Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays

Abstract

Background: Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published.Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49).Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.

The utility of cfDNA in TGCT patient management: a systematic review

“…The biology of TGCT itself could be another potential issue, with molecular profiling by NGS being among the main approaches of cfDNA applications in patient management. 27 , 29 This results in two challenges. First, an adequate amount of cfDNA has to be extracted from the LB for the analysis to be reliable.…”

“… 61 , 65 The advent of digital droplet PCR has already sparked a renewed interest in various aspects of ctDNA research. 15 , 29 , 83 However, if sequencing is to be employed, perhaps efforts focused on amplification of chromosome 12 (i12p), as a near-universal TGCT marker, should be investigated. 3 , 9 …”

“…Instead of focusing on incremental technical improvements, a different approach would be to take into account the specific biology of TGCT, focusing on biologically more significant non-genetic markers, such as cfDNA methylation or fragmentation. 18 , 27 , 29 Methylation of cfDNA is