Skip Navigation


Japanese Journal of Clinical Oncology Advance Access originally published online on November 28, 2008
Japanese Journal of Clinical Oncology 2009 39(1):62-69; doi:10.1093/jjco/hyn129
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
39/1/62    most recent
hyn129v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Koga, Y.
Right arrow Articles by Matsumura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koga, Y.
Right arrow Articles by Matsumura, Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© 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


   Abstract
TOP
 Abstract
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
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


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
Colorectal cancer (CRC) is one of the most common malignancies worldwide. In Japan, CRC is the third leading cause of cancer-related mortality and is the second leading cause of cancer-related incidence (1). However, CRC can be cured if diagnosed early and resected surgically at an early stage (2). Thus, to reduce the mortality rate of CRC, development of a screening test to detect the disease at an early stage is necessary.

To date, the fecal occult blood test (FOBT) has been used widely as a screening test for CRC (3–9). However, three recent large-scale studies made clear that the sensitivity of the FOBT ranged from 12.9 to 23.9% using total colonoscopy in all subjects as a reference standard (10–12). In addition, the FOBT is not so specific to detect surgically resectable CRCs.

Alternatively, several screening methods for CRC using stool DNA have been reported based on mutated DNA (13–18), cancer-related methylation (19–25) or DNA integrity (26,27). However, these methods are time-consuming and are not sufficiently sensitive. The major reason for this inaccuracy is the fact that nucleic acids in feces are derived from an enormous number and variety of bacteria and normal cells. Accordingly, the proportion of genes derived from cancer cells in feces is as low as 1%, at most. This makes the application of gene-detecting methods difficult in clinical practice.

We previously reported that cancer cells are alive in feces and can be isolated from naturally evacuated feces using the Percoll centrifugation method (28). Then, to improve cell retrieval rates from feces, we developed a new cell isolation method using immuno-magnetic beads (29). After extracting DNA from isolated cells from feces, the DNA was examined for mutations in CRC-related genes. Consequently, we obtained a 71% (82 of 116) sensitivity and an 88% (73 of 83) specificity (29). We are currently modifying this diagnosing method to enable its use in a future randomized mass screening test. Recently, for that purpose, the maximum storage time and the optimal storage temperature for fecal samples after defecation were determined (30). Since, this result was, however, analyzed only by PCR successful rate, it was necessary to measure the quantity of the extracted DNA or RNA of colonocytes from feces under several storage conditions and analyze whether the gene alterations of colonocytes from feces could be detected under several storage conditions.

In the present study, we clarified the optimal storage condition for exfoliated colonocytes in feces using quantitative real-time PCR and investigated the gene alterations originated from exfoliated cancer cells in feces stored under different conditions after evacuation.


   PATIENTS AND METHODS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
Patients
The study population consisted of 12 CRC patients. Patients had undergone curative surgery at the National Cancer Center Hospital (Tokyo, Japan) between February 2007 and July 2007. Stools were obtained from all the 12 patients before surgery but surgically resected CRC tissues were collected from only six of the 12 patients. All patients were thoroughly informed about, and gave written consent to participate in the study, which was approved by the Institutional Review Board of the National Cancer Center, Japan.

Fecal Samples and Isolation of Exfoliated Cells
Naturally evacuated fecal samples were obtained from 12 CRC patients before surgical resection. Each 42 g fecal sample was divided into 21 pieces of 2 g each, and then randomly allocated into one of the seven groups (0, 1A, 1B, 2A, 2B, 3A and 3B), i.e. three pieces per group. The fecal samples of Group 0 were prepared for the next step immediately after arriving in our laboratory. The other samples were kept in Stomacher Lab Blender bags (Seward, Thetford, UK) under different storage conditions and were then processed for examination (Table 1). The storage conditions of each group were as follows: Groups 1A (4°C, 24 h), 1B (37°C, 24 h), 2A (4°C, 48 h), 2B (37°C, 48 h), 3A (4°C, 72 h) and 3B (37°C, 72 h). The fecal samples were prepared as described previously (29). Briefly, the fecal samples were homogenized with a buffer (40 ml) consisting of Hanks solution, 10% fetal bovine serum (FBS) and 25 mM HEPES buffer (pH 7.35) at 200 times per minute for 1 min using a Stomacher system (Seward, Thetford, UK). The homogenized solutions were filtered through a nylon filter (pore size; 512 µm), 80 µl of Dynabeads Epithelial Enrich (Dynal, Oslo, Norway) was added to each solution, and the mixtures were incubated for 30 min under gentle rolling conditions at room temperature. The mixtures on the magnet were incubated on a shaking platform for 15 min at room temperature. The supernatant was then removed, and colonocytes were stored at –80°C until the extraction of DNA and RNA.


