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

项目摘要 有大量关于视网膜母细胞瘤的遗传、基因组和表观基因组改变的研究。 (RB)，一种在幼儿发育中的视网膜形成的原发眼癌。然而，这些研究是 对晚期RB的手术摘除(去核)眼的肿瘤组织进行手术，因为肿瘤活检不是 可能是由于肿瘤有眼外扩散的真实风险。因此，RB肿瘤DNA以前从未 除了这些去核标本之外，人们对分子的了解也很有限 可能导致肿瘤行为的改变。此外，分子诊断的任何应用或使用 体内个体化药物的预后生物标记物受限于诊断或诊断时肿瘤组织的缺乏 在治疗期间。因此，需要一种液体活检法，以克服这种严重的肿瘤组织缺乏。 治疗这种疾病。在NCI K08的支持下，我们证明了房水(AH)，一种眼内液体， 是肿瘤来源的无细胞DNA(CfDNA)的丰富来源。我们开发了一种液体活组织检查方法来检测 沙门氏菌RB1抑癌基因的体细胞拷贝数改变和致病变异 单份100型的L的血液标本。我们发现来自AH的基因组改变是高度一致的(&gt；94%) 与在摘除眼球的肿瘤中发现的相同。我们确定了潜在的候选生物标记物，染色体6p AHcfDNA中获得和/或局部MYCNa与治疗失败风险增加16.5倍相关 需要手术摘除眼睛的。我们证明了AH cfDNA肿瘤部分(TFX)的变化与 治疗有效，TFX升高提示复发或微小的眼内残留疾病。 这表明TFX单独可以作为治疗反应的可靠的实时生物标志物。我们也 论证了使用AH评估肿瘤甲基化特征的可行性，从而促进了更好的 了解可能预测肿瘤行为和潜在治疗反应的肿瘤生物标志物。基座 在这些结果中，我们假设AH cfDNA可以用于体内RB肿瘤的分子表征 基于有效的基因组和表观基因组生物标记物，为眼科挽救的诊断和预后提供信息。至 为了验证这一假设，我们现在提出了一项多中心、多组学、前瞻性研究，以表征预后 前瞻性和纵向确定治疗结果的生物标志物。 这项研究的好处将包括增进对疾病任何阶段的过程的了解，提供 指导治疗的生物标志物，并为未来基于分子的精确医学临床试验奠定基础 对于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.