Validation of an aqueous humor liquid biopsy for molecular prognostication and monitoring of children with retinoblastoma.

项目摘要 有一个显着的机构的研究，遗传，基因组和表观基因组的改变视网膜母细胞瘤 (RB)这是一种原发性眼癌，在幼儿视网膜发育中形成。然而，这些研究 对手术切除（摘除）的晚期RB眼睛的肿瘤组织进行了检查，因为肿瘤活检不是 可能是由于肿瘤眼外扩散的真实的风险。因此，RB肿瘤DNA以前从未被 除了这些去核标本外，对这些细胞的分子结构的了解有限。 可能驱动肿瘤行为的改变。此外，分子诊断的任何应用或分子诊断的使用都是可能的。 用于体内个性化药物的预后生物标志物受到诊断时缺乏肿瘤组织的限制， 在治疗期间。因此，需要一种液体活检方法来克服这种严重缺乏肿瘤组织的情况 治疗这种疾病在NCI K 08的支持下，我们证明了眼内液体， 是肿瘤来源的无细胞DNA（cfDNA）的富集来源。我们开发了一种液体活检检测方法 体细胞拷贝数改变（SCNAs）和致病性变异的RB 1肿瘤抑制基因从一个 单个100 μ l AH样品。我们发现AH的基因组改变高度一致（>94%）。 与眼球摘除肿瘤中发现的相同我们确定了潜在的候选生物标志物，染色体6 p AH cfDNA中的增益和/或局灶性MYCNa与治疗失败风险增加16.5倍相关 需要手术摘除眼睛我们证明了AH cfDNA肿瘤分数（TFX）的变化与肿瘤细胞的生长相关。 治疗反应，TFx增加表明复发或轻微残留眼内疾病。 这表明单独的TFX可以作为治疗反应的可靠实时生物标志物。我们也 证明了使用AH评估肿瘤甲基化谱的可行性，从而促进了更好的 了解可以预测肿瘤行为和潜在治疗反应的肿瘤生物标志物。基于 在这些结果中，我们假设AH cfDNA可用于体内RB肿瘤的分子表征 基于经验证的基因组和表观基因组生物标志物，为眼挽救提供诊断和预后信息。到 为了验证这一假设，我们现在提出一项多中心、多组学、前瞻性研究来描述预后特征 前瞻性和纵向评估所有生物标志物，以确定治疗结局。 这项研究的好处将包括在任何阶段推进对疾病过程的了解， 生物标志物来指导治疗，并为未来基于分子的精准医学临床试验奠定基础 关于RB

Project Summary There is a significant body of research into the genetic, genomic and epigenomic alterations of retinoblastoma (RB), a primary eye cancer that forms in the developing retina in young children. However, these studies were done on tumor tissues from surgically removed (enucleated) eyes with advanced RB, as tumor biopsy is not possible due to the real risk of tumor extraocular dissemination. As a result, RB tumor DNA was never previously accessible aside from these enucleated specimens, and there is limited understanding of the molecular alterations that may drive tumor behavior. Furthermore, any application of molecular diagnostic or use of prognostic biomarkers for personalized medicine in vivo is limited by the lack of tumor tissue at diagnosis or during therapy. Thus, a liquid biopsy approach, which overcomes this critical lack of tumor tissue, was needed for this disease. With support of an NCI K08, we demonstrated that the aqueous humor (AH), an intraocular fluid, is an enriched source of tumor-derived cell-free DNA (cfDNA). We developed a liquid biopsy assay to detect somatic copy number alterations (SCNAs) and pathogenic variants in the RB1 tumor suppressor gene from a single 100 l sample of AH. We identified that the genomic alterations from the AH are highly concordant (>94%) with those found in the tumor of enucleated eyes. We identified potential candidate biomarkers, chromosome 6p gain and/or focal MYCNa in the AH cfDNA that are associated with a 16.5-fold increased risk of treatment failure requiring surgical removal of the eye. We demonstrated that changes in AH cfDNA tumor fraction (TFx) correlate with treatment response, with increases in TFx indicative of recurrence or minimal residual intraocular disease. This suggests that TFx alone may serve as a reliable real-time biomarker for treatment response. We also demonstrated the feasibility of evaluating tumor methylation profiles using the AH, thereby facilitating a better understanding of tumor biomarkers that may predict tumor behavior and potentially treatment response. Based in these results, we hypothesize that AH cfDNA can be used for molecular characterization of in vivo RB tumors to inform diagnosis and prognosis for eye salvage based on validated genomic and epigenomic biomarkers. To test this hypothesis, we now propose a multi-center, multi-omics, prospective study to characterize prognostic AH biomarkers prospectively and longitudinally to determine treatment outcomes. Benefits from this study will include advancing knowledge about the course of the disease at any stage, providing biomarkers to guide treatment, and forming the basis for future molecular-based, precision medicine clinical trials for RB.