Abstract
Purpose :
Detection of small fragments of DNA [circulating free DNA (cfDNA)] is a promising tool to monitor and predict tumor progression, as well as treatment response. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions e.g.extracellular vesicles (EV). Our group recently demonstrated that cfDNA is clinically relevant as a biomarker of tumor development and progression in uveal melanoma (UM), the most common intraocular tumor in adults. However, the biology of cfDNA in UM is unknown. We assessed the release pattern of cfDNA and its role in a xenograft model.
Methods :
Human 92.1 and MP41 UM cell lines were used. Apoptosis was induced using Roscovite, APO2L, and RU486 and necrosis with heat. Annexin V validated cell death. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit. EV-associated-DNA was obtained using QIAamp Mini kit using 100 mL ultra-centrifugated supernatant. cfDNA from plasma were obtained from rabbits xenografted with 92.1 (N=5) and MP41(N=4) cells before inoculation, at tumor observation, and week 12 post-injection. Digital PCR was used to assess cfDNA through wildtype (WT) and mutation (MUT) of GNAQ/11.
Results :
WT GNAQ/11 was detected in all cfDNA. The MUT cfDNA level was 4.3 (MP41) and 2.4 (92.1) in normal conditions, a higher level was detected under APO2L [15.6 (MP41) and 4 (92.1) copies/mL] and a dramatically higher level under roscovite [913 copies/mL (MP41)]. cfDNA release correlated to RU486 concentration: 10uM= 9, 20uM=28.6, and 40uM=380 copies/mL. Not cfDNA increasment under heat-induced necrosis. EV-associated DNA showed a homogenous released of WT vs. MUT cfDNA while WT cfDNA were higher than MUT in cfDNA derived from control cultured media. In xenograft model, ctDNA levels correlated with ophthalmology observations; none detectable cfDNA levels were found before inoculation, while cfDNA ranging from 0.36-0.63 (92.1) and 0.3-2.5 (MP41) were detected at the time of tumors development, and a range of 0.6-1.3 (92.1) and 1.3-7.1 (MP41) copies/mL at 12 weeks post-injection.
Conclusions :
Our work is the first study focused on understanding the pattern of release of cfDNA using in vitro and in vivo models. Knowing the biology of cfDNA can help to understand its role in UM development and treatment response, and how best to utilize this marker clinically, especially post cytotoxic agents that induce different mechanisms of cell death.
This is a 2020 ARVO Annual Meeting abstract.