Berry, J.L., Xu, L., Murphree, A.L., et al.
活组织检查 房水 视网膜
摘要：重要性：视网膜母细胞瘤（Rb）是第一个具有已知遗传病因的肿瘤之一。然而，由于这种肿瘤的活检是禁忌的，因此不可能确定继发性遗传改变对疾病进程的影响。目的：旨在研究Rb眼房水（AH）是否具有足够的肿瘤来源DNA来进行肿瘤基因分析。设计，设置和研究对象：这项调查是在一家三级医院（洛杉矶儿童医院）----一家大型Rb治疗中心进行的一项病例系列研究。从3例Rb患儿（其中2例原发性摘除，1例多次玻璃体注射美法仑治疗玻璃体播种）的6个AH样本中分离出无细胞DNA（cfDNA）。样本采集于2014年12月至2015年9月。主要预后指标：AH中可测量的核酸水平和AH中肿瘤来源的DNA拷贝数变异。使用Qubit高灵敏度试剂盒分析AH，DNA和RNA。从AH中分离无细胞DNA，并对测序数据库进行优化。在Illumina平台上进行浅全基因组测序，接着进行全基因组染色体拷贝数变异分析以评估3名患者的AH cfDNA中肿瘤DNA部分的存在。结果：从3例患儿（男2例，女1例;分别在7,20,28个月时诊断）的3只Rb眼获得了6例AH标本。确定了AH cfDNA染色体拷贝数变异谱与来自去核样品的肿瘤来源的DNA之间的确定模式。另外，1名儿童玻璃体注射美法仑期间获得的AH样品中也确定了无义RB1突变（Lys→STOP），其与肿瘤样品二次去核相匹配。结果与相关性：本研究评估了玻璃体内注射美法仑对Rb眼进行补救性治疗的AH患者的AH核酸进行了评估，结果提示当Rb肿瘤组织不可用时，AH可以用作替代肿瘤活组织检查。这种新方法将允许分析中正在进行挽救疗法但尚未去核的Rb眼肿瘤来源的DNA。
Importance:Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course.Objective:To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations.Design, Setting, and Participants:This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015.Main Outcomes and Measures:Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation.Results:Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation.Conclusions and Relevance:In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated