Presence of tumor DNA in aqueous humor is correlated with high risk uveal melanoma

Abstract

Metastatic risk stratification is critical for uveal melanoma (UM) management, as approximately up to half of patients develop metastatic disease. Current prognostication for patients undergoing eye-preserving therapies relies on tumor staging and molecular analysis of tumor tissue obtained through potentially invasive biopsy, which can be challenging. While liquid biopsy using cell-free DNA (cfDNA) has emerged as a less invasive alternative for other cancers, studies have shown limited utility of blood-derived cfDNA in UM due to low tumor DNA fractions. This study investigates the potential of aqueous humor (AH) and vitreous body (VB) aspirates as alternative sources of tumor DNA for molecular prognostication in UM patients at the time of diagnosis. In this prospective study, AH and/or VB samples were collected from 96 consecutive UM patients undergoing enucleation, transretinal endoresection or transretinal biopsy. DNA was extracted from the ocular fluids and analyzed for the presence of tumor-derived DNA using deep amplicon sequencing targeting mutations in GNAQ and GNA11. This approach achieved an average read depth of 120,000, enabling highly sensitive detection of tumor-specific variants. Tumor DNA was detected in at least one ocular fluid (AH or VB) in 43 of 88 evaluable patients (49%), with variant allele fractions (VAFs) ranging from 0.3 to 50%. Of these positive cases, tumor DNA was identified in VB only in 22 patients, AH only in 5 patients, and both fluids in 16 patients. Importantly, tumor DNA in AH was almost exclusively observed in patients with monosomy 3 UM. No significant correlation was found between the presence of tumor DNA in either ocular fluid and primary tumor size or location. Liquid biopsy of AH and VB offers a promising, minimally invasive strategy for obtaining tumor DNA in nearly half of UM patients at diagnosis. The strong association between detectable tumor DNA in AH and monosomy 3 status warrants further investigation and may offer valuable insights into UM biology and dissemination mechanisms. This approach may improve risk stratification and inform personalized treatment strategies for patients with UM.

Keywords: Uveal melanoma; Aqueous humor; Biomarkers; CfDNA; Liquid biopsy; Ocular liquids; Prognostic testing; Vitreous body.

Fig. 1

Presence (positive) or absence (negative) of tumor-derived DNA in aqueous humor or vitreous body aspirate samples as determined by targeted GNAQ or GNA11 deep amplicon NGS in ocular fluids of 88 UM patients.

Fig. 2

Schematic overview of patients included in this study grouped according to the presence or absence of tumor-derived DNA in the ocular fluids tested. Bubble grid chart (lower part) showing the variant allele fraction (in %) in paired liquid biopsy samples of individual patients. Absence of circle, missing data due to insufficient or missing material. Empty circle, absence of tumor derived DNA in the sample.

Fig. 3

The distribution of the VAF of oncogenic GNAQ or GNA11 mutations in vitreous body and aqueous humor samples that were scored positive for the presence of tumor DNA.

Fig. 4

The distribution of tumor size (LBD and tumor height) is plotted for tumors showing either presence (positive) or absence (negative) of tumor-DNA in the aqueous humor or the vitreous body. (LBD, pearson correlation coefficient (PCC) = -1.34, p = 0.19; tumor height, PCC = 0.86, p = 0.39).

Fig. 5

Oncoplot (A) and mosaic plot (B) showing presence of tumor-derived DNA in liquid biopsy samples stratified according to the chromosome 3 status of the primary tumor. D3: disomy 3, M3: monosomy 3, unknown: chromosome 3 status not available, ns: not significant. **: significant association between number of samples containing tumor DNA and chromosome 3 status (p = 0.0016).

Fig. 6

Presence of tumor-dervied DNA correlated with the tumor location. Bargraph showing the percentage of samples with presence (positive) or absence (negative) of tumor-DNA in relation to the tumor location given as either ciliary body involvement (left part) or pre/postequatorial location (right part).