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 Table of Materials). Select and treat the fecal material by performing the extraction according to the manufacturer&#8217;s instructions. Amplify the purified DNA directly or store at -20 &#176;C for subsequent analysis.</li>
</ol>
2. Preparation of positive control, standards, spike-in DNA, and clinical samples
<ol>
	<li>Preparation of standards and samples
	<ol>
		<li>To prepare the positive control, standards, spike-in DNA, and all clinical samples, centrifuge an aliquot of positive control, standards, and spike-in DNA, then resuspend each reagent by adding the correct amount of provided water (see below). Then, carefully vortex the positive control, standard, and spike-in DNA, then centrifuge for 10 s. To achieve a complete resuspension of the dry reagents, store the liquid reagents at room temperature (RT) for 30 min before use.
		<ol>
			<li>The positive control is human DNA in a dry format. Resuspend each aliquot with 750 &#181;L of water.</li>
			<li>The spike-in DNA is salmon (Oncorhynchus keta) DNA, which is used as an exogenous internal control to verify the possible presence of inhibitors in DNA samples extracted from stool. Resuspend each aliquot with 100 &#181;L of water.</li>
		</ol>
		</li>
		<li>To prepare the standard curve, produce four 1:5 dilutions starting from the stock solution. The standard points must be 10 ng/reaction, 2 ng/reaction, 0.4 ng/reaction, and 0.08 ng/reaction.</li>
	</ol>
	</li>
	<li>Preparation of the 1x spike-in DNA
	<ol>
		<li>Prepare the spike-in DNA control directly before use.</li>
		<li>Prepare the 1x spike-in DNA control by mixing 5 &#181;L of FL-DNa spike with 20 &#181;L of sterile water. The number of 1x spike-in DNA control samples will be prepared according to the number of the samples to be analyzed, plus the positive control.</li>
	</ol>
	</li>
	<li>Preparation of samples
	<ol>
		<li>Mix 75 &#181;L of the samples (clinical samples or positive control) with 25 &#181;L of 1x spike-in DNA, yielding a total volume of 100 &#181;L.</li>
	</ol>
	</li>
</ol>
3. Amplification and determination of the FL-DNA value using qPCR Easy PGX
NOTE: Complete amplification mixtures containing specific primers and probes targeting the human DNA and the internal control are provided in a lyophilised format in 8 well strips for FL-DNA Mix A and FL-DNA Mix B. Standards, positive and negative controls, and samples must be amplified with both lyophilized mixes. Clinical samples only must only be amplified in duplicate with both lyophilized mixes.
<ol>
	<li>See the Table of Materials for qPCR instrument and operating software.
	<ol>
		<li>Open the operating software and set up the plate and thermal profile:
		<ol>
			<li>Set up the plate as shown in Table 1.
			<ol>
				<li>Set the well type for all eight positions in column 1 as Standard.</li>
				<li>Set the well type for the A2 and B2 wells as NTC.</li>
				<li>Set the well type for C2 and D2 (the positive controls) as Unknown.</li>
				<li>Set the well type for all other positions as Unknown.</li>
				<li>Select all 96 positions, and add the Dyes FAM and HEX. Click Sync Plate.</li>
			</ol>
			</li>
			<li>Set the thermal profile according to Table 2.</li>
		</ol>
		</li>
		<li>Centrifuge the needed number of strips for 10 s to bring the contents to the bottom of the tube.</li>
		<li>Gently remove the seals from the strips, while paying attention to retain the contents, and add to the respective strips: negative control: 20 &#181;L of water; sample: 20 &#181;L of DNA; standard curve: 20 &#181;L of standard 1, 2, 3, or 4; positive control: 20 &#181;L of positive control.</li>
		<li>Close carefully all the strips using the 8 strip flat optical caps and vortex for few seconds.</li>
		<li>Centrifuge the strips for 10 s and load them into the instrument. Then, start the run.</li>
