Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma

Abstract

Background Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Future perspectives of uveal melanoma blood based biomarkers

“…ctDNA predicted response to targeted therapy and increasing ctDNA preceded radiological progression with 4–10-week lead time. Bustamante [ 115 ] ddPCR PG or SST 2 ≤1 2000/20 → 2000/20 Q 25 14/14 pUM 8/16 naevi pUM 3.75 (0.7–31.4) Naevi 2.3 (1–13.3) ctDNA levels correlated with malignancy. In naevi, ctDNA levels correlated with clinical risk factors.…”

“…In primary UM, a recent study ctDNA was detected in 100% of primary UM, and 50% of ocular naevi, with ctDNA levels correlating with malignancy [ 115 ] using droplet digital PCR (ddPCR) to detect mutation in GNAQ , GNA11 , PLCβ4 , and CYSLTR2 . In contrast, in our previous study ctDNA was only detectable in 26% of patients with primary UM prior to localised therapy [ 85 ] using ddPCR, and did not correlate with clinical or histological markers of disease.…”

Is Tissue Still the Issue? The Promise of Liquid Biopsy in Uveal Melanoma

“…Moreover, naevi can harbor the same primary driver mutations in GNAQ or GNA11 as UMs do, without malignant transformation. Elevated levels of mutated cfDNA are highly correlated to malignancy and clinical risk factors for UM [ 75 ]. Therefore, circulating DNA harboring primary driver mutations could provide information of malignant transformation of these lesions and can be used for follow-up of naevi at risk.…”

Uveal Melanoma

“…21 Perhaps the most sensitive marker, CtDNA was detected in the blood of 100% of patients with uveal melanoma, but also detected in 50% of patients with ocular nevi. 29 CmiRNA was used to differentiate benign uveal nevi from uveal melanoma with 93% sensitivity and 100% specificity. 30 However, the studies examining the diagnostic value of ctDNA and cmiRNA have been limited and the number of participants have been low, so further validation is required in order for these to become relevant.…”