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


METHODS
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.
Institut Curie, Paris, France.
Jordan
J
Madic
Sophie
S
Piperno-Neumann
Vincent
V
Servois
Aurore
A
Rampanou
Maud
M
Milder
Bénédicte
B
Trouiller
David
D
Gentien
Stéphanie
S
Saada
Franck
F
Assayag
Aurélie
A
Thuleau
Fariba
F
Nemati
Didier
D
Decaudin
François-Clément
FC
Bidard
Laurence
L
Desjardins
Pascale
P
Mariani
Olivier
O
Lantz
Marc-Henri
MH
Stern
D000818:Animals; D004247:DNA / Q000097:blood*; D006801:Humans; D008545:Melanoma / Q000097:blood / Q000556:secondary; D051379:Mice; D009374:Neoplasms, Experimental / Q000097:blood / Q000556:secondary; D009360:Neoplastic Cells, Circulating; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014604:Uveal Neoplasms / Q000097:blood / Q000556:secondary; D000098943:Uveal Melanoma
D004247:DNA