Background

Detection of germline RB1 mutations is critical for risk assessment of retinoblastoma (RB) patients. Assessment of somatic copy number alterations (SCNAs) is also critically important because of their prognostic significance. Herein we present a refined approach for the simultaneous identification of RB1 variants and SCNAs in the aqueous humor (AH) of RB eyes.

Materials and Methods

Subjects included 7 eyes of 6 RB patients that underwent AH extraction, and 4 matched tumor samples. Cell-free DNA (cfDNA) was isolated and sequenced to assess genome-wide SCNAs. The same sequencing libraries then underwent targeted resequencing and mutation detection using a custom hybridization panel that targets RB1 and MYCN. Illumina paired-end 2x150bp sequencing was used to characterize single-nucleotide variants (SNVs) and loss of heterozygosity (LOH). Results were compared to peripheral blood RB1 testing. Tumor fraction (TFx) was calculated using ichorCNA.

Results

Four of 7 AH samples contained clinically significant SCNAs. Of the 3 other samples, 1 showed focal MYCN amplification and 1 showed focal RB1 deletion. All 4 enucleated tumors contained SCNAs. Mutational analysis of tumor DNA identified all first hits (2 germline RB1 SNVs, 2 germline CNAs) and second hits (4 RB1 SNVs). RB1 variants in AH were concordant with those obtained from corresponding tumor tissue and blood. In AH samples without paired tumor, both RB1 hits were identified with high variant allele frequency, even in the absence of SCNAs.

Conclusions

AH liquid biopsy is a minimally invasive, in vivo alternative to tissue analysis for the simultaneous identification of RB1 variants and SCNAs in RB eyes.

中文翻译：

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同时鉴定视网膜母细胞瘤眼房水中临床相关的RB1突变和拷贝数变化

背景

种系RB1突变的检测对于视网膜母细胞瘤（RB）患者的风险评估至关重要。体细胞拷贝数改变（SCNA）的评估也具有至关重要的意义，因为它们具有预后意义。在这里，我们提出了一种用于同时识别RB眼房水（AH）中RB1变体和SCNA的改进方法。

材料和方法

受试者包括接受AH提取的6例RB患者的7眼，以及4个匹配的肿瘤样品。分离无细胞DNA（cfDNA）并测序以评估全基因组SCNA。然后，使用针对RB1和MYCN的定制杂交小组，对相同的测序文库进行有针对性的重测序和突变检测。Illumina的配对末端2x150bp测序用于表征单核苷酸变异（SNV）和杂合性缺失（LOH）。将结果与外周血RB1测试进行比较。使用ichorCNA计算肿瘤分数（TFx）。

结果

7个AH样本中有四个含有临床上显着的SCNA。在其他3个样本中，有1个显示局灶性MYCN扩增，另外1个显示局灶性RB1缺失。所有4例去核肿瘤均包含SCNA。肿瘤DNA的突变分析确定了所有第一击（2个种系RB1 SNV，2个种系CNA）和第二击（4个RB1 SNV）。AH中的RB1变异与从相应的肿瘤组织和血液获得的变异相一致。在没有配对肿瘤的AH样本中，即使没有SCNA ，两个RB1命中都具有较高的等位基因频率。

结论

AH液体活检是一种微创的，体内替代组织分析的方法，用于同时识别RB眼中的RB1变体和SCNA。