Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma.
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.
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Claudia
C
Pfitzner
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Isabel
I
Schröder
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Cornelia
C
Scheungraber
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Askin
A
Dogan
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Ingo Bernhard
IB
Runnebaum
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Matthias
M
Dürst
Department for Gynaecology and Obstetrics, Jena University Hospital, Friedrich Schiller University, Germany.
Norman
N
Häfner
D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000235:genetics; D052161:Human papillomavirus 18 / Q000235:genetics; D006801:Humans; D009360:Neoplastic Cells, Circulating; D009856:Oncogene Proteins, Viral / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000821:virology*
D014408:Biomarkers, Tumor; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins