Kim Mary E, Xu Liya, Prabakar Rishvanth K, Shen Lishuang, Peng Chen-Ching, Kuhn Peter, Gai Xiaowu, Hicks James, Berry Jesse L
The Vision Center, Children's Hospital Los Angeles; USC Roski Eye Institute, Keck School of Medicine of the University of Southern California.
The Vision Center, Children's Hospital Los Angeles; USC Roski Eye Institute, Keck School of Medicine of the University of Southern California; Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, University of Southern California; The Saban Research Institute, Children's Hospital Los Angeles.
J Vis Exp. 2021 Sep 7(175). doi: 10.3791/62939.
There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is prohibited in these patients. The aqueous humor (AH) forms in a separate compartment from the tumor but is enclosed within the same ocular space. Thus, it is an enriched source of eye-specific tumoral genomic information that can be used as a liquid biopsy or surrogate to tumor biopsy for this disease. This manuscript details a methodology for safely extracting the AH from retinoblastoma eyes via clear corneal paracentesis. Additionally, the steps for genomic analysis, including cell-free DNA isolation and purification, next-generation sequencing, somatic copy number alteration (SCNA) analysis, RB1 single nucleotide variant (SNV) mutation identification, and tumor fraction estimation are presented. The pre-analytical, analytical, and early clinical validity of the AH liquid biopsy platform have been evaluated; however, it is not without limitations. These are largely a consequence of the quantity of cell-free DNA that is required for certain steps of the assay. Compared to other blood-based liquid biopsy platforms currently under investigation for retinoblastoma, an AH-based platform is limited by the volume of biofluid (and thus the quantity of DNA) that can be extracted from the eye; the benefit is that AH is eye-specific. The platform discussed here is unique in that it detects circulating tumor DNA in the AH via two mechanisms (SCNAs and RB1 SNVs), yielding a higher sensitivity for identifying tumoral genomic information. The AH liquid biopsy has the potential for direct clinical application to precision oncology for retinoblastoma patients, with particular importance for patients with bilateral disease as the AH is specific to the tumors in each eye. There is ongoing research with applications of this platform to patients with other ocular tumors as well.
鉴于视网膜母细胞瘤患者禁止进行直接肿瘤活检，液体活检平台在视网膜母细胞瘤中的应用具有显著的潜在临床效用。房水（AH）在与肿瘤分开的腔室中形成，但被封闭在同一眼内空间内。因此，它是眼部特异性肿瘤基因组信息的丰富来源，可作为该疾病的液体活检或肿瘤活检的替代物。本手稿详细介绍了一种通过透明角膜穿刺术从视网膜母细胞瘤眼中安全提取AH的方法。此外，还介绍了基因组分析的步骤，包括游离DNA的分离和纯化、下一代测序、体细胞拷贝数改变（SCNA）分析、RB1单核苷酸变异（SNV）突变鉴定和肿瘤分数估计。已经评估了AH液体活检平台的分析前、分析和早期临床有效性；然而，它并非没有局限性。这些主要是由于检测某些步骤所需的游离DNA数量所致。与目前正在研究的用于视网膜母细胞瘤的其他基于血液的液体活检平台相比，基于AH的平台受到可从眼中提取的生物流体体积（以及因此DNA的数量）的限制；好处是AH是眼部特异性的。这里讨论的平台的独特之处在于，它通过两种机制（SCNA和RB1 SNV）检测AH中的循环肿瘤DNA，在识别肿瘤基因组信息方面具有更高的灵敏度。AH液体活检有可能直接应用于视网膜母细胞瘤患者的精准肿瘤学，对于双侧疾病患者尤为重要，因为AH对每只眼中的肿瘤具有特异性。目前也在研究将该平台应用于其他眼部肿瘤患者。
