Pfitzner Claudia, Schröder Isabel, Scheungraber Cornelia, Dogan Askin, Runnebaum Ingo Bernhard, Dürst Matthias, Häfner Norman
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Sci Rep. 2014 Feb 5;4:3970. doi: 10.1038/srep03970.
The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts.
检测癌症患者循环肿瘤细胞（CTC）可能有助于治疗监测和复发预测。基于对宫颈癌细胞具有高度特异性的人乳头瘤病毒（HPV）癌基因转录本建立了一种灵敏的检测方法。数字直接逆转录聚合酶链反应（DD-RT-PCR）以低通量数字PCR形式将Ficoll分离、ThinPrep固定和一步法RT-PCR相结合，无需进行RNA分离即可直接分析和检测单个CTC。实验样本显示，在500,000个HPV阴性细胞中能检测到一个HPV阳性细胞。在每毫升乙二胺四乙酸（EDTA）血液中加入低至5个HPV阳性细胞的加标实验，通过PCR和免疫细胞化学得到了一致的阳性结果。10例宫颈癌（CxCa）患者中有3例的血样在5至15×10⁵个单核细胞（MNBC）中各自含有一个表达HPV癌基因转录本的CTC。7例局部病变患者中只有1例有CTC，但3例全身性疾病女性中有2例有CTC。这种用于检测CTC的高灵敏度DD-RT-PCR也可应用于表达肿瘤特异性转录本的其他肿瘤实体。
