Paraneoplastic thrombocytosis in ovarian cancer

Abstract

Background: The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear.

Methods: We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained.

Results: Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis.

Conclusions: These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Figure 1. Clinical Significance of and Thrombopoietic Cytokines Associated with Paraneoplastic Thrombocytosis in Patients with Epithelial Ovarian Cancer

Panel A shows Kaplan–Meier survival curves for progression-free survival according to platelet count at initial diagnosis among patients with epithelial ovarian cancer, and Panel B shows curves for overall survival. Panel C shows mean plasma levels of interleukin-6 in 104 patients with epithelial ovarian cancer and normal platelet counts and 46 patients with epithelial ovarian cancer and thrombocytosis, and Panel D shows mean plasma levels of thrombopoietin in the same patients. T bars indicate standard deviations. Thrombocytosis was defined as a platelet count of more than 450,000 per cubic millimeter.

Figure 2. Underlying Mechanism of Paraneoplastic Thrombocytosis in Epithelial Ovarian Cancer

Panel A shows mean platelet counts in non–tumor-bearing (NTB) mice, tumor-bearing (TB) mice, and TB mice that lacked functional hepatocyte interleukin-6 receptors (TB IL-6R–null mice). T bars indicate standard errors; for NTB mice, the standard error is too small to be shown. Panel B shows the proposed paracrine signaling pathway that mediates paraneoplastic thrombocytosis in epithelial ovarian cancer. Interleukin-6 secreted by ovarian cancer cells stimulates hepatic thrombopoietin (TPO) production. This drives thrombopoiesis in the bone marrow, giving rise to thrombocytosis. Panel C shows Kaplan–Meier survival curves for overall survival according to plasma interleukin-6 levels. Median overall survival among patients with plasma interleukin-6 levels of more than 10 pg per milliliter was 3.38 years, as compared with 5.99 years among those with levels of 10 pg per milliliter or less.

Figure 3. Functional Role of Interleukin-6 and Thrombo-poietin in Paraneoplastic Thrombocytosis

Panel A shows the effect of silencing interleukin-6 alone, thrombopoietin alone, and the two in combination on paraneoplastic thrombocytosis in mice with epithelial ovarian cancer. The P value is for the difference in platelet counts between combination and control siRNA treatment. Panel B shows the effect of siltuximab treatment (every 2 weeks) on platelet counts in patients with epithelial ovarian cancer. I bars indicate standard deviations. The term siRNA denotes small interfering RNA.

Figure 4. Antitumor Effects of Blocking Paraneoplastic Thrombocytosis

Panel A shows the mean tumor weight in mice after treatment with either antiplatelet antibody (APA) or control IgG. Representative macroscopic appearances of tumors at necropsy are shown below the histogram. Panel B shows the effect of APA versus control IgG on tumor-cell viability (upper photomicrographs) and proliferation (lower photomicrographs). The corresponding histograms show the mean necrotic tumor area and the number of cells that were positive for Ki67, normalized to the total cell count per high-power field (HPF). T bars indicate standard errors.

Figure 5. Platelets in the Tumor Microenvironment

Panel A shows the method used for detecting extravasated platelets. Platelets isolated from female transgenic C57BL/6 mice that express yellow fluorescent protein (YFP) under the control of the promoter of platelet factor 4 (PF4) were transfused by tail-vein injections into tumor-bearing mice. After 1 hour, ascites was removed by paracentesis, and intravital fixation was performed before the resection of tumor specimens. Panel B shows extravascular YFP platelets in solid tumor (left: CD31, red; nuclei, blue) and in ascites (right: tumor cells, red). Panel C shows representative photomicrographs of tumor, normal peritoneum, and peritoneum after the induction of peritonitis, with immunofluorescence staining for CD31 and CD42b antigens to label the vasculature (red) and endogenous platelets (green), respectively. The arrowheads indicate colocalization of CD31 and CD42b, indicative of intravascular platelets. The bar graph depicts the number of extravascular platelets per five random high-power fields (HPF) for each condition. T bars indicate standard errors.