for improved patient response and a minor decrease in viable cell counts. CONCLUSIONS: Flow cytometric CEC analysis based on CD45, CD31, and CD146 requires careful discrimination between blood cell populations with overlapping phenotype showing hallmarks of activated T cells and large platelets. However, these three cell populations show distinct regulation during cancer therapy, and their concomitant analysis may offer extended prognostic and predictive information.

Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

BACKGROUND: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. METHODS: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. RESULTS: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). CONCLUSION: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

Myelosuppression of thrombocytes and monocytes is associated with a lack of synergy between chemotherapy and anti-VEGF treatment.

PURPOSE: Chemotherapeutic agents that have shown improved patient outcome when combined with anti-vascular endothelial growth factor (VEGF) therapy were recently identified to induce the mobilization of proangiogenic Tie-2-expressing monocytes (TEMs) and endothelial progenitor cells (EPCs) by platelet release of stromal cell-derived factor 1α (SDF-1α). VEGF blockade was found to counteract cell mobilization. We aimed to determine why agents like gemcitabine do not elicit TEM and EPC recruitment and may therefore lack synergy with anti-VEGF therapy. EXPERIMENTAL DESIGN: Locally advanced pancreatic cancer patients (n = 20) were monitored during 16 weeks of neoadjuvant therapy. Treatment was based on gemcitabine with or without the addition of bevacizumab. Blood levels of proangiogenic cell populations and angiogenesis factors were determined in 2-week intervals. RESULTS: The lack of EPC mobilization during gemcitabine therapy was associated with severe thrombocytopenia and reduced SDF-1α blood concentrations. Furthermore, myelosuppression by gemcitabine correlated significantly with loss of TEMs. With respect to angiogenic factors stored and released by platelets, plasma levels of the angiogenesis inhibitor thrombospondin 1 (TSP-1) were selectively decreased and correlated significantly with thrombocytopenia in response to gemcitabine therapy. CONCLUSIONS: A thorough literature screen identified thrombocytopenia as a common feature of chemotherapeutic agents that lack synergy with anti-VEGF treatment. Our results on gemcitabine therapy indicate that myelosuppression (in particular, with respect to thrombocytes and monocytes) interferes with the mobilization of proangiogenic cell types targeted by bevacizumab and may further counteract antiangiogenic therapy by substantially reducing the angiogenesis inhibitor TSP-1.