View this table:
[in this window]
[in a new window]

  		Table 1. Storage conditions of fecal sample

 
Cancer Tissue Samples
Patients had undergone curative surgery at the National Cancer Center Hospital (Tokyo, Japan). Fresh tissue samples were obtained from the surgically resected specimens of the patients. The samples were frozen in liquid nitrogen within 20 min of their arrival at the pathologic specimen reception area and stored in liquid nitrogen until the extraction of DNA and RNA.

Extraction of Genomic DNA and Total RNA
Genomic DNA and total RNA were extracted from colonocytes isolated from feces using an Allprep mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Fresh tissue samples were put into tubes containing ceramic beads and homogenized using a Percellys 24 device (Bertin Technologies, Saint-Quentin-en-Yvelines Cedex, France) at 6500 rpm for 50 s. Genomic DNA and total RNA were also extracted from each homogenized tissue sample using an Allprep mini kit (Qiagen).

cDNA Synthesis
cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). The reaction mixture consisted of 4 µl of total RNA, 2 µl of 10 x RT buffer, 2 µl of 10 x random primer, 0.8 µl of 25 x dNTPs (100 mM), 1 µl of RNase Inhibitor (20 U/µl) and 1 µl of MultiScribe Reverse Transcriptase (50 U/µl).

Real-Time Quantitative PCR Analysis
For genomic DNA analysis, we targeted a consensus sequence of human Alu-repeats. The sequences for the Alu primers and probe used in this study were as follows: forward primer, 5'-TAGTAGAGACGGGGTTTCACCTTG-3'; reverse primer, 5'-AGCTTGCAGTGAGCCGAGAT-3'; probe, 5'-GAGAATGGCGTGAA-3'. The reporter dye at the 5'-end of the probe was 6-carboxyfluorescein (FAM), and the quencher dye at the 3'-end was minor groove binder (MGB).

The reaction mixture for genomic DNA analysis consisted of 4 µl of a template DNA, 10 µl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems), 500 nM of forward and reverse primers and 250 nM of probe in a total reaction volume of 20 µl. Real-time PCR amplification was performed with precycling heat activation at 95°C for 20 s, followed by 25 cycles of denaturation at 95°C for 3 s and annealing/extension at 62°C for 30 s in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The absolute quantification of genomic DNA in each sample was determined using a standard curve with serial dilutions (10 ng–100 fg) of TaqMan Control Genomic DNA (Applied Biosystems). A negative control (without template) was run in each reaction plate.

For total RNA analysis, we targeted 18S ribosomal RNA, and used TaqMan Eukaryotic 18S rRNA primers and probe mixture (Applied Biosystems). The reporter dye at the 5'-end of the probe was FAM, and the quencher dye at the 3'-end was MGB.

The reaction mixture for total RNA analysis consisted of 4 µl of a template cDNA, 10 µl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and 1 µl of 20 x primers/probe mixture in a total reaction volume of 20 µl. Real-time PCR amplification was performed with precycling heat activation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s and annealing/extension at 62°C for 30 s in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The relative quantification of total RNA in each sample was determined using the comparative Ct method. In this analysis, the calculating formulae of the relative quantifications of each group were as follows: (dCt of each group) = (Ct of each group) – (Ct of group 0), and (Relative Quantification of each group) = 2–(dCt of each group). A negative control (without template) was run in each reaction plate.

