Detection of the DNA point mutation of colorectal cancer cells isolated from feces stored under different conditions.
BACKGROUND
We reported on a novel diagnostic method for colorectal cancer (CRC) using a DNA-based analysis of isolated colonocytes from feces. The aim of the present study was to investigate with real-time PCR and direct sequencing analysis whether the cancer cells could be detected in feces stored under different conditions after evacuation.


METHODS
Feces were collected from patients with CRC. Feces were divided into 21 pieces and each piece was manipulated at time after arrival (zero time) and after storage of 24, 48 and 72 h at 4 or 37 degrees C. Colonocytes were isolated from each separate fecal sample, and DNA and RNA were extracted from the colonocytes. We investigated the relationship between storage conditions and content of extracted DNA or RNA with real-time PCR. We also clarified the gene alterations regarding APC and p53 genes under different storage conditions with direct sequence analysis.


RESULTS
Though the amount of genomic DNA and total RNA recovered from colonocytes isolated from each fecal piece decreased significantly at 37 degrees C at any storage time compared with 0 h, the gene alterations were detected independent of any storage conditions.


CONCLUSIONS
The colonocytes recovery rate from feces was unchanging for 3 days as long as the feces were kept at 4 degrees C. However, the identical point mutation to one obtained in cancer tissue was detected in the corresponding exfoliated colonocytes even after storage for 72 h at 37 degrees C, which suggests that exfoliated CRC cells maintain their configuration in feces at least 3 days after evacuation.
Koga
Yoshikatsu
Y
Investigative Treatment Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan.
Yasunaga
Masahiro
M
Moriya
Yoshihiro
Y
Akasu
Takayuki
T
Fujita
Shin
S
Yamamoto
Seiichiro
S
Baba
Hideo
H
Matsumura
Yasuhiro
Y
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics*; D003106:Colon / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000235:genetics*; D005243:Feces / Q000737:chemistry / Q000166:cytology*; D006801:Humans; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D025601:Adenomatous Polyposis Coli Protein; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53