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 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.

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 time^1,2. Despite CRC'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 ....

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

1. Use a kit to prepare stool samples (see

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.......

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 lesions^{7,8,9,10,11,12}. Molecular, noninvasive screening tests have been developed over the years for the detection of colorectal can.......

The authors have no acknowledgments.