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Japanese Journal of Clinical Oncology Advance Access published online on November 28, 2008
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyn129
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© The Author (2008). Published by Oxford University Press. All rights reserved

Detection of the DNA Point Mutation of Colorectal Cancer Cells Isolated from Feces Stored Under Different Conditions

Yoshikatsu Koga1,3, Masahiro Yasunaga1, Yoshihiro Moriya2, Takayuki Akasu2, Shin Fujita2, Seiichiro Yamamoto2, Hideo Baba3 and Yasuhiro Matsumura1

1 Investigative Treatment Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba
2 Department of Surgery, National Cancer Center Hospital, Tokyo
3 Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

For reprints and all correspondence: Yasuhiro Matsumura, Investigative Treatment Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa 277-8577, Japan. E-mail: yhmatsum{at}east.ncc.go.jp

Received September 8, 2008; accepted October 15, 2008

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°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°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°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°C, which suggests that exfoliated CRC cells maintain their configuration in feces at least 3 days after evacuation.

Key Words: colorectal cancer • colonocytes • fecal storage conditions • direct sequence • cancer diagnosis


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