	</ol>
	</li>
</ol>
4. Data analysis
NOTE: Data analysis can be performed automatically or manually depending on the software (see Table of Materials).
<ol>
	<li>At the end of the run, select columns A, C, E, G for &#34;FAM: FL-DNA-A&#34;&#160;and &#34;HEX: IC&#34;, and columns B, D, F, H for &#34;FAM: FL-DNA-B&#34;&#160;and &#34;HEX: IC&#34;.</li>
	<li>Set the following for the Standard Quantities Starting Amount: 10 ng/reaction for A1 and B1 wells, 2 ng/reaction for C1 and D1, 0.4 ng/reaction for E1 and F1, and 0.08 ng/reaction for G1 and H1.</li>
	<li>Set Threshold Fluorescence values to 150 for both FAM (FL-DNA A and FL-DNA B) and HEX (IC) channels.</li>
	<li>In the box Result Table, click Column Options | Select All | Ok to obtain the results in both channels with their respective Cq (&#8710;R) and &#8710;R last values. 
	NOTE: These values are supplied by the Real Time PCR instrument software. &#916;R last corresponds to the fluorescence value normalized to the last amplification cycle.</li>
	<li>In the box Result Table, right-click on the table to open the context menu and click Send to Excel to export the raw data.</li>
	<li>Check the values of the standards to verify the suitability of the standard curve.</li>
	<li>For each FL-DNA mix, check the R2 [&#34;R&#178; (&#8710;R)&#34;&#160;column] and efficiency [&#34;Efficiency (%)&#34;&#160;column] values. If they are in an acceptable range, it is possible to proceed with analysis accordingly to manufacturer&#8217;s instructions (Table 3).</li>
	<li>If the results of the FAM channel are not in the expected range, omit one point of the standard curve and reanalyze the run.</li>
	<li>Determine the values of the negative and positive controls with the following formula, considering the &#34;No Cq&#34;&#160;values as zero: 
	<img alt="Equation 1" src="/files/ftp_upload/59426/59426eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/59426/59426eq2.jpg" /></li>
	<li>Compare the obtained values with those reported in Table 4.</li>
	<li>If the reaction controls are in the range of expected values, proceed with analysis of the samples. 
	NOTE: Verify that the Cq values obtained are generated from a real amplification reaction (sigmoidal fluorescence curve) and not from an artifact (linear fluorescence curve).</li>
	<li>To analyze suitability of the sample for each FL-DNA mix, compare the Cq values of the HEX channel. If the value is &#8805;16, proceed with analysis of the samples. If the value is &#60;16 or there is no Cq, it is likely due to a dispensing error of the FL-DNA Spike. Therefore, it is not possible to analyze the samples.</li>
	<li>Calculate the average of the Cq values in the &#34;HEX&#34;&#160;channel of the positive control using the following formula: 
	<img alt="Equation 3" src="/files/ftp_upload/59426/59426eq3.jpg" /></li>
	<li>Calculate the average of the Cq values in the &#34;HEX&#34;&#160;channel of the sample replicates using the following formula: 
	<img alt="Equation 4" src="/files/ftp_upload/59426/59426eq4.jpg" /></li>
	<li>Calculate the &#8710;CqHEX values according to the following formula: 
	<img alt="Equation 5" src="/files/ftp_upload/59426/59426eq5.jpg" /></li>
	<li>Compare the &#8710;CqHEX values of the samples with those reported in Table 5.&#160;</li>
	<li>For each mix (Mix A and Mix B), compare the Cq values of the FAM channel with those reported in Table 6.</li>
	<li>To determine the FL-DNA value of each suitable sample, use the following formula, considering the &#34;No Cq&#34;&#160;values as zero: 
	<img alt="Equation 6" src="/files/ftp_upload/59426/59426eq6.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/59426/59426eq2.jpg" /> 
	NOTE: Colorectal cancer risk and prevalence is a function of iFOBT and FL-DNA evaluations according to the Fagan nomogram results obtained by Rengucci et al.12 (Table 7).</li>
</ol>

