Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma.

PURPOSE To develop a molecular tool to detect circulating tumor-derived DNA (ctDNA) in the plasma from patients with uveal melanoma as a marker of tumor burden and monitor treatment efficacy. EXPERIMENTAL DESIGN A real-time PCR was developed on the basis of bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) for the quantification of ctDNA using 3'blocked primer pairs specific for the 3 recurrent mutually exclusive mutations of Gα subunits GNAQ and GNA11. RESULTS Sensitivity and specificity of bi-PAP were assessed on serial dilutions of tumor DNA in normal DNA for the 3 recurrent mutations. Each assay could detect a single mutated molecule per reaction, whereas 10(4) copies of normal DNA were not detected. The ctDNA was readily detected in plasma of mice bearing uveal melanoma xenografts in amounts proportional to circulating human DNA. Finally, plasma was almost always found positive (20 of 21 tested patients) in a prospective analysis of patients with metastatic uveal melanoma. CONCLUSIONS Bi-PAP assays detect and quantify ctDNA in patients with metastatic uveal melanoma. A prospective study is ongoing to assess the clinical usefulness of ctDNA level in uveal melanoma.