Abstract
The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5'-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.
Highlights
Our results reveal that ck19/20 expression occurs too frequently in the peripheral blood of healthy volunteers to be clinically useful in the assessment of colorectal cancer patients
The median number of nucleated blood cells (NBC) in individual blood samples was 3.4 × ± 1.7 × 106. dThe large standard deviation is due to the one healthy volunteer expressing high copy numbers of guanylyl cyclase C (GCC). eThe large standard deviation is due to one patient expressing 7 × copies of GCC per ml blood
Since all previous reports base their claimed sensitivity on spiking experiments that use tissue culture cells, our results suggest that their use overestimates the sensitivity of the RT-polymerase chain reaction (PCR) assay
Summary
RNeasy Mini Kits (Qiagen Ltd) were used to extract approximately 25 μg of total RNA from 106 T84 cells, a human colorectal cancer cell line. This RNA was stored at – 70°C at a concentration of 50 ng μl. RNA from tumour tissue was extracted using RNeasy Mini kits (Qiagen, Crawley, UK) according to manufacturer’s instructions and stored at – 70°C at a concentration of 50 ng μl. For each 3-ml blood sample, total RNA was extracted in 2 × 1.5 ml aliquots using the RNeasy Blood Mini Kit (Qiagen Ltd, Crawley, UK). The downstream primer contains two mismatches with the ck pseudogene and the probe contains a further three
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