As the amount of genomic DNA or total RNA of colonocytes isolated from 2-g feces varies individually to determine the optimal storage condition, we used the recovery rate of genomic DNA or total RNA. In each piece, amount of genomic DNA or total RNA at 0 h (Group 0) was considered 100%. The calculating formula of the recovery rate of each group was as follows: (Recovery rate of each group, %) = [Amount of genomic DNA (or total RNA) of each group]/[Amount of genomic DNA (or total RNA) of Group 0] x 100.

Direct Sequence Analysis
Direct sequencing was conducted to identify mutations in the APC codon 1270–1594, in Exons 5, 6, 7 and 8 of the p53 gene and in the K-ras codon 12.

The PCR primers used in this study were as follows: APC (5'-AAGATGGGCAAGACCCAAACAC-3', 5'-TTCAACACAATACACCCGTGGC-3'), p53 (5'-AACTCTCTCTAGCTCGCTAG-3', 5'-TCCATCGTAAGTCAAGTAGC-3') and K-ras (5'-CTGGTGGAGTATTTGATAGTG-3', 5'-CCCAAGGAAAGTAAAGTTC-3').

The sequence primers used in this study were as follows: APC (5'-AAACACCTCAAGTTCCAACCAC-3', 5'-CAAAAGGCTGCCACTTGCAAAG-3', 5'-GAATCAGCCAGGCACAAAGC-3', 5'-AAAATAAAGCACCTACTGCTG-3', 5'-CATATTGGAGTATCTTCTACAC-3', 5'-TTTGGAGGGAGATTTCGCTC-3', 5'-TACTGCAGCTTGCTTAGGTC-3', 5'-GGTAATTTTGAAGCAGTCTGGGC-3'), p53 Exon 5 (5'-GCCGTCTTCCAGTTGCTTTAT-3', 5'-CCAAATACTCCACACGCAAAT-3'), p53 Exon 6 (5'-CATGAGCGCTGCTCAGATAG-3', 5'-TGCACATCTCATGGGGTTATAG-3'), p53 Exon 7 (5'-CTTGGGCCTGTGTTATCTCCTA-3', 5'-AGAAATCGGTAAGAGGTGGG-3'), p53 Exon 8 (5'-ACCTCTTAACCTGTGGCTTC-3', 5'-TACAACCAGGAGCCATTGTC-3') and K-ras (5'-CTGGTGGAGTATTTGATAGTG-3', 5'-CCCAAGGAAAGTAAAGTTC-3'). Each fragment was sequenced directly using BigDye Terminator v3.1 (Applied Biosystems).

All obtained sequences were aligned with previously published sequences [The National Center for Biotechnology Information (NCBI) GenBank accession No. M74088 [GenBank] (APC), X54156 [GenBank] (p53) and M54968 [GenBank] (K-ras)] for each of the target genes and were analyzed using SEQUENCHER Ver. 4.7 for Windows (Gene Codes Corporation, Ann Arbor, MI, USA). The presence and nature of each mutation were confirmed by repeated PCR and sequencing.

Statistical Analysis
Differences in the genomic DNA and total RNA concentrations for the same individual between Group 0 and each of the other groups were analyzed by two-sided Fisher's exact test. Statistical analyses were performed using StatView Ver. 5 for Windows (Abacus Concepts, Inc., Berkeley, CA, USA). A value of P < 0.05 was considered to denote statistical significance.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 Funding
 Acknowledgements
 References
 
Sensitivity and Accuracy of the Alu-Real-Time PCR
As the amount of genomic DNA or total RNA of isolated colonocytes from 2-g feces was very small, the concentration of genomic DNA or total RNA was analyzed using real-time PCR method. For genomic DNA analysis, we targeted a consensus sequence of human Alu-repeats. Figure 1A shows the sensitivity of Alu-real-time PCR using serial dilutions (10 ng–100 fg) of TaqMan Control Genomic DNA (Applied Biosystems) and the negative control (distilled water). From 10 ng to 100 fg of DNA could be detected in this study, and no DNA could be detected in the negative control. Figure 1B shows the accuracy of Alu-real-time PCR, and there was an equilateral correlation between the DNA concentration and Ct values (R2 = 0.9982). This result shows that Alu-real-time PCR analysis is useful even for a very small amount of isolated cells from feces.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Sensitivity and accuracy of Alu-real-time PCR. (A) Sensitivity of Alu-real-time PCR using several concentrations of a control genomic DNA. Line 1, 10 ng/µl; line 2, 1 ng/µl; line 3, 100 pg/µl; line 4, 10 pg/µl; line 5, 1 pg/µl; line 6, 100 fg/µl. Negative control (distilled water) was not detected. The green line represents the threshold. (B) Accuracy of Alu-real-time PCR using a control genomic DNA. An equilateral correlation is seen between DNA concentration and Ct values (R2 = 0.9982).

