A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood.
BACKGROUND
The detection of occult carcinoma cells in patients with breast cancer has been shown to predict disease recurrence and metastasis.


METHODS
To improve on molecular detection of breast carcinoma cells in blood, we have developed a sensitive and quantitative assay using real-time quantitative RT-PCR identifying transcripts of the cytokeratin-19 (CK19) gene.


RESULTS
This real-time quantitative RT-PCR is sensitive, accurate and has a high reproducibility within a wide dynamic range, which permits simultaneous quantitative analysis of samples with varying input concentrations. Furthermore, the procedure offers several technical advantages over classic quantitative PCR methods (competitive RT-PCR, Northern blotting) such as decreased likelihood of contamination due to absence of post-PCR manipulations, high sample throughput because of absence of post-PCR processing time (no agarose gel electrophoresis). In this pilot study, we detected significantly elevated CK19 transcript levels in < 10% of the volunteers, in +/- 30% of stage I-IIIa patients preoperatively and in > 70% of the and stage IV breast cancer patients.


CONCLUSIONS
Analyses using this real time quantitative RT-PCR for CK19 mRNA may prove to have clinical implications in the assessment of circulating tumour cells in peripheral blood, micrometastases in bone marrow or lymph nodes in breast cancer patients. Application of this technique in a clinical population may improve diagnosis and monitoring of metastatic breast cancer and its validation is currently ongoing.
Experimental Laboratory Medicine, University Hospital Gasthuisberg, Catholic University Leuven, Belgium. joeri.aerts@uz.kuleuven.ac.be
J
J
Aerts
W
W
Wynendaele
R
R
Paridaens
M R
MR
Christiaens
W
W
van den Bogaert
A T
AT
van Oosterom
F
F
Vandekerckhove
D000328:Adult; D001483:Base Sequence; D001943:Breast Neoplasms / Q000175:diagnosis / Q000473:pathology*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D007633:Keratins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis / Q000175:diagnosis; D009360:Neoplastic Cells, Circulating; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013997:Time Factors; D014158:Transcription, Genetic
D007633:Keratins