Therapy with Bevacizumab Is Associated with Reduced Expression of KLF2 in Peripheral Blood Mononuclear Cells: A Potential Prothrombotic Mechanism

Background: Bevacizumab, the first approved anti-angiogenic agent for metastatic colon cancer, is associated with an increased risk of arterial thromboembolism (ATE) and potentially venous thromboembolism (VTE). The molecular mechanisms underlying this association are incompletely understood. The transcription factor Kruppel-like factor 2 (KLF2) is a master regulator of cellular inflammatory responses, and decreased expression of KLF2 is associated with a prothrombotic phenotype. KLF2 is particularly important in regulating inflammatory and prothrombotic genes in endothelial cells and monocytes.

Objective: To determine whether bevacizumab therapy was associated with alterations in the expression of KLF2 or other genes potentially associated with thrombosis in peripheral blood mononuclear cells.

Methods: We conducted a prospective observational cohort study of consecutive patients initiating bevacizumab therapy for metastatic colorectal, lung, or brain cancer. Patients with prior history of VTE or on anticoagulation were excluded. Blood was collected at baseline (prior to initiation of treatment) and every 4 weeks (± 1 week) for up to 16 weeks while on Bevacizumab therapy. Total RNA was isolated and levels of KLF2 mRNA as well as mRNA encoding 18 other genes potentially associated with thrombosis were measured using a customized qPCR microarray (Qiagen RT² Profiler). Changes in gene expression levels were standardized through inclusion of housekeeping genes, RT, and PCR controls on each array. Associations with clinical events were assessed by stepwise linear regression.

Results: The study population comprised 25 patients who received from one to four courses of bevacizumab. Primary sites of cancer included colorectal adenocarcinoma (N=18), non-small cell lung cancer (N=2), primary brain tumor (N=4) and small bowel adenocarcinoma (N=1). The average patient age was 58 years. Average length of study participation was 71.8 days. PCR analyses demonstrated a significant decrease in KLF2 mRNA after the first cycle of bevacizumab, and levels of KLF2 remained suppressed in patients who remained on study through at least three cycles of therapy ( p = 0.0015, 0.0139, 0.011, respectively). No significant change in the expression of other prothrombotic genes was demonstrated. Levels of KLF2 correlated inversely with levels of D-dimer (p = 0.008).

Conclusions: Therapy with Bevacizumab was associated with significant and durable decreases in the expression of KLF2 in peripheral blood mononuclear cells. These findings suggest a potentially new mechanism which may contribute to the prothrombotic phenotype of these patients through loss of thromboprotective gene expression in monocytes. Enhancement of KLF2 expression may provide a potential strategy to explore for circumventing thrombosis in patients treated with bevacizumab.

Acknowledgment: This work was supported by funds from the Scott Hamilton Cares Initiative, the Stephen Hardis Chair in Oncology Research, and the V Foundation.