 
Recovery Rate of Genomic DNA or Total RNA
To evaluate the optimal fecal storage conditions for genomic DNA, the concentration of DNA from isolated colonocytes was analyzed using real-time PCR targeted Alu-repeat. The concentration of genomic DNA of each storage group summarized at Table 1 was compared with Group 0. Figure 2A shows the recovery rate of genomic DNA compared with Group 0. The recovery rates of Groups 0, 1A, 1B, 2A, 2B, 3A and 3B were 100 ± 56 (average ± SD), 87 ± 128, 13 ± 20, 130 ± 216, 24 ± 58, 113 ± 174 and 14 ± 32%, respectively. There were no significant differences between Group 0 and Groups 1A, 2A and 3A, but there were significant differences between Group 0 and Groups 1B, 2B and 3B (P < 0.01). This result showed that fecal sample could be stored for 72 h at 4°C, but not stored anytime at 37°C in terms of the amount of genomic DNA.


Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Recovery rate of genomic DNA and total RNA. Storage conditions for each fecal sample are shown in Table 1. (A) Recovery rate of genomic DNA from isolated cells stored at several conditions compared with Group 0 (prepared on Day 0). (B) Recovery rate of total RNA from isolated cells stored at several conditions compared with Group 0 (prepared on Day 0). *Significant differences can be seen between group 0 and groups 1B, 2B and 3B (P < 0.01).

 
Also we evaluated the optimal fecal storage conditions for total RNA using real-time PCR targeted 18S ribosomal RNA. Figure 2B shows the recovery rate of total RNA compared with Group 0. The recovery rates of Groups 0, 1A, 1B, 2A, 2B, 3A and 3B were 112 ± 54 (average ± SD), 120 ± 175, 26 ± 30, 140 ± 147, 43 ± 87, 137 ± 184 and 20 ± 31%, respectively. There were no significant differences between Group 0 and Groups 1A, 2A and 3A, but there were significant differences between Group 0 and Groups 1B, 2B and 3B (P < 0.01). This result also showed that fecal sample could be stored for 72 h at 4°C, but not stored anytime at 37°C in terms of the amount of total RNA.

Direct Sequence Analysis
Though the optimal fecal storage condition was kept at 4°C in terms of recovery rate of genomic DNA or total RNA, we evaluated whether the same gene alterations in the cancer tissue were conserved in exfoliated colonocytes stored at severe condition. Gene alterations of APC and p53 gene were detected in only four of the six patients. No gene alteration of K-ras gene was detected in this study. Therefore, gene alterations in exfoliated colonocytes were examined precisely in those four patients.

In the direct sequence analysis for DNA extracted from colonocytes under several storage conditions, the same gene alterations of APC or p53 genes were analyzed. For example, the genetic alteration of APC gene (C3602A) was observed in the colon cancer tissue of the patient no. 1 (Pt1) and was not observed in the normal colon mucosa of the Pt1 (Fig. 3). The same gene alterations were observed in isolated cells of Groups 0, 1B, 2A, 2B and 3B. Three samples per each group were analyzed because the detection of the same gene alteration was depended on the fecal sample.


Figure 3
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. The genetic alteration of APC gene in patient no. 1 shown in Table 2. The point mutation of APC (C3602A) is observed in colon cancer tissue. The amino acid of APC is altered from serin (TCA) to stop codon (TAA) by this point mutation. The point mutation is not observed in normal colon mucosa. The same point mutations are observed in the exfoliated cells of group 0, 1B, 2A, 2B and 3B. The arrow heads are detections of the point mutation. The red line, blue line, green line, and black line show the nucleotide of T (thymine), C (cytosine), A (adenine) and G (guanine), respectively.

