High Fragmentation Characterizes Tumour-Derived Circulating DNA

Abstract

BackgroundCirculating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments.MethodologyIn order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients.Conclusion/SignificanceFragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60–100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16).

Circulating DNA as a Strong Multimarker Prognostic Tool for Metastatic Colorectal Cancer Patient Management Care

“…We observed a higher ccfDNA fragmentation level from colorectal cancer patients as compared with healthy individuals buttressing the notion that fragmentation could be an interesting parameter for diagnosis (35,36). In regard to prognosis, patients with a higher fragmentation level showed, in this study, a lower median OS of 17 months as compared with 23 months for patients with a lower fragmentation level.…”

Potentials, challenges and limitations of a molecular characterization of circulating tumor DNA for the management of cancer patients

“…Size profiles were highly correlated with the amount of tumor DNA in the circulation, whereas the abundance of shorter fragments was associated with larger amounts of tumor DNA [37]. Similar observations were already made in 2011, when the Thierry group demonstrated the presence of a higher proportion of cfDNA fragments below 100 bp particularly in samples from cancer [38]. However, data from our group indicated that an inefficient clearance of nucleosomal derived DNA may lead to multiples of mono-nucleosomal DNA, which correlated with higher fractions of tumor DNA [33,39].…”

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

“…Similarly, cell‐free circulating DNA has a half‐life of approximately 14 h and is rapidly cleared from blood, if not replenished from apoptotic/necrotic cells every few hours 42. Moreover, the vast majority of cell‐free DNA fragments are between 180 and 200 bp,43, 44 and cannot be amplified by our DBS assays, which targets regions between 379 and 597 bp in length. Thus, neither circulating tumor cells nor cell‐free tumor DNA can account for the observed EMRs > 1% or single CpG hypermethylation > 2%.…”