Development of a surrogate liquid biopsy from the aqueous humor in retinoblastoma eyes.

摘要 视网膜母细胞瘤是一种发生在儿童眼中的原发性眼内癌症。肿瘤发生开始 是由RB 1基因突变引起的，这是第一个被描述的肿瘤抑制基因。研究肿瘤 由RB 1调控的抑制子途径为研究 肿瘤发生，不仅是视网膜母细胞瘤，而且几乎所有的人类癌症。具体评价 突变、癌基因和抑制基因、肿瘤标志物和基因表达谱已经彻底改变了 乳腺癌、肺癌、前列腺癌等癌症患者的护理。然而，尽管事实上与视网膜母细胞瘤相关 研究启动了癌症遗传学领域，利用这一知识来个性化护理， 视网膜母细胞瘤患者在很大程度上是难以捉摸的，因为我们不能安全地活检这种肿瘤， 眼外扩散的担忧因此，我们目前还不能将遗传和基因组变化联系起来， 肿瘤水平与临床结果。识别这些基因组变化，并提供临床相关性 对于视网膜母细胞瘤患者来说是非常需要的。这项提案的目的是开发一种替代肿瘤 使用房水进行视网膜母细胞瘤的活检，房水是眼睛独立部分中的透明液体， 肿瘤形成的地方。从2012年开始，房水的提取已经被常规和安全地完成， 将化疗药物注射到眼睛玻璃体腔的标准程序部分;没有病例 已经报道了眼外扩散。我们的初步数据从评估的水显示， 存在肿瘤来源的无细胞DNA，全基因组测序显示染色体DNA谱。 证实肿瘤变化的得失。本研究的具体目的是测试 以下假设：水可以作为替代肿瘤活检-更准确地说，水 含有肿瘤来源的遗传物质，其可以被测定并与临床结果相关。目标1是 以确定在水相中高度复发的染色体变化，例如2 p增益/MYCN扩增， 已知发生在视网膜母细胞瘤肿瘤中。目的2是鉴定导致糖尿病的特异性RB 1突变， 肿瘤的发生。最后，目标3将评估RNA表达谱，以确定 亚组在水，并将这些与临床结果，如肿瘤复发。目前 实现这些目标的唯一方法是对肿瘤组织进行评估，只有当眼睛 移除（例如，摘除）。然而，通过定义这些遗传变化的水，我们将，第一， 时间，能够在治疗过程中原位表征肿瘤。这将有助于发展预后 治疗反应的标志物，并在未来，可能指导治疗。使用房水作为 替代肿瘤活检具有巨大的潜力，从直接影响我们管理视网膜母细胞瘤的方式， 患者，最终允许为这种致盲的儿童癌症开发个性化药物。 ! !

Abstract Retinoblastoma is a primary intraocular cancer that develops in the eyes of children. Tumorigenesis is initiated by a mutation in the RB1 gene, which was the first tumor suppressor gene described. Investigating the tumor suppressor pathway regulated by RB1 has provided unprecedented insights into the genetic mechanisms of tumorigenesis, not only for retinoblastoma, but virtually all human cancers. The evaluation of specific mutations, oncogenes and suppressors, tumor markers and gene expression profiles have revolutionized the care of breast, lung, prostate and other cancer patients. However, despite the fact that retinoblastoma-related research launched the field of cancer genetics, leveraging this knowledge to personalize the care for retinoblastoma patients has been elusive in large part because we cannot safely biopsy this tumor due to concerns of extraocular spread. Thus, we currently cannot correlate the genetic and genomic changes at the level of the tumor with clinical outcomes. Identifying these genomic changes, and providing clinical correlation is critically needed for retinoblastoma patients. The objective of this proposal is to develop a surrogate tumor biopsy for retinoblastoma using the aqueous humor, which is the clear fluid in a separate part of the eye from where the tumor forms. Starting in 2012, extractions of aqueous humor have been routinely and safely done as a standard part of the procedure to inject chemotherapy into the vitreous cavity of the eye; no cases of extraocular spread have been reported. Our preliminary data from evaluation of the aqueous revealed that tumor-derived cell-free DNA is present and whole genome sequencing demonstrates a profile of chromosomal gains and losses that corroborate changes in the tumor. The specific aims of this study are designed to test the following hypothesis: the aqueous can serve as a surrogate tumor biopsy – more precisely, the aqueous harbors tumor-derived genetic material which can be assayed and correlated with clinical outcomes. Aim 1 is to define highly recurrent chromosomal changes in the aqueous, such as 2p gain/MYCN amplification, that are known to occur in retinoblastoma tumors. Aim 2 is to identify the specific RB1 mutation(s) that underlie tumorigenesis from the aqueous humor. Finally, Aim 3 will evaluate RNA expression profiles to define subgroups in the aqueous, and correlate these with clinical outcomes such as tumor recurrence. Currently, the only way any of these Aims are possible is from evaluation of tumor tissue, which is only available if an eye is removed (e.g. enucleated). However, by defining these genetic changes in the aqueous we will, for the first time, be able to characterize tumors in situ during therapy. This will allow for the development of prognostic markers for therapeutic response and, in the future, may guide therapy. Use of the aqueous humor as a surrogate tumor biopsy holds tremendous potential, from directly impacting the way we manage retinoblastoma patients, to ultimately allowing for the development of personalized medicine for this blinding childhood cancer. ! !