DNA Integrity as a Potential Marker for Stool-based Detection of Colorectal Cancer

Abstract

Background: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa. Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity. Results: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group. Conclusions: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.

Pilot Study of the Sensitivity and Specificity of the DNA Integrity Assay for Stool-based Detection of Colorectal Cancer in Malaysian Patients

“…While these tests may prove useful for research, they are not feasible for mass screening purposes. Interestingly, there were studies where DNA integrity assay (DIA) was used alone as a single marker and the sensitivity reached to 56-86 % with 81-97 % specificity (Boynton et al, 2003;Calistri et al, 2004;Calistri et al, 2009;Kalimutho et al, 2011). These studies suggest that DIA-based tests may potentially be employed for mass screening without a significant loss of sensitivity and specificity.…”

“…These studies suggest that DIA-based tests may potentially be employed for mass screening without a significant loss of sensitivity and specificity. With exception of one study (Boynton et al, 2003), the remaining three employed the use of fluorescent primers in quantitative fluorescence PCR (Calistri et al, 2004(Calistri et al, : 2009 and quantitative-denaturing high performance liquid chromatography (Kalimutho et al, 2011) which makes these tests expensive, and technically demanding.…”

“…Furthermore, the DNA recovered from the CRC patients stool was of high molecular weight, unlike fragmented apoptotic DNA recovered from the stool samples of colonoscopy negative individuals (Boynton et al, 2003). Increased amount of high molecular weight DNA (often referred as long DNA or L-DNA) in the stool of CRC patients may result from the decreased apoptosis of the intestinal epithelial cells and/or increased exofoliation of cancerous cells from the tumor(s) (Boynton et al, 2003;Abbaszadegan et al, 2007). Therefore, the differential presence of L-DNA in the stools of CRC patients may be used as a diagnostic marker for CRC (Boynton et al, 2003;Abbaszadegan et al, 2007).…”

“…Increased amount of human genomic DNA is found in the stool of CRC patients as compared to the healthy individuals (Klassen et al, 2002). Furthermore, the DNA recovered from the CRC patients stool was of high molecular weight, unlike fragmented apoptotic DNA recovered from the stool samples of colonoscopy negative individuals (Boynton et al, 2003). Increased amount of high molecular weight DNA (often referred as long DNA or L-DNA) in the stool of CRC patients may result from the decreased apoptosis of the intestinal epithelial cells and/or increased exofoliation of cancerous cells from the tumor(s) (Boynton et al, 2003;Abbaszadegan et al, 2007).…”

“…Increased amount of high molecular weight DNA (often referred as long DNA or L-DNA) in the stool of CRC patients may result from the decreased apoptosis of the intestinal epithelial cells and/or increased exofoliation of cancerous cells from the tumor(s) (Boynton et al, 2003;Abbaszadegan et al, 2007). Therefore, the differential presence of L-DNA in the stools of CRC patients may be used as a diagnostic marker for CRC (Boynton et al, 2003;Abbaszadegan et al, 2007). The presence of the L-DNA can easily and reliably be detected by polymerase chain reaction (PCR), making this a sensitive, specific, and affordable CRC test for mass screening.…”