Name: evaluation of colorectal cancer risk and prevalence by stool dna integrity detection
Uploaded: 2020-06-08T12:30:00
Description: Watch this Scientific Journal Video about Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection at JoVE.com

The authors have no acknowledgments.

Patients were recruited at the Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) of Meldola (FC, Italy) between 2013 and 2015. Enrolled patients were into protocol IRSTB002, approved by the Ethics Committee of IRST - IRCCS AVR (25/10/2012, ver. 1). All methods were performed in accordance with relevant guidelines and regulations. Written informed consent was obtained from all patients.
1. DNA extraction from stool
<ol>
	<li>Use a kit to prepare stool samples (see <stro...

<ol>
	<li>Fearon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Genetics+of+Colorectal+Cancer.">Molecular Genetics of Colorectal Cancer.</a> Annual Review of Pathology. 6, 479-507 (2011).</li><li>Sears, C. L., Garrett, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbes,+Microbiota,+and+Colon+Cancer.">Microbes, Microbiota, and Colon Cancer.</a> Cell Host and Microbe. 15, 317-328 (2014).</li><li>Levin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+Surveillance+for+the+Early+Detection+of+Colorectal+Cancer+and+Adenomatous+Polyps,+2008:+A+Joint+Guideline+From+the+American+Cancer+Society,+the+US+Multi-Society+Task+Force+on+Colorectal+Cancer,+and+the+American+College+of+Radiology.">Screening and Surveillance for the Early Detection of Colorectal Cancer and Adenomatous Polyps, 2008: A Joint Guideline From the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology.</a> Gastroenterology. 134, 1570-1595 (2008).</li><li>Bosch, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+tests+for+colorectal+cancer+screening.">Molecular tests for colorectal cancer screening.</a> Clinical Colorectal Cancer. 10, 8-23 (2011).</li><li>Ahlquist, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+colorectal+neoplasia.">Molecular detection of colorectal neoplasia.</a> Gastroenterology. 138, 2127-2139 (2010).</li><li>Calistri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+multiple+molecular+tests+to+detect+colorectal+cancer+in+stool.">Fecal multiple molecular tests to detect colorectal cancer in stool.</a> Clinical Gastroenterology and Hepatology. 1, 377-383 (2003).</li><li>Calistri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+colorectal+cancer+by+a+quantitative+fluorescence+determination+of+DNA+amplification+in+stool.">Detection of colorectal cancer by a quantitative fluorescence determination of DNA amplification in stool.</a> Neoplasia. 6, 536-540 (2004).</li><li>Calistri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescence+determination+of+long-fragment+DNA+in+stool+as+a+marker+for+the+early+detection+of+colorectal+cancer.">Quantitative fluorescence determination of long-fragment DNA in stool as a marker for the early detection of colorectal cancer.</a> Cellular Oncology. 31, 11-17 (2009).</li><li>Calistri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+DNA+for+noninvasive+diagnosis+of+colorectal+cancer+in+immunochemical+fecal+occult+blood+test-positive+individuals.">Fecal DNA for noninvasive diagnosis of colorectal cancer in immunochemical fecal occult blood test-positive individuals.</a> Cancer Epidemiology Biomarkers and Prevention. 19, 2647-2654 (2010).</li><li>De Maio, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+and+stool+nucleic+acid+analysis+for+colorectal+cancer+diagnosis.">Circulating and stool nucleic acid analysis for colorectal cancer diagnosis.</a> World Journal of Gastroenterology. 20, 957-967 (2014).</li><li>Rengucci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+stool+DNA+integrity+method+for+early+colorectal+cancer+diagnosis.">Improved stool DNA integrity method for early colorectal cancer diagnosis.</a> Cancer Epidemiology Biomarkers and Prevention. 23, 2553-2560 (2014).</li></ol>

Previous studies have demonstrated that DNA integrity analysis of stools extracted by manual and semi-automatic approaches can represent an alternative tool for the early detection of colorectal lesions7,8,9,10,11,12. Molecular, noninvasive screening tests have been developed over the years for the detection of colorectal can...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/59426/evaluation-colorectal-cancer-risk-prevalence-stool-dna-integrity">Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection</a>. An&#160;affiliation&#160;was&#160;updated.
The first&#160;affiliation was updated from:
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST)
to:
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

An erratum was issued for:&#160;<a href="https://www.jove.com/t/59426/evaluation-colorectal-cancer-risk-prevalence-stool-dna-integrity">Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection</a>. An&#160;affiliation&#160;was&#160;updated.