<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>

Maura Menghi is full-time employee of Diatech Pharmacogenetics srl.

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 cancer, but the widespread diffusion of these methods is limited due to time-consuming approaches and poor cost-effectiveness compared to other screening tests.
This approach is relatively cheap and not too time-consuming. It also has increased accuracy in detecting colorectal lesions due to a new procedure requiring few manual steps. The approach described here is fast with fewer manual steps, and it is able to be easily performed on many samples per week. The DNA extraction does not present particular issues and can be performed through easy manual steps or use of an automatic instrument. In the latter case, it is necessary to determine the automatic DNA extraction instrument, allowing for the most reproducible results. The most critical step of DNA extraction is the collection of stool and method of its storage before DNA extraction. It is advisable to keep the stool frozen and proceed with &#160;extraction as soon as possible.
Another critical issue is represented by possible inhibitors of the amplification reactions. The fecal extraction is not able to purify the genomic DNA, as impurities compromise the correct amplification reaction. In this regard, the protocol requires the use of spike-in DNA for verifying the presence/absence of reaction inhibitors.
Until recently, the fecal occult blood test is the main approach used to detect colorectal lesions in screening programs; although, it presents some limits in terms of accuracy. An alternative strategy for the diagnosis of colorectal cancer is based on the analysis of DNA from exfoliated cells excreted in stool. Several approaches have been evaluated in the past year, but none are available for use in screening programs.
The FL-DNA integrity value can be a useful alternative, which in combination with the standard screening iFOBT test value, can predict the presence of tumors and/or high-risk adenomas11,12 by a Fagan Nomogram approach10 (Table 7). This approach estimates the combined test probability of neoplastic lesion presence, improving diagnostic accuracy compared to the standard approach used alone.
This information may help clinicians in planning diagnostic tests in addition to an appropriate colonoscopy and personalizing its surveillance. Indeed, the FL-DNA kit simplifies the manual steps and ensures stable and consistent results. However, some issues remain to be clarified to improve diagnostic accuracy of the method. For instance, the frequency at which the tests should be performed and number of stool samples that need to be analyzed at specific timepoints for each individual should be carefully selected. Given that this test must be performed concurrently with iFOBT, a method that requires collection every 2 years, as is standard in numerous screening protocols, is necessary to verify the effectiveness of this test as a replacement or alternative to the current screening tests.

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.

