使用新型微滴数字PCR技术对循环肿瘤细胞进行经济高效的靶向DNA突变和基因表达变化分析

Introduction: Liquid Biopsies provide a less invasive method for cancer diagnosis and prognosis when compared to conventional tissue biopsies. Over the past two decades, fragmented tumor derived DNA (ctDNA) found in blood has been studied extensively and shown to contain tumor-specific gene mutations and distinct fragment patterns compared to non-tumor cfDNA, which are now employed to monitor the molecular status of patients with cancer. Similarly, Circulating tumor cells (CTCs) have recently evolved as promising biomarkers whose existence and quantity in blood have shown a close correlation to disease progression and recurrence potential. While initial studies on CTCs relied on their protein marker expression and enumeration, recent CTC studies at the molecular level revealed genomic mutations in Estrogen receptor 1 (ESR1) for breast cancer, and epidermal growth factor gene (EGFR) across various cancers. Along with ctDNA studies, detection of mutations and gene expression changes in CTCs can offer additional insights into drug efficacy evaluations and prognosis. However, the rarity of CTCs and lack of streamlined protocols utilizing cost-effective technologies for clinics present a crucial challenge. Methods: For lung cancer, three non-small cell lung cancer cell lines with different EGFR mutations were prepared. For breast cancer, one epithelial breast cancer cell line (MCF7) with/without acquired tamoxifen resistance was prepared. Each CTC sample was prepared by spiking cancer cells into a total of 7.5 ml of whole blood from healthy donor at a concentration of up to 100 cells/ml. The Genesis Cell Isolation System efficiently captured CTCs based on size and deformability and the enriched CTCs were subsequently processed for simultaneous DNA and RNA extraction using the SingleShot Cell Lysis Kit. Either extracted gDNA or total RNA was used for mutation detection using ddPCR or gene expression studies upon cDNA synthesis, respectively. To determine the presence of specific EGFR/ESR1 mutations within the genomic DNA of captured cells, Droplet Digital PCR was performed using assays for EGFR/ESR1 mutation variants as well as known mutations PIK3CA p.E542K and GATA3 p.D336fs*17. For gene expression changes in breast cancer upon tamoxifen resistance, 6/7-multiplex gene expression assays with targeted representative genes were performed using a QX600 or a QX700, respectively. Results: The streamlined combined protocol using the Genesis System and ddPCR facilitated sensitive detection of specific genomic DNA mutations as well as gene expression changes in CTCs upon drug treatment.Discussion and Conclusions: ddPCR is a cost-effective approach for unlocking molecular information carried by CTCs in blood for liquid biopsy. Further studies using clinical specimens with larger sample sizes would facilitate the clinical use of the suggested protocol.