 
Table 2 shows all cases of the gene alterations of APC or p53 gene observed in isolated colonocytes. The same gene alterations were detected in isolated colonocytes independent of storage conditions. Though the number of samples analyzed in this study was small and the statistical differences could not be analyzed, the gene alterations could be detected more in the fecal samples kept under 4°C than that kept under 37°C. The gene alteration could not be detected in all samples of Pt4 whose cancer located in cecum.


View this table:
[in this window]
[in a new window]

  		Table 2. Gene alterations of colorectal cancer-related genes in exfoliated colonocytes and clinicopathological characteristics

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
Funding
 Acknowledgements
 References
 
In an attempt to detect CRC in the early stage, we previously reported a new methodology for isolating colonocytes from feces and detecting a gene alteration in DNA purified from the cells (29). We are currently refining and improving this DNA analysis, which will enable us to conduct a mass screening in the near future. So far, we have been considering genomic DNA or total RNA analysis of isolated colonocytes from feces, or cytology of isolated colonocytes as a tool for mass screening. Whichever method we will finally adopt, it would be important to know how soon the preparation of the stool samples should be started after evacuation. In the present study, we examined the retrieval rate of genomic DNA and total RNA using real-time PCR, and the mutation detection rate using direct sequence in colonocytes isolated from feces after storage under different conditions of time and temperature. Since genomic DNA of apoptotic cells was truncated into short size fragments (31), the presence of large size DNA and the same mutation to corresponding cancer tissue using direct sequencing would indicate that the cancer cells were still conserved.

Alu sequences are ~300 bp long and are classified as short-interspersed elements (SINE). The Alu repeat is the most abundant repeated sequence in the human genome, with 1.4 million copies per genome (32,33), and that accounts for >10% of the human genome (34). Because of its high sensitivity, Alu-real-time PCR has been reported as the detection tool of choice for small amounts of human genomic DNA (27,35). The number of isolated colonocytes from 2 g of feces is estimated to range from 1 x 101 to 1 x 103. Therefore, we used Alu-real-time PCR, the sensitivity and accuracy of which are high, to estimate the concentration of small amounts of DNA from isolated colonocytes from the fecal samples.

In the present study, the recovery rates of genomic DNA and total RNA stored at 4°C for 24–72 h did not differ from that of genomic DNA and total RNA purified on Day 0. On the other hand, the recovery rates of genomic DNA and total RNA stored at 37°C for any time were significantly reduced compared with that of genomic DNA and total RNA purified on Day 0. These results show that, to collect almost same amount of genomic DNA and total RNA compared with that prepared on Day 0, the fecal sample should be stored at 4°C for up to 72 h, but not at 37°C for any time.

The direct sequencing analysis of isolated cells from feces in the present study showed that the detections of the gene alteration under each fecal storage condition were storage-condition independent. Even under the most severe condition such as Group 3B (37°C, 72 h duration), the same gene alteration was detected as well as under the other conditions. This is the first report which has showed that the gene alteration identical to one obtained in cancer tissue was detected in the corresponding isolated colonocytes even after 3 days storage at 37°C.

Though we were able to detect the same gene alteration of isolated colonocytes from feces even after severe storage conditions of both temperature and time, the gene alterations could be detected more in the fecal samples kept under 4°C than that kept under 37°C. In general, the amount of DNA, RNA and the number of cells should be large enough for cancer diagnosis, and the fecal samples should be prepared on Day 0 or can be prepared after storage at 4°C for a maximum of 72 h.

In the present study, we can conclude that the colonocytes recovery rate from naturally evacuated feces is high enough up to 72 h compared to 0 time after evacuation as long as fecal sample is kept at 4°C. Though we can also speculate that exfoliated cancer cells maintain their configuration in feces at least for 3 days even at 37°C, the fecal samples should be prepared after storage at 4°C up to 72 h.


   Funding
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Funding
Acknowledgements
 References
 
This work was supported by a Grant-in-Aid for the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Mitsui Life Social Welfare Foundation. The author, Yoshikatsu Koga, is an awardee of a research resident fellowship from the Foundation for Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control.

Conflict of interest statement

None declared.