<table><tbody><tr><td>1.5 mL and 2 mL polypropylene twist-lock tubes (DNase-, RNase-, DNA-, PCR inhibitor-free)</td><td></td><td></td><td>Consumables required for DNA extraction and Real Time PCR</td></tr><tr><td>Absolute Ethanol (quality of analytical degree)</td><td></td><td></td><td>Reagent required for DNA extraction</td></tr><tr><td>Benchtop centrifuge&amp;nbsp;</td><td></td><td></td><td>Maximum speed of 20000 x g. Instrument required for DNA extraction</td></tr><tr><td>EasyPGX analysis software version 2.0.0</td><td>Diatech Pharmacogenetics</td><td>RT800-SW</td><td>Analysis software&amp;nbsp;</td></tr><tr><td>EasyPGX centrifuge/vortex 8-well strips</td><td>Diatech Pharmacogenetics</td><td>RT803</td><td>Instrument recommended for the Real Time PCR assay</td></tr><tr><td>EasyPGX qPCR instrument 96&amp;nbsp;</td><td>Diatech Pharmacogenetics</td><td>RT800-96</td><td>Instrument recommended for the Real Time PCR assay</td></tr><tr><td>EasyPGX ready FL-DNA</td><td>Diatech Pharmacogenetics</td><td>RT029</td><td>Kit required for the Real Time PCR assay</td></tr><tr><td>Micropipettes (volumes from 1 to 1.000 &amp;micro;L)</td><td></td><td></td><td>Consumables required for DNA extraction and Real Time PCR</td></tr><tr><td>Powder-free disposable gloves</td><td></td><td></td><td>Consumables required for DNA extraction and Real Time PCR</td></tr><tr><td>QIAamp Fast DNA Stool</td><td>Qiagen</td><td>51604</td><td>Kit recommended for the DNA extraction and purification from stool</td></tr><tr><td>Sterile filter tips DNase-, RNase-free (volumes from 1 to 1.000 &amp;micro;L)</td><td></td><td></td><td>Consumables required for DNA extraction and Real Time PCR</td></tr><tr><td>Thermal block e.g. EasyPGX dry block</td><td>Diatech Pharmacogenetics</td><td>RT801</td><td>Instrument required for DNA extraction</td></tr><tr><td>Vortex e.g. EasyPGX centrifuge/vortex 1.5 ml&amp;nbsp;</td><td>Diatech Pharmacogenetics</td><td>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 is an example of a suitable positive sample. The sample signal on the HEX channel is within the acceptable range. The positive signal is above the threshold on the FAM channel. (B) Shown is an example of a suitable negative/not positive sample. The sample signal is within the acceptable range on the HEX channel. The negative control signal is below the threshold on the FAM channel. (C) Shown is an example of a not-suitable sample. The sample signal is not within the acceptable range on the HEX channel; thus, a potential inhibition can be assumed. This sample must be repeated, starting from the extraction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59426/59426fig1.jpg" /> 
Figure 1: Workflow for FL-DNA quantification. <a href="https://www.jove.com/files/ftp_upload/59426/59426fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59426/59426fig2.jpg" /> 
Figure 2: Fluorescence curves showing the amplification of Mix A (or Mix B) target genes (channel FAM) and internal control (channel HEX). (A) FL-DNA positive sample. (B) FL-DNA negative sample. (C) Inhibition of sample amplification. Red curve: positive control; green curve: negative control; black curve: clinical sample. <a href="https://www.jove.com/files/ftp_upload/59426/59426fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:font-size="7px" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
			<td>9</td>
			<td>10</td>
			<td>11</td>
			<td>12</td>
			<td></td>
			<td></td>
			<td>X&#160;</td>
		</tr>
		<tr>
			<td>A</td>
			<td>Standard 1</td>
			<td>WATER</td>
			<td>DNA2</td>
			<td>DNA4</td>
			<td>DNA6</td>
			<td>DNA8</td>
			<td>DNA10</td>
			<td>DNA12</td>
			<td>DNA14</td>
			<td>DNA16</td>
			<td>DNA18</td>
			<td>DNA20</td>
			<td></td>
			<td>1</td>
			<td>FL-DNA Mix A</td>
		</tr>
		<tr>
			<td>B</td>
			<td>Standard 1</td>
			<td>WATER</td>
			<td>DNA2</td>
			<td>DNA4</td>
			<td>DNA6</td>
			<td>DNA8</td>
			<td>DNA10</td>
			<td>DNA12</td>
			<td>DNA14</td>
			<td>DNA16</td>
			<td>DNA18</td>
			<td>DNA20</td>
			<td></td>
			<td>2</td>
			<td>FL-DNA Mix B</td>
		</tr>
		<tr>
			<td>C</td>
			<td>Standard 2</td>
			<td>POS</td>
			<td>DNA2</td>
			<td>DNA4</td>
			<td>DNA6</td>
			<td>DNA8</td>
			<td>DNA10</td>
			<td>DNA12</td>
			<td>DNA14</td>
			<td>DNA16</td>
			<td>DNA18</td>
			<td>DNA20</td>
			<td></td>
			<td>3</td>
			<td>FL-DNA Mix A</td>
		</tr>
		<tr>
			<td>D</td>
			<td>Standard 2</td>
			<td>POS</td>
			<td>DNA2</td>
			<td>DNA4</td>
			<td>DNA6</td>
			<td>DNA8</td>
			<td>DNA10</td>
