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Prisca Bustamante, Thupten Tsering, Boli Fan, Sonia Callejo, Miguel N Burnier, Julia Burnier; GNAQ and GNA11 in circulating tumor DNA as a novel liquid biopsy-based biomarker for Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2019;60(9):956. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Uveal melanoma (UM) is the most common intraocular tumour in adults and is characterized by an early initiating mutation in either Gαq (GNAQ) or Gα11 (GNA11) gene. Liquid biopsy involves the analysis of fragments of DNA released by tumor cells, called circulating tumor DNA (ctDNA). Fluctuations in ctDNA can be used to monitor tumor formation and progression. Thus, ctDNA is a potential marker to assess tumor-specific genetic features and tumor burden. Our aim is to evaluate the GNAQ/11 expression to detect ctDNA in UM in vitro and in blood of patients.
Culture media of four human UM cell lines (MP41, 92.1, Mel270 and Mel285) was used as a source of ctDNA. In addition, blood from patients with choroidal nevi (n=6) and from healthy cancer-free control subjects (n=7) were analysed. Nevi patients were assessed clinically for risk factors for malignant transformation. ctDNA was isolated from 2 mL of plasma and 4 mL of culture media. The presence of four hotspot mutations [GNAQ (Q209P and Q209L) and GNA11 (Q209P and Q209L) were analyzed in 1 ng of ctDNA using hydrolysis probes in a QX200 PCR platform for Digital Droplet PCR. gBlocks gene fragments were used as a positive control.
Genomic DNA analysis from the 4 different cell lines confirmed the expected mutations: GNA11 Q209P in MP41, GNAQ Q209P in 92.1, and GNAQ Q209L in Mel270; Mel285 are wildtype for GNAQ/11. In the ctDNA analysis, wildtype GNAQ/11 was detected in all ctDNA derived from cell-free cultured medium of UM cells and derived from plasma. In addition, GNAQ Q209L (26 copies/µl) in Mel270, GNAQ Q209P (72 copies/µl) in 92.1, and GNA11 Q209 (270 copies/µl) in MP41 culture medium were seen. As expected, Mel285 supernatant tested negative for mutations. Low risk nevus patients (n = 3) and all cancer-free control subjects (n = 7) tested negative for GNAQ/11 mutations. However, a mutation in GNAQ Q209L (0.14 copies/µl) was observed in the nevus patient with 4 risk factors for malignant transformation.
This is the first study assessing expression of ctDNA in vitro in UM. Our data validates the potential of cell culture models to study ctDNA. ctDNA through a liquid biopsy is a powerful tool to detect and monitor UM: it is minimally invasive, and its analysis is sensitive and specific. Detectable levels of mutant GNAQ/11 in blood derived ctDNA may indicate nevi patients undergoing malignant transformation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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