   Acknowledgements
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
References
 
We thank Ms S. Miyaki, Mr K. Inoue, Ms J. Izumisawa and Ms Y. Ishihara for their technical assistance and Ms K. Shiina for her secretarial assistance. Y. Koga is an awardee of research resident fellowship from the Foundation for Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control.


   References
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
1 The Editorial Board of the Cancer Statics in Japan. Cancer Statistics in Japan 2005. Available at: http://www.fpcr.or.jp.

2 Bosman FT. Prognostic value of pathological characteristics of colorectal cancer. Eur J Cancer (1995) 31A:1216–21.[CrossRef]

3 Mandel JS, Church TR, Bond JH, Ederer F, Geisser MS, Mongin SJ, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. N Engl J Med (2000) 343:1603–7.[Abstract/Free Full Text]

4 Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-update based on new evidence. Gastroenterology (2003) 124:544–60.[CrossRef][Web of Science][Medline]

5 Steele RJ. Fecal occult blood test screening in the United Kingdom. Am J Gastroenterol (2006) 101:216–8.[CrossRef][Web of Science][Medline]

6 Fraser CG, Matthew CM, Mowat NA, Wilson JA, Carey FA, Steele RJ. Immunochemical testing of individuals positive for guaiac faecal occult blood test in a screening programme for colorectal cancer: an observational study. Lancet Oncol (2006) 7:127–31.[CrossRef][Web of Science][Medline]

7 Smith A, Young GP, Cole SR, Bampton P. Comparison of a brush-sampling fecal immunochemical test for hemoglobin with a sensitive guaiac-based fecal occult blood test in detection of colorectal neoplasia. Cancer (2006) 107:2152–9.[CrossRef][Web of Science][Medline]

8 Levi Z, Rozen P, Hazazi R, Vilkin A, Waked A, Maoz E, et al. A quantitative immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med (2007) 146:244–55.[Abstract/Free Full Text]

9 Lee KJ, Inoue M, Otani T, Iwasaki M, Sasazuki S, Tsugane S. Colorectal cancer screening using fecal occult blood test and subsequent risk of colorectal cancer: a prospective cohort study in Japan. Cancer Detect Prev (2007) 31:3–11.[CrossRef][Web of Science][Medline]

10 Lieberman DA, Weiss DG. One-time screening for colorectal cancer with combined fecal occult-blood testing and examination of the distal colon. N Engl J Med (2001) 345:555–60.[Abstract/Free Full Text]

11 Sung JJ, Chan FK, Leung WK, Wu JC, Lau JY, Ching J, et al. Screening for colorectal cancer in Chinese: comparison of fecal occult blood test, flexible sigmoidoscopy, and colonoscopy. Gastroenterology (2003) 124:608–14.[CrossRef][Web of Science][Medline]

12 Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. N Engl J Med (2004) 351:2704–14.[Abstract/Free Full Text]

13 Ahlquist DA, Skoletsky JE, Boynton KA, Harrington JJ, Mahoney DW, Pierceall WE, et al. Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multitarget assay panel. Gastroenterology (2000) 119:1219–27.[CrossRef][Web of Science][Medline]

14 Dong SM, Traverso G, Johnson C, Geng L, Favis R, Boynton K, et al. Detecting colorectal cancer in stool with the use of multiple genetic targets. J Natl Cancer Inst (2001) 93:858–65.[Abstract/Free Full Text]

15 Rengucci C, Maiolo P, Saragoni L, Zoli W, Amadori D, Calistri D. Multiple detection of genetic alterations in tumors and stool. Clin Cancer Res (2001) 7:590–3.[Abstract/Free Full Text]

16 Traverso G, Shuber A, Olsson L, Levin B, Johnson C, Hamilton SR, et al. Detection of proximal colorectal cancers through analysis of faecal DNA. Lancet (2002) 359:403–4.[CrossRef][Web of Science][Medline]

17 Traverso G, Shuber A, Levin B, Johnson C, Olsson L, Schoetz DJ Jr, et al. Detection of APC mutations in fecal DNA from patients with colorectal tumors. N Engl J Med (2002) 346:311–20.[Abstract/Free Full Text]

