Nucleic acids from intact epithelial cells as a target for stool-based molecular diagnosis of colorectal cancer.
Stool-based molecular techniques may improve strategies for colorectal cancer screening. Molecular methods have successfully been applied to detect tumour DNA in stool from patients diagnosed for colorectal carcinoma. In these assays human DNA has to be analyzed against a background of excess nucleic acids from bacteria and dietary waste products. More recently a different diagnostic approach has been described characterizing intact cells isolated from stool. In this study we combine both approaches preparing nucleic acids from isolated epithelial cells to evaluate if: a) tumour cell-specific RNA can be analyzed since cellular RNA molecules are prevented from early digestion by an intact cell membrane; and b) specificity or sensitivity of established DNA-based methods can be improved when epithelial cells are separated from other stool components. Comparing different protocols we found cell isolation using epithelium-specific antibodies to be more effective and reproducible than a technique using density gradient centrifugation. A detection limit of 10(4) cells per ml stool was determined when samples from healthy volunteers were spiked with epithelial cells. Amplification of human sequences from total stool DNA was more efficient than a correspondent amplification of DNA extracted from isolated cells, so that an improvement of DNA-based methods cannot be expected by introducing cell isolation procedures. RNA detection was successful in 1 of 5 patients with confirmed diagnosis of colorectal cancer. The authors suggest that low numbers of detectable cells might rather be a biological than an analytical problem limiting a routinely performed method for colorectal cancer diagnosis.
Spethmann
Sebastian
S
Institute of Clinical Chemistry, University Clinic Hamburg-Eppendorf, D-20251 Hamburg, Germany.
Fischer
Carsten
C
Wagener
Christoph
C
Streichert
Thomas
T
Tschentscher
Peter
P
D001483:Base Sequence; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification*; D004847:Epithelial Cells / Q000737:chemistry; D005243:Feces / Q000737:chemistry / Q000166:cytology; D016158:Genes, p53; D006801:Humans; D007633:Keratins / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D007633:Keratins