Erratum: Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Colorectal cancer (CRC) derives from a multi-step process in which healthy epithelium slowly develops into adenomas or polyps, which progress into malignant carcinomas over time1,2. Despite CRC&#39;s high incidence rate, a downward trend in the percentage of deaths has been observed over the past decade3. Indeed, early diagnostic tools adopted in screening programs have led to early detection and removal of pre-neoplastic adenomas or polyps4. However, due to the different technical limits, none of these methods is optimal. Indeed, in order to improve sensitivity and specificity, many stool DNA tests have been proposed alone or in combination with current routine diagnostic tests5,6.
Typically, healthy mucosa sheds into the fecal stream apoptotic colonocytes, whereas diseased mucosa exfoliates non-apoptotic colonocytes. Fragments of 200 bp or more in length characterize non-apoptotic DNA. This DNA is called long DNA (L-DNA) and has become a utilizable biomarker for CRC early diagnosis. The L-DNA can be isolated from stool specimen and quantified by qPCR using an in vitro diagnostic FL-DNA kit7,8,9,10,11,12.
The test consists of two assays for the detection of FL-DNA fragments ranging from 138 bp to 339 bp. Each assay allows the amplification of FL-DNA (FAM) as well as spike-in DNA (HEX). To ensure optimal amplification of all fragments, the test has been divided into two assays (named &#34;A&#34;&#160;and &#34;B&#34;). The A assay detects two regions of exon 14 of the APC gene (NM_001127511) and a fragment of exon 7 of the TP53 gene (NM_001276760). The B assay detects a fragment of&#160; exon 14 of the APC gene (NM_001127511) and two regions of exons 5 and 8 of the TP53 gene (NM_001276760). The assays do not distinguish between the regions detected. The spike-in DNA corresponds to the Oncorhynchus keta salmon DNA and enables verification that the procedure has been done properly and checks for the possible presence of inhibitors, which may yield false negative results. The FL-DNA concentration is evaluated by absolute quantification using the standard curve method and is expressed as ng/reaction.
The FL-DNA method is a non-invasive and inexpensive stool DNA test that, combined with the immunochemical-based fecal occult blood test (iFOBT), is currently used in CRC screening programs and allows for better predictions of CRC and/or high-risk adenoma lesions12.

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	<tbody>
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			<td height="63" style="height:63px;width:267px;">1.5 mL and 2 mL polypropylene twist-lock tubes (DNase-, RNase-, DNA-, PCR inhibitor-free)</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td style="width:525px;">Consumables required for DNA extraction and Real Time PCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Absolute Ethanol (quality of analytical degree)</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td>Reagent required for DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Benchtop centrifuge&#160;</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td>Maximum speed of 20000 x g. Instrument required for DNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">EasyPGX analysis software version 2.0.0</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td>RT800-SW</td>
			<td>Analysis software&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">EasyPGX centrifuge/vortex 8-well strips</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td style="width:119px;">RT803</td>
			<td>Instrument recommended for the Real Time PCR assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">EasyPGX qPCR instrument 96&#160;</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td style="width:119px;">RT800-96</td>
			<td>Instrument recommended for the Real Time PCR assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">EasyPGX ready FL-DNA</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td style="width:119px;">RT029</td>
			<td>Kit required for the Real Time PCR assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Micropipettes (volumes from 1 to 1.000 &#181;L)</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td>Consumables required for DNA extraction and Real Time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Powder-free disposable gloves</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td>Consumables required for DNA extraction and Real Time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">QIAamp Fast DNA Stool</td>
			<td style="width:140px;">Qiagen</td>
			<td style="width:119px;">51604</td>
			<td>Kit recommended for the DNA extraction and purification from stool</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Sterile filter tips DNase-, RNase-free (volumes from 1 to 1.000 &#181;L)</td>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td>Consumables required for DNA extraction and Real Time PCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Thermal block e.g. EasyPGX dry block</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td style="width:119px;">RT801</td>
			<td>Instrument required for DNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Vortex e.g. EasyPGX centrifuge/vortex 1.5 ml&#160;</td>
			<td style="width:140px;">Diatech Pharmacogenetics</td>
			<td style="width:119px;">RT802</td>
			<td>Instrument required for DNA extraction</td>
		</tr>
	</tbody>
</table>

evaluation of colorectal cancer risk and prevalence by stool dna integrity detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The workflow of this protocol is shown in Figure 1. The workflow provides two control steps and different actions according to these step results. First, if a sample presents unsuitable controls, the amplification must be repeated. Second, if the amplification is inhibited, the sample must be reprocessed from the beginning or classified as not valuable.
Figure 2 shows the fluorescence curves produced by positive and negative samples. (A) Shown...

Watch this Scientific Journal Video about Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection at JoVE.com

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

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