Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Tokyo, Japan.
Department of Pediatrics, Yokohama City University Graduate School of Medicine, Kanagawa, Japan.
Genes Chromosomes Cancer. 2022 Nov;61(11):662-669. doi: 10.1002/gcc.23073. Epub 2022 Jun 24.
Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.
液体活检是一种利用血液样本检测基因组改变的方法，作为手术组织活检的非侵入性替代方法，最近引起了关注。我们尝试进行定量分析，以检测神经母细胞瘤（NB）的预后因素中所需的 MYCN 扩增（MYCNamp）和 11q 杂合性丢失（11qLOH）。在这项研究中，从 24 名 NB 患者的诊断血浆样本中提取无细胞 DNA（cfDNA）。通过液滴数字 PCR（ddPCR）对 MYCN 和 NAGK 基因的拷贝数进行定量分析。通过检测 11q 区域杂合单核苷酸多态性的等位基因失衡，也评估了 11qLOH。将获得的结果与肿瘤组织标本的结果进行比较。cfDNA 中 MYCN 拷贝数的相关系数为 0.88（p<0.00001）。cfDNA 也能准确检测到 11qLOH，除了一例局限性 NB 病例。鉴于液体活检的高准确性，为了研究 cfDNA 的成分，通过检查 cfDNA 中肿瘤特异性突变的变异等位基因频率，估计肿瘤衍生 DNA 的比例。cfDNA 中肿瘤衍生 DNA 的比例为 42.5%（范围为 16.9%-55.9%），表明液体活检对 NB 具有足够的灵敏度。总之，ddPCR 分析可定量分析血浆 cfDNA 中的 MYCN 拷贝数和 11qLOH。这些结果表明，血浆 cfDNA 可以替代肿瘤 DNA，也可以用于全面的基因组分析。
