Vahid Afshar-Kharghan

Paraneoplastic Thrombocytosis in Ovarian Cancer

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.).

Platelet activation leads to activation and propagation of the complement system

Inflammation and thrombosis are two responses that are linked through a number of mechanisms, one of them being the complement system. Various proteins of the complement system interact specifically with platelets, which, in turn, activates them and promotes thrombosis. In this paper, we show that the converse is also true: activated platelets can activate the complement system. As assessed by flow cytometry and immunoblotting, C3 deposition increased on the platelet surface upon cell activation with different agonists. Activation of the complement system proceeded to its final stages, which was marked by the increased generation of the anaphylotoxin C3a and the C5b-9 complex. We identified P-selectin as a C3b-binding protein, and confirmed by surface plasmon resonance binding that these two proteins interact specifically with a dissociation constant of 1 μM. Using heterologous cells expressing P-selectin, we found that P-selectin alone is sufficient to activate the complement system, marked by increases in C3b deposition, C3a generation, and C5b-9 formation. In summary, we have found that platelets are capable of activating the complement system, and have identified P-selectin as a receptor for C3b capable of initiating complement activation. These findings point out an additional mechanism by which inflammation may localize to sites of vascular injury and thrombosis.

Platelets increase the proliferation of ovarian cancer cells

Platelets promote metastasis and angiogenesis, but their effect on tumor cell growth is uncertain. Here we report a direct proliferative effect of platelets on cancer cells both in vitro and in vivo. Incubation of platelets with ovarian cancer cells from murine (ID8 and 2C6) or human (SKOV3 and OVCAR5) origin increased cell proliferation. The proliferative effect of platelets was not dependent on direct contact with cancer cells, and preincubation of platelets with blocking antibodies against platelet adhesion molecules did not alter their effect on cancer cells. The proliferative effect of platelets was reduced by fixing platelets with paraformaldehyde, preincubating platelets with a TGF-β1-blocking antibody, or reducing expression of TGF-βR1 receptor on cancer cells with siRNA. Infusing platelets into mice with orthotopic ovarian tumors significantly increased the proliferation indices in these tumors. Ovarian cancer patients with thrombocytosis had higher tumor proliferation indices compared with patients with normal platelet counts.

Leukocyte adhesion and thrombosis.

Purpose of the reviewThe consequences of arterial thrombosis such as myocardial infarction, stroke and peripheral vascular occlusion are the leading causes of morbidity and mortality. A high leukocyte count and an elevation in inflammatory markers are identified as significant risk factors for thrombosis. Leukocytes form the front line in defense against infection and are the first cells arriving at the site of inflammation. This review summarizes the cellular and molecular mechanisms by which adherent leukocytes can induce a prothrombotic state. Recent findingsCirculating tissue factor has been recognized as a potential prothrombotic factor initiating thrombosis after vascular injury. The tissue factor is present on microvesicles originated from activated leukocytes. Leukocytes generate tissue factor containing microvesicles following stimulation with cytokines and following platelet adhesion via P-selectin. Additionally, activated leukocytes release several mediators, such as cathepsin G and elastase, which can activate both the coagulation cascade and platelets. Furthermore, new roles for leukocytes have been identified in vascular injury in sickle cell anemia, in vascular occlusion following the rupture of atherosclerotic plaque, and in thrombotic complications of myeloproliferative diseases. SummaryLeukocyte adhesion to endothelium and platelets plays an important role in the activation of the coagulation cascade. An excessive activation of leukocytes during the inflammatory process may induce a systemic procoagulant state. Elucidation of critical steps in activation of coagulation by leukocytes may offer a new therapeutic target for antithrombotic therapy based on blocking leukocyte adhesion.

Autocrine Effects of Tumor-Derived Complement

SUMMARY We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.

Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome

Complement dysregulation leads to atypical hemolytic uremic syndrome (aHUS), while ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. We investigated whether genetic variations in the ADAMTS13 gene partially explain the reduced activity known to occur in some patients with aHUS. We measured complement activity and ADAMTS13 function, and completed mutation screening of multiple complement genes and ADAMTS13 in a large cohort of aHUS patients. In over 50% of patients we identified complement gene mutations. Surprisingly, 80% of patients also carried at least 1 nonsynonymous change in ADAMTS13, and in 38% of patients, multiple ADAMTS13 variations were found. Six of the 9 amino acid substitutions in ADAMTS13 were common single nucleotide polymorphisms; however, 3 variants-A747V, V832M, and R1096H- were rare, with minor allele frequencies of 0.0094%, 0.5%, and 0.32%, respectively. Reduced complement and ADAMTS13 activity (<60% of normal activity) were found in over 60% and 50% of patients, respectively. We concluded that partial ADAMTS13 deficiency is a common finding in aHUS patients and that genetic screening and functional tests of ADAMTS13 should be considered in these patients.

