Laboratory of Analytical Chemistry, University of Athens, and Medical Oncology Unit, Helena Venizelou Hospital, Athens, Greece.
Clin Chem. 2011 Mar;57(3):421-30. doi: 10.1373/clinchem.2010.154328. Epub 2011 Jan 18.
Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs.
mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin-phycoerythrin. We quantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals.
The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively.
The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis.
循环肿瘤细胞（CTC）的分子特征对于确定新的诊断和治疗靶点以实现个体化治疗至关重要。我们开发了一种多重聚合酶链反应-液相珠阵列来检测 CTC 中多个基因的表达。
用免疫磁珠分离富集的 CTC 中的 mRNA，进行多重聚合酶链反应，以检测 KRT19（角蛋白 19；也称为 CK19）、ERBB2[v-erb-b2 红细胞白血病病毒致癌基因同源物 2，神经/胶质母细胞瘤衍生致癌基因同源物（禽类）；也称为 HER2]、SCGB2A2（分泌球蛋白 2A，成员 2；也称为 MGB1，乳球蛋白 A）、MAGEA3（黑色素瘤抗原家族 A，3）、TWIST-1[扭转型蛋白 1（果蝇）]和 HMBS（羟甲基胆素合酶；也称为 PBGD）的表达。生物素化的扩增子与携带基因特异性捕获探针的荧光微球杂交，并与链霉亲和素-藻红蛋白孵育。我们定量捕获的标记扩增子，并通过 Luminex 流式细胞术对珠解码。该测定方法经过了检测限、特异性和与反转录定量聚合酶链反应（RT-qPCR）的比较验证，并在 64 例可手术乳腺癌患者、20 例转移患者和 17 例健康个体中评估了其临床性能。
该测定方法在复杂的多重格式下对每种基因均具有特异性，并且能够检测到单个 SK-BR-3 细胞水平的每种基因的表达。该测定方法产生的结果与每种基因的 RT-qPCR 结果相当。在健康供体的 CTC 部分均未检测到测试的基因。我们分别在 26.6%、12.5%、18.7%、10.9%和 31.2%的可手术乳腺癌患者中检测到 KRT19、ERBB2、MAGEA3、SCGB2A2 和 TWIST1，在 65%、20%、30%、20%和 20%的经证实转移的患者中分别检测到相应的基因。
CTC 中 6 个基因的表达可以同时可靠地测量，从而节省宝贵的样本并减少分析的成本和时间。
