Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients.
Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients.
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DOI：
10.21037/tlcr-20-841
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发表时间：
2020-10
影响因子：
4
通讯作者：
Reuben JM
中科院分区：
文献类型：
--
作者：
Cohen EN;Jayachandran G;Gao H;Qiao W;Liu S;He J;Qiao Y;Yao L;Lin SH;Reuben JM
通过对癌症患者的血液进行采样进行液体活检的各种方法允许获得极少量的肿瘤，这些肿瘤可以在整个治疗过程中容易地重复采样。循环肿瘤细胞（CTC）代表脱落的肿瘤细胞，其可以通过成像或分子技术使用可靠的富集平台来表征。在此，我们验证了Hitachi Chemical Micro Cavity Array（MCA）用于富集诊断为III期非小细胞肺癌（NSCLC）患者血液中的CTC。MCA是一种半自动过滤系统，其基于大小和膜变形性而不是表面抗原的偏倚选择来富集CTC。从放化疗开始时诊断为III期NSCLC的38例患者的外周血中富集CTC。从每例患者中采集两管EDTA血液，并通过MCA平行处理。在第一个试管中，CTC被鉴定为泛细胞角蛋白（CK）+CD 45 −有核细胞并计数。在通过MCA富集之前，使用CD 45抗体包被的磁性微珠耗尽第二管的白细胞，然后进行定量逆转录聚合酶链反应（qRT-PCR）以询问CTC富集的裂解物中来自一组上皮、间充质、干细胞样和癌症信号相关基因的16种靶mRNA的表达。使用来自18名健康供体的类似制备的外周血样品的CTC富集的裂解物来确定阳性基因表达。通过成像在38个患者样品中的30个（79%）中鉴定CTC。在接受分子表征的25份患者样本中，有23份（92%）至少有1个靶基因呈阳性表达。CTC计数≥7与不良无进展生存期（PFS）[风险比（HR）4.24，95%置信区间（CI），1.73-10.40，P=0.020]和不良总生存期（HR 8.17，95% CI，2.87-23.26，P<0.001）相关。MCA富集的CTC表达BCL 2与PFS较差相关（HR 3.11，95% CI，1.18-8.22，P=0.022）。在多变量分析中，单独地，CTC计数和BCL 2表达各自仍然是疾病进展和总生存的统计学显著预测因子。这是第一次证明MCA富集的CTC的裂解物适合于分子表征。MCA富集的CTC是NSCLC的独立预后标志物。
Various methods of liquid biopsy through the sampling of blood in cancer patients allow access to minuscule amounts of tumor that can easily be sampled repeatedly throughout therapy. Circulating tumor cells (CTCs) represent shed tumor cells that can be characterized by imaging or molecular techniques using an amenable enrichment platform. Here we validate the Hitachi Chemical Micro Cavity Array (MCA) for the enrichment of CTCs from the blood of patients diagnosed with stage III non-small cell lung cancer (NSCLC). MCA is a semi-automated filtration system that enriches CTCs on the basis of size and membrane deformability rather than a biased selection of surface antigens. CTCs were enriched from the peripheral blood of 38 patients diagnosed with stage III NSCLC at the start of chemoradiation. Two tubes of EDTA blood were collected from each patient and processed through MCA in parallel. In the first tube, CTCs were identified as pan-cytokeratin (CK)+ CD45− nucleated cells and enumerated. The second tube was depleted of leukocytes using CD45 antibody-coated magnetic microbeads before enrichment by MCA, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to interrogate CTC-enriched lysates for expression of 16 target mRNAs from a panel of epithelial, mesenchymal, stem-like, and cancer signaling-related genes. CTC-enriched lysates from similarly prepared peripheral blood samples from 18 healthy donors were used to define positive gene expression. CTCs were identified by imaging in 30 of 38 patient samples (79%). At least 1 target gene was positively expressed in 23 of 25 (92%) patient samples that was subjected to molecular characterization. A CTC count of ≥7 was associated with poor progression-free survival (PFS) [hazard ratio (HR) 4.24, 95% confidence interval (CI), 1.73–10.40, P=0.020] and poor overall survival (HR 8.17, 95% CI, 2.87–23.26, P<0.001). Expression of BCL2 by MCA-enriched CTCs was associated with poor PFS (HR 3.11, 95% CI, 1.18–8.22, P=0.022). Individually, CTC count and expression of BCL2 each remained statistically significant predictors of disease progression and overall survival in multivariate analysis. This is the first demonstration that lysates of MCA-enriched CTCs are amenable to molecular characterization. CTCs enriched by MCA are an independent prognostic marker in NSCLC.