Nell R J, Versluis M, Menger N V, Gelmi M C, Vu T H K, Verdijk R M, Luyten G P M, Jager M J, van der Velden P A
Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands.
J Exp Clin Cancer Res. 2025 Apr 17;44(1):124. doi: 10.1186/s13046-025-03374-y.
Uveal melanoma is an aggressive ocular malignancy. Early molecular characterisation of primary tumours is crucial to identify those at risk of metastatic dissemination. Although tumour biopsies are being taken, liquid biopsies of ocular fluids may form a less invasive but relatively unexplored alternative. In this study, we aim to evaluate the DNA content of vitreous fluid from eyes with a uveal melanoma to obtain molecular tumour information.
DNA was isolated from 65 vitreous fluid samples from enucleated eyes with a uveal melanoma and studied using digital PCR. Primary and additional driver mutations (in GNAQ, GNA11, PLCB4, CYSLTR2, BAP1, SF3B1 and EIF1AX) were investigated using accustomed targeted and drop-off assays. The copy numbers of chromosome 3p and 8q were measured using multiplex and single-nucleotide polymorphism-based assays. Our findings were compared to the molecular profile of matched primary tumours and to the clinicopathological tumour characteristics.
Almost all (63/65) vitreous fluids had measurable levels of DNA, but melanoma-cell derived DNA (containing the primary driver mutation) was detected in 45/65 samples (median proportion 15.5%, range 0.03-94.4%) and was associated with a larger tumour prominence, but not with any of the molecular tumour subtypes. Among the vitreous fluids with melanoma-cell derived DNA, not all samples harboured (analysable) other mutations or had sufficient statistical power to measure copy numbers. Still, additional mutations in BAP1, SF3B1 and EIF1AX were detected in 15/17 samples and chromosome 3p and 8q copy numbers matched the primary tumour in 19/21 and 18/20 samples, respectively. Collectively, a clinically-relevant molecular classification of the primary tumour could be inferred from 29/65 vitreous fluids.
This proof-of-concept study shows that substantial amounts of DNA could be detected in vitreous fluids from uveal melanoma patients, including melanoma-cell derived DNA in 69% of the samples. Prognostically-relevant genetic alterations of the primary tumour could be identified in 45% of the patients. A follow-up study is needed to evaluate our approach in a prospective clinical context. Additionally, our work highlights improved possibilities to sensitively analyse scarce and heterogeneous tumour biopsies, with potential application in other malignancies.
葡萄膜黑色素瘤是一种侵袭性眼部恶性肿瘤。原发性肿瘤的早期分子特征对于识别有转移扩散风险的肿瘤至关重要。尽管正在进行肿瘤活检，但眼内液体的液体活检可能是一种侵入性较小但相对未被充分探索的替代方法。在本研究中，我们旨在评估葡萄膜黑色素瘤患者眼内玻璃体的DNA含量，以获取分子肿瘤信息。
从65例摘除的葡萄膜黑色素瘤患者的眼内玻璃体样本中分离DNA，并使用数字PCR进行研究。使用常规靶向和脱落分析研究主要和其他驱动基因突变（在GNAQ、GNA11、PLCB4、CYSLTR2、BAP1、SF3B1和EIF1AX中）。使用多重和基于单核苷酸多态性的分析方法测量3号染色体短臂和8号染色体长臂的拷贝数。将我们的研究结果与匹配的原发性肿瘤的分子特征以及临床病理肿瘤特征进行比较。
几乎所有（63/65）玻璃体样本都有可测量的DNA水平，但在45/65个样本中检测到黑色素瘤细胞衍生的DNA（包含主要驱动基因突变）（中位数比例为15.5%，范围为0.03 - 94.4%），并且与更大的肿瘤突出相关，但与任何分子肿瘤亚型均无关。在含有黑色素瘤细胞衍生DNA的玻璃体样本中，并非所有样本都存在（可分析的）其他突变或具有足够测量拷贝数所需的统计效力。尽管如此，在15/17个样本中检测到BAP1、SF3B1和EIF1AX的其他突变，并且在19/21和18/20个样本中，3号染色体短臂和8号染色体长臂的拷贝数分别与原发性肿瘤匹配。总体而言，从29/65个玻璃体样本中可以推断出原发性肿瘤的临床相关分子分类。
这项概念验证研究表明，在葡萄膜黑色素瘤患者的玻璃体中可以检测到大量DNA，包括69%的样本中存在黑色素瘤细胞衍生的DNA。在45%的患者中可以识别出原发性肿瘤的预后相关基因改变。需要进行后续研究以在临床前瞻性背景下评估我们的方法。此外，我们的工作突出了更灵敏地分析稀缺且异质性肿瘤活检样本的可能性增加，这在其他恶性肿瘤中可能有应用。
