肺腺癌软脑膜转移患者的脑脊液和血液中不同的基因突变谱:基于循环肿瘤DNA的液体活检

Different Gene Mutation Spectrum of the Paired CSF and Plasma Samples in Lung Adenocarcinoma with Leptomeningeal Metastases: the Liquid Biopsy Based on Circulating Tumor DNA

背景与目的软脑膜转移(leptomeningeal metastasis,LM)是晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的一种严重并发症,通常预后较差。对于驱动基因阳性的NSCLC患者,表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitors,EGFR-TKIs)是治疗的首选,但常伴随着无法解决的耐药问题。本研究旨在探讨NSCLC-LM患者的脑脊液(cerebrospinal fluid,CSF)和血液中不同的基因突变谱及突变丰度,筛查相关耐药基因,以期能精确指导个体化的治疗。方法采用二代基因测序(next generation sequencing,NGS)的液体活检技术,检测、对比分析TKI耐药后NSCLC患者同期的外周血及脑脊液中的循环肿瘤DNA(circulating tumor DNA,ct DNA)。结果共纳入18例NSCLC伴有LM的患者,基础突变中11例(61.11%)为EGFR,6例(33.33%)为间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK),1例(5.56%)为原癌基因酪氨酸蛋白激酶ROS(ROS protooncogene 1,receptor tyrosine kinase,ROS1),伴随突变中肿瘤蛋白P53(tumor protein p53 gene,TP53)、间质-上皮细胞转化因子(mesenchymal-epithelial transition factor,MET)检出率相对较高。CSF样本的靶向基因突变检出率明显高于外周血(100.00%vs 66.67%,P=0.006),且基础突变基因的CSF丰度均明显高于血浆样本(P<0.001)。CSF样本检测出丰富的单核苷酸变异(single-nucleotide variations,SNV)和拷贝数变异(copy number variants,CNV),数量均多于血液样本;且相较于只接受过单一TKI治疗的患者,使用多种TKI后脑脊液和血液中会产生更多的SNV突变。结论NSCLC-LM患者CSF中的ct DNA,相较于外周血,能更加准确、全面地反映出LM的真实基因突变状态,在指导用药、耐药监测、预后评估等方面具有广泛的应用前景,可作为液体活检的优选。

Background and objective Leptomeningeal metastasis(LM)are a severe complication of non-small cell lung cancer(NSCLC),and normally accompanied by poor prognosis.For the patients with targetable mutations,epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)are the preferred treatment,but the acquired TKI resistance is inextricable.The aim of this study is to analyze the different gene mutation spectrum and mutation frequency of the cerebrospinal fluid(CSF)and plasma in NSCLC patients with LM,and screen out the drug-resistant mutations so as to guide the choice of treatment accurately.Methods The paired CSF and plasma samples were collected from the NSCLC-LM patients with acquired TKI resistance.Next generation sequencing(NGS)was used to detect the gene variations of circulating tumorDNA(ct DNA).Results A total of 18 NSCLC patients with LM were collected.Of the basic mutations,11 cases(61.11%)were EGFR,6 cases(33.33%)were anaplastic lymphoma kinase(ALK),and 1 case(5.56%)was ROS proto-oncogene 1,receptor tyrosine kinase(ROS1).Tumor protein p53 gene(TP53)and mesenchymal-epithelial transition factor(MET)were the two most frequently accompanying mutated genes in CSF ct DNA.The detected mutation rate of CSF samples was higher than that of plasma samples(100.00%vs 66.67%,P=0.006),and the maximum allelic fractions were all higher in CSF than in plasma(P<0.001).Abundant single-nucleotide variations(SNV)and copy number variants(CNV)were detected in CSF,the amount of both of which were more than in blood.In addition,the CSF and plasma samples of patients treated with several TKIs had more SNV mutations than patients who received only a single TKI treatment.Conclusion For the patients of NSCLC,ct DNA in CSF could reveal genomic alterations of LM more exactly and overally than it in plasma,thus could be an optimal source of liquid biopsy for guiding therapy,monitoring therapeutic effect,and predicting prognosis.