一种由大体积恶性胸腔积液中分离肿瘤细胞的方法初探

A Method for Isolating Tumor Cells from Large Volume of Malignant Pleural Effusion and Its Efficacy Evaluation

背景与目的恶性胸腔积液(malignant plural effusion,MPE)是液体活检基因检测的常用标本来源,本研究评估一种从大体积MPE中分离肿瘤细胞方法的分离效果及其在基因检测中的应用前景。方法收集20例伴MPE的晚期肺癌患者一次胸腔积液引流的全部MPE(>500 mL),联合运用细胞分离介质Percoll和Ficoll分离肿瘤细胞,统计分离情况。对其中既往行组织基因检测的肺腺癌患者,分别使用胸腔积液上清游离DNA(tumor derived DNA from pleural effusion supernatant,etDNA),总细胞DNA及分离肿瘤细胞DNA(DNA from tumor cells in pleural effusion,ETC-DNA)3种成分进行二代基因测序,比较检测情况。结果从MPE中分离得到细胞中位数量8.50×104个(上下四分位间距9.25×103-3.75×105),肿瘤细胞纯度85.50%±5.80%。对10例既往行组织表皮生长因子受体(epidermal growth factor receptor,EGFR)基因检测的患者,etDNA、总细胞DNA及ETC-DNA EGFR基因突变检出率分别为70.00%、50.00%、70.00%。ETC-DNA基因检测阳性率与组织(P>0.999,kappa=1.000)和etDNA(P>0.999,kappa=1.000)一致性均良好。etDNA、总细胞DNA、ETC-DNAEGFR基因突变中位丰度分别为16.05%(4.78%-43.06%)、1.09%(0.00%-2.39%)和33.02%(18.50%-76.70%)。ETC-DNA检测丰度倾向高于etDNA,但差异无统计学意义。结论该提取方法可以有效地从大体积MPE中分离出大量高纯度肿瘤细胞,利用提取出的肿瘤细胞进行基因检测可能可以提高基因检测效能,值得进一步研究。

Background and objective Malignant plural effusion(MPE)is one of the most common specimen for liquid biopsy gene detection.This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection.Methods Pleural effusions(>500 mL)from 20 advanced lung cancer patients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll.Cell number and purity were calculated.DNA was extracted from the supernatant(etDNA),total cells and isolated tumor cells of pleural effusion(ETC-DNA)to detect the mutation of tumor-related genes by next-generation sequencing.Results The median number of cells isolated from malignant pleural effusion was 8.50×10~4(interquel range:9.25×10~3-3.75×10~5),85.50%±5.80%of the cells were identified as tumor cells.The detection rates of epidermal growth factor receptor(EGFR)gene mutation of etDNA,total cell DNA and ETC-DNA were 70.00%,50.00%and 70.00%,reseparately,while the median EGFR mutation abundance in 3 components was 16.05%(4.78%-43.06%),1.09%(0.00%-2.39%),and 33.02%(18.50%-76.70%),respectively.ETC-DNA had good consistency with tissue DNA(P>0.999,kappa=1.000)and et DNA(P>0.999,kappa=1.000).ETC-DNA inclined to have higher EGFR mutation than et DNA,but the result was not statistically significant.Conclusion Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity.Using ETC-DNA as specimen improves the efficacy of gene detection,thus is worth further study.