Abstract：
OBJECTIVE: Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies.
METHODS: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene.
RESULTS: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable.
CONCLUSIONS: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
METHODS: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene.
RESULTS: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable.
CONCLUSIONS: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
摘要：
目的：视网膜母细胞瘤（RB）是儿童年龄组中最常见的眼内肿瘤。遗传学在预测生存预后中的作用已被探索，但是它在预测全球救助中的作用仍未得到探索。我们在此旨在从RB眼中的房水（AH）中分离无细胞DNA（cfDNA），并验证其在遗传研究中的用途。
方法：AH来自26只接受摘除（A组）或玻璃体内化疗（B组）的眼睛。使用QIAamp®循环核酸试剂盒进行cfDNA的分离，cfDNA用于RB1基因的靶向测序。
结果：我们可以在所有眼睛中分离cfDNA（单侧72％和双侧28％），分布峰在140至160bp之间，A臂的平均浓度为27.75ng/μl，B臂的平均浓度为14ng/μl。对四个样品进行的靶向测序显示RB1基因突变，即,内框删除(c.78-80del,p.Pro29del),起始缺失突变(c.1A>T，p.Met1？），无意义突变(c.2236G>T，p.Glu746Ter)，(c.1659T>A，p.Cys553Ter)，和(c.2065C>T，p.Gln689Ter)，和新的错义突变(c.672C>A，p.Asp224Glu）和c.692C>T（p。Pro231Leu)。从AH提取的cfDNA和来自肿瘤组织的基因组DNA的遗传图谱具有可比性。
结论：我们的研究支持以前的报道，即AH可以用作肿瘤来源的cfDNA来源。这是南亚关于从RB眼AH中分离和遗传分析cfDNA的第一份报告，因此,在奠定肿瘤遗传学在RB中的作用方面向前迈出了一大步。需要进一步的研究来阐明肿瘤和AH遗传谱之间的一致性。
方法：AH来自26只接受摘除（A组）或玻璃体内化疗（B组）的眼睛。使用QIAamp®循环核酸试剂盒进行cfDNA的分离，cfDNA用于RB1基因的靶向测序。
结果：我们可以在所有眼睛中分离cfDNA（单侧72％和双侧28％），分布峰在140至160bp之间，A臂的平均浓度为27.75ng/μl，B臂的平均浓度为14ng/μl。对四个样品进行的靶向测序显示RB1基因突变，即,内框删除(c.78-80del,p.Pro29del),起始缺失突变(c.1A>T，p.Met1？），无意义突变(c.2236G>T，p.Glu746Ter)，(c.1659T>A，p.Cys553Ter)，和(c.2065C>T，p.Gln689Ter)，和新的错义突变(c.672C>A，p.Asp224Glu）和c.692C>T（p。Pro231Leu)。从AH提取的cfDNA和来自肿瘤组织的基因组DNA的遗传图谱具有可比性。
结论：我们的研究支持以前的报道，即AH可以用作肿瘤来源的cfDNA来源。这是南亚关于从RB眼AH中分离和遗传分析cfDNA的第一份报告，因此,在奠定肿瘤遗传学在RB中的作用方面向前迈出了一大步。需要进一步的研究来阐明肿瘤和AH遗传谱之间的一致性。
