Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

Abstract

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS.We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

SFRPTumour Suppressor Genes Are Potential Plasma-Based Epigenetic Biomarkers for Malignant Pleural Mesothelioma

“…We were also able to detect SFRP1 and SFRP4 methylation in plasma samples; however, there was no distinctive separation of patients and controls. There are many studies reporting detection of biomarkers in noninvasive samples from cancer patients, including sputum (lung cancer) [ 40 ], urine (bladder cancer) [ 41 , 42 ], plasma (breast cancer) [ 43 ], and stool (colon cancer) [ 44 , 45 ]. Although we have demonstrated that methylated SFRPs can be detected in plasma of MPM patients, the small sample size means that we are not able to make a definitive conclusion about whether we can use this finding as a diagnostic marker for MPM.…”

Mutations by Next Generation Sequencing in Stool DNA from Colorectal Carcinoma Patients - A Literature Review and our Experience with this Methodology

“…Recently, Russo et al [44] described the feasibility and high sensitivity of single molecule third generation sequencing for the detection of CRC mutations in stool DNA. They applied single molecule real time circular consensus sequencing (SMRT-CCS) to detect APC gene mutations at a very low frequency of 0.5% without encountering any false positives.…”

“…An alternative approach was recently described by Russo et al [44]; they applied single molecule third generation sequencing for detection of APC gene mutations in DNA from paired stool and tissue samples and claimed that it was possible to detect mutations at very low levels (0.5%).…”

The Translational Status of Cancer Liquid Biopsies

“…Where shorter (< 10 kb) reads suffice, templates can be read multiple times, allowing computation of highly accurate circular consensus sequences (CCS) from individual single passes (subreads). CCS-based sequencing has already been used to detect mutations at frequencies below 0.5% in the stool of CRC patients [64].…”