Paraneoplastic thrombocytosis: the secrets of tumor self-promotion

Paraneoplastic thrombocytosis is associated with many solid tumors and often correlates with reduced survival. Recent studies suggest that a pathogenic feed back loop may be operative between platelets and tumor cells, with reciprocal interactions between tumor growth/metastasis and thrombocytosis/platelet activation. Specific molecular pathways have been identified in which tumors can stimulate platelet production and activation; activated platelets can, in turn, promote tumor growth and metastasis. Taken together, these findings provide exciting new potential targets for therapeutic intervention.

Role of Kozak sequence polymorphism of platelet glycoprotein Ibα as a risk factor for coronary artery disease and catheter interventions

OBJECTIVES We sought to determine the role of the -5T/C polymorphism of the platelet glycoprotein (GP) Ibalpha as a potential risk factor for coronary artery disease (CAD) and adverse events complicating a coronary catheter intervention. BACKGROUND The platelet GP Ib-IX-V receptor complex plays a crucial role in arterial thrombus formation. The -5T/C polymorphism of GP Ibalpha is associated with increased receptor density. METHODS We genotyped 1,000 patients with angiographically confirmed CAD, as well as 1,000 age- and gender-matched control subjects, for this polymorphism by polymerase chain reaction/restriction fragment length polymorphism. Among the patients with CAD, 269 underwent percutaneous transluminal coronary angioplasty (PTCA), 103 underwent directional coronary atherectomy and 278 underwent stenting. This intervention group was followed for a 30-day composite end point of target vessel revascularization, myocardial infarction or death. RESULTS Carriers of the -5C allele were significantly over-represented in the group of patients developing acute coronary syndromes (relative risk [RR] 1.43, 95% confidence interval [CI] 1.05 to 1.95, p = 0.02). The -5C allele furthermore predicted an increased risk for developing complications after PTCA (RR 3.75, 95% CI 1.15 to 12.27, p = 0.029). CONCLUSIONS The -5C allele of the GP Ibalpha Kozak polymorphism may represent a risk factor in clinical conditions in which thrombosis plays an important role, such as in acute coronary syndromes and in complications after PTCA.

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

Differential platelet levels affect response to taxane-based therapy in ovarian cancer

Purpose: We hypothesized that platelet levels during therapy could serve as a biomarker for response to therapy and that manipulation of platelet levels could impact responsiveness to chemotherapy. Experimental Design: The medical records of patients with recurrent or progressive ovarian cancer were retrospectively queried for changes in platelet and CA-125 levels during primary therapy. In vitro coculture experiments and in vivo orthotopic models of human ovarian cancer in mice were used to test the effect of modulating platelet levels on tumor growth and responsiveness to docetaxel. Results: Thrombocytosis at the diagnosis of ovarian cancer was correlated with decreased interval to progression (P = 0.05) and median overall survival (P = 0.007). Mean platelet levels corrected during primary therapy and rose at recurrence. Contrary to treatment-responsive patients, in a cohort of patients refractory to primary therapy, platelet levels did not normalize during therapy. In A2780, HeyA8, and SKOV3-ip1 ovarian cancer cell lines, platelet coculture protected against apoptosis (P < 0.05). In orthotopic models of human ovarian cancer, platelet depletion resulted in 70% reduced mean tumor weight (P < 0.05). Compared with mice treated with docetaxel, mice treated with both docetaxel and platelet-depleting antibody had a 62% decrease in mean tumor weight (P = 0.04). Platelet transfusion increased mean aggregate tumor weight 2.4-fold (P < 0.05), blocked the effect of docetaxel on tumor growth (P = 0.55) and decreased tumor cell apoptosis. Pretransfusion aspirinization of the platelets blocked the growth-promoting effects of transfusion. Conclusions: Platelet-driven effects of chemotherapy response may explain clinical observations. Clin Cancer Res; 21(3); 602–10. ©2014 AACR.