Gynaecological Cancer Research Group, School of Women's and Children's Health, Faculty of Medicine and Health, University of New South Wales, Australia.
Gynaecological Oncology Department, Royal Hospital for Women, Sydney, Australia.
Gynecol Oncol. 2021 Sep;162(3):720-727. doi: 10.1016/j.ygyno.2021.06.028. Epub 2021 Jul 5.
Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content.
Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating.
The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h.
cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.
恶性腹水是卵巢癌的常见临床特征，代表了肿瘤细胞和肿瘤游离 DNA（cftDNA）易于获取的样本。本研究旨在从大小谱、稳定性和 cftDNA 含量等方面描述腹水游离 DNA（cfDNA）的特征。
从 18 例卵巢癌患者的腹水中收集细胞球体、疏松细胞和无细胞液体。分离 cfDNA 并通过电泳评估其大小，通过荧光计评估浓度，通过 HOXA9 和 IFFO1 启动子区域的甲基化特异性 qPCR 和靶向测序评估 cftDNA 含量。在将腹水于 4°C 储存 24 和 72 小时后进行分离之前，评估稳定性。
腹水 cfDNA 浓度范围为 6.6 至 300ng/ml。cfDNA 大小分布与血浆衍生 cfDNA 相似，主要峰对应于单和二核小体 DNA 片段。在 18 例患者中的 7 例中观察到高分子量 cfDNA，似乎与细胞外囊泡有关。cfDNA 中的 IFFO1 和 HOXA9 甲基化比例高于球体和疏松细胞部分，在健康原代细胞中未观察到。与单个细胞和来自腹水的球体相比，cfDNA 的变异等位基因频率最高。尽管接受化疗的 1 例复发性腹水患者的腹水中癌细胞数量接近零，但仍可采样 cftDNA。cfDNA 的大小、浓度和肿瘤含量在 72 小时内保持稳定。
卵巢癌腹水中的 cfDNA 表现出个体间变异性，但始终是 cftDNA 的丰富来源，是一种稳定的底物。这支持腹水在卵巢癌分子分析中的更广泛临床应用。
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