18 Diehl F, Schmidt K, Durkee KH, Moore KJ, Goodman SN, Shuber AP, et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Gastroenterology (2008) 135:489–98.[CrossRef][Web of Science][Medline]

19 Lenhard K, Bommer GT, Asutay S, Schauer R, Brabletz T, Goke B, et al. Analysis of promoter methylation in stool: a novel method for the detection of colorectal cancer. Clin Gastroenterol Hepatol (2005) 3:142–9.[CrossRef][Web of Science][Medline]

20 Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, et al. Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene. J Natl Cancer Inst (2005) 97:1124–32.[Abstract/Free Full Text]

21 Itzkowitz SH, Jandorf L, Brand R, Rabeneck L, Schroy PC 3rd, Sontag S, et al. Improved fecal DNA test for colorectal cancer screening. Clin Gastroenterol Hepatol (2007) 5:111–7.[CrossRef][Web of Science][Medline]

22 Zou H, Harrington J, Rego RL, Ahlquist DA. A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening. Clin Chem (2007) 53:1646–51.[Abstract/Free Full Text]

23 Model F, Osborn N, Ahlquist D, Gruetzmann R, Molnar B, Sipos F, et al. Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease. Mol Cancer Res (2007) 5:153–63.[Abstract/Free Full Text]

24 Lees NP, Harrison KL, Hall CN, Margison GP, Povey AC. Human colorectal mucosal O6-alkylguanine DNA-alkyltransferase activity and DNA-N7-methylguanine levels in colorectal adenoma cases and matched referents. Gut (2007) 56:380–4.[Abstract/Free Full Text]

25 Huang ZH, Li LH, Yang F, Wang JF. Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions. World J Gastroenterol (2007) 13:950–4.[Web of Science][Medline]

26 Boynton KA, Summerhayes IC, Ahlquist DA, Shuber AP. DNA integrity as a potential marker for stool-based detection of colorectal cancer. Clin Chem (2003) 49:1058–65.[Abstract/Free Full Text]

27 Zou H, Harrington JJ, Klatt KK, Ahlquist DA. A sensitive method to quantify human long DNA in stool: relevance to colorectal cancer screening. Cancer Epidemiol Biomarkers Prev (2006) 15:1115–9.[Abstract/Free Full Text]

28 Yamao T, Matsumura Y, Shimada Y, Moriya Y, Sugihara K, Akasu T, et al. Abnormal expression of CD44 variants in the exfoliated cells in the feces of patients with colorectal cancer. Gastroenterology (1998) 114:1196–205.[CrossRef][Web of Science][Medline]

29 Matsushita H, Matsumura Y, Moriya Y, Akasu T, Fujita S, Yamamoto S, et al. A new method for isolating colonocytes from naturally evacuated feces and its clinical application to colorectal cancer diagnosis. Gastroenterology (2005) 129:1918–27.[CrossRef][Web of Science][Medline]

30 Onouchi S, Matsushita H, Nomura S, Minowa T, Matsumura Y. PCR-based assessment of the recovery rate of exfoliated colonocytes or cancer cells from fecal samples depends on the storage conditions after defecation. J Gastrointestin Liver Dis (2007) 16:369–72.[Web of Science][Medline]

31 Giacona MB, Ruben GC, Iczkowski KA, Roos TB, Porter DM, Sorenson GD. Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas (1998) 17:89–97.[Web of Science][Medline]

32 Hwu HR, Roberts JW, Davidson EH, Britten RJ. Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution. Proc Natl Acad Sci USA (1986) 83:3875–9.[Abstract/Free Full Text]

33 Gu Z, Wang H, Nekrutenko A, Li WH. Densities, length proportions, and other distributional features of repetitive sequences in the human genome estimated from 430 megabases of genomic sequence. Gene (2000) 259:81–8.[CrossRef][Web of Science][Medline]

34 Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature (2001) 409:860–921.[CrossRef][Medline]

35 Umetani N, Kim J, Hiramatsu S, Reber HA, Hines OJ, Bilchik AJ, et al. Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats. Clin Chem (2006) 52:1062–9.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
39/1/62    most recent
hyn129v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Koga, Y.
Right arrow Articles by Matsumura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koga, Y.
Right arrow Articles by Matsumura, Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?