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

Abstract

Purpose: To develop a molecular tool to detect circulating tumor-derived DNA (ctDNA) in the plasma from patients with uveal melanoma (UM) as a marker of tumor burden and monitor treatment efficacy. Experimental Design: A real-time PCR was developed based on bi-directional 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, while 104 copies of normal DNA were not detected. CtDNA was readily detected in plasma of mice bearing UM xenografts in amounts proportional to circulating human DNA. Finally, plasma were almost always found positive (20 out of 21 tested patients) in a prospective analysis of metastatic UM patients. Conclusions: Bi-PAP assays detect and quantify ctDNA in metastatic UM patients. A prospective study is ongoing to assess the clinical usefulness of ctDNA level in UM.