Abstract
Purpose :
Uveal melanoma (UM) is the most common intraocular tumor in adults and is
characterized by an early initiating mutation in either the GNAQ or GNA11 gene. Our group has previously shown that circulating tumor DNA (ctDNA) from plasma in a biomarker of UM development and progression. Currently there are no studies that assess the presence of ctDNA in aqueous humor (AH). AH may be an alternative liquid biopsy that could be used to detect ctDNA and monitor tumor response to therapy and metastasis. This study aims to evaluate ctDNA through GNAQ mutations from AH in an UM animal model.
Methods :
Five albino rabbits were inoculated into the subchroidal space with human 92.1 UM cells (GNAQ-mutant) as a source of ctDNA. AH was extracted by paracentesis from the anterior chamber using an insulin-like syringe. No dilation of the eye was needed and local anesthesia with Proparacaine was administered to the eye. The extraction was at two time points: 100ul before cell inoculation (Time 0) and 300ul at the end of the study (Time 1). ctDNA was extracted using the QIAamp Circulating Nucleic acid kit. The presence of GNAQ (Q209L) hotspot mutation was analyzed from 1.5 ng of DNA using hydrolysis probes in a QX200 PCR platform for Digital Droplet PCR. gBlocks gene fragments were used as a positive control.
Results :
Ocular tumors were found in all rabbits (week 5). Paracentesis was successful in all rabbits at both time points. One rabbit had a capsular rupture; no signs of a cataract or complications were found. The rupture resolved without treatment. Total DNA from AH was quantified to be 6.4-10.32 ng at Time 0 and 5.56-8.08ng at Time 1. Before cell inoculation, AH samples were found to be negative for GNAQ-mutant ctDNA. In contrast, 4/5 rabbits showed detectable levels of ctDNA after tumor formation, with a range of 0.09-0.48 copies/ul. The rabbit with a higher level of ctDNA had 5 multifocal pigmented masses (4 extending to the nasal side and 1 inferior), while the other rabbits only had one mass.
Conclusions :
To the best of our knowledge, this was the first study to detect ctDNA in AH in an UM animal model. Our data demonstrate that GNAQ mutations are detectable in the AH of UM eyes, and that AH may serve as a less invasive “surrogate tumor biopsy”. While additional studies are required, these results show that AH has potential as a source of tumor derived material that can be used for the diagnosis and monitoring of UM.
This is a 2020 ARVO Annual Meeting abstract.