			<td>DNA12</td>
			<td>DNA14</td>
			<td>DNA16</td>
			<td>DNA18</td>
			<td>DNA20</td>
			<td></td>
			<td>4</td>
			<td>FL-DNA Mix B</td>
		</tr>
		<tr>
			<td>E</td>
			<td>Standard 3</td>
			<td>DNA1</td>
			<td>DNA3</td>
			<td>DNA5</td>
			<td>DNA7</td>
			<td>DNA9</td>
			<td>DNA11</td>
			<td>DNA13</td>
			<td>DNA15</td>
			<td>DNA17</td>
			<td>DNA19</td>
			<td>DNA21</td>
			<td></td>
			<td>5</td>
			<td>FL-DNA Mix A</td>
		</tr>
		<tr>
			<td>F</td>
			<td>Standard 3</td>
			<td>DNA1</td>
			<td>DNA3</td>
			<td>DNA5</td>
			<td>DNA7</td>
			<td>DNA9</td>
			<td>DNA11</td>
			<td>DNA13</td>
			<td>DNA15</td>
			<td>DNA17</td>
			<td>DNA19</td>
			<td>DNA21</td>
			<td></td>
			<td>6</td>
			<td>FL-DNA Mix B</td>
		</tr>
		<tr>
			<td>G</td>
			<td>Standard 4</td>
			<td>DNA1</td>
			<td>DNA3</td>
			<td>DNA5</td>
			<td>DNA7</td>
			<td>DNA9</td>
			<td>DNA11</td>
			<td>DNA13</td>
			<td>DNA15</td>
			<td>DNA17</td>
			<td>DNA19</td>
			<td>DNA21</td>
			<td></td>
			<td>7</td>
			<td>FL-DNA Mix A</td>
		</tr>
		<tr>
			<td>H</td>
			<td>Standard 4</td>
			<td>DNA1</td>
			<td>DNA3</td>
			<td>DNA5</td>
			<td>DNA7</td>
			<td>DNA9</td>
			<td>DNA11</td>
			<td>DNA13</td>
			<td>DNA15</td>
			<td>DNA17</td>
			<td>DNA19</td>
			<td>DNA21</td>
			<td></td>
			<td>8</td>
			<td>FL-DNA Mix B</td>
		</tr>
	</tbody>
</table>
Table 1: Set-up plate with distribution of control, curve, and samples. Column X indicates the number imprinted on the top of the strip.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature and time</td>
		</tr>
		<tr>
			<td>Hot Start (1 Cycle)</td>
			<td>95 &#176;C for 5 min</td>
		</tr>
		<tr>
			<td>Amplification (40 cycles)</td>
			<td>95 &#176;C for 15 s 
			54 &#176;C for 15 s 
			60 &#176;C for 45 s (Data Collection)</td>
		</tr>
	</tbody>
</table>
Table 2: Thermal profile for DNA amplification.
<img alt="Table 3" src="/files/ftp_upload/59426/59426tbl3v2.jpg" /> 
Table 3: Range of HEX and FAM channel values of the standards to verify suitability of the standard curve.
<img alt="Table 4" src="/files/ftp_upload/59426/59426tbl4v2.jpg" /> 
Table 4: Range of HEX and FAM channel values of negative and positive controls to verify suitability of the run.
<img alt="Table 5" src="/files/ftp_upload/59426/59426tbl5v3.jpg" /> 
Table 5: Range of HEX and FAM channel values of samples to verify suitability of the sample analysis.
<img alt="Table 6" src="/files/ftp_upload/59426/59426tbl6v2.jpg" /> 
Table 6: Mix A and Mix B Cq values of the FAM channel to verify suitability of FL-DNA analysis.
<img alt="Table 7" src="/files/ftp_upload/59426/59426tbl7v2.jpg" /> 
Table 7: Cancer risk evaluation as a function of iFOBT and FL-DNA values. According to the relationship between iFOBT and FL-DNA values, the table estimates the probability of colorectal neoplastic lesions.

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 ...

evaluation-colorectal-cancer-risk-prevalence-stool-dna-integrity

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.9K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 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.

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

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Quantification of  Colonic Stem Cell Mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

Developmental Biology

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Immunology and Infection

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Biology

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and &#946;-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 &plusmn; 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops ...

Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining

Source: Maliszewska-Olejniczak et al. <a href="https://www.jove.com/v/61399"> Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells.</a> J. Vis. Exp. (2020).
This video describes a protocol to detect radiation-induced DNA repair foci in the colon cancer cell lines using immunofluorescence staining. DNA repair proteins get activated at the DNA damage site; therefore, we can detect them using specific antibodies and visualize them under a fluorescence microscope.

Ocena ognisk uszkodzenia DNA: Identyfikacja ognisk uszkodzenia DNA wywołanych promieniowaniem w linii komórek nowotworowych za pomocą barwienia immunofluorescencyjnego

Source: Maliszewska-Olejniczak et al.  Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells. J. Vis. Exp. (2020).
This ...