Method development to quantify Bv8 expression in circulating CD11b+ cells in patients with neovascular age-related macular degeneration (nvAMD) exhibiting Anti-VEGF refractoriness

• A technique of assaying gene expression in sets of circulating cells in nvAMD patients is developed.
• The assay is shown to have good reproducibility and low intra-patient variability.
• A link between Bv8 expression in CD11b + cells and nvAMD treatment response is investigated.

A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. Prokineticin-2 (Bv8) expression in CD11b+ cells has been linked to anti-VEGF response. We have developed a reproducible method to quantify gene expression in circulating CD11b + cells. Utilizing this method we tested the hypothesis that high Bv8 expression in circulating CD11b+ cells is associated with anti-VEGF refractoriness in nvAMD patients. Two groups of nvAMD subjects undergoing treatment with anti-VEGF agents were recruited and classified as refractory or non-refractory to anti-VEGF treatment (n = 33 for each group). Two blood draws were obtained from each subject 1–9 months apart. Peripheral blood mononuclear cells (PBMCs) were isolated and CD11b+ cells were purified via magnetic bead separation. RNA was purified, and relative expression of Bv8 among the subjects was compared via quantitative PCR analysis. Utilizing this approach no significant difference was detected in the mean LogRQ values between the first and second blood draws (t-test, p = 0.826) indicating low intra-patient variability and demonstrating good reproducibility of the assay. There was no significant difference in Bv8 expression between nvAMD subjects classified as refractory versus non-refractory. We were unable to find a correlation between Bv8 expression in CD11b + cells and anti-VEGF refractoriness in human nvAMD subjects. Relatively high expression in Bv8 in these subjects did not correlate with clinical treatment history, as measured by the frequency of injections. Utilizing this well characterized technique, studies are underway to examine alternative gene expression profiles in various circulating cell populations that may contribute to anti-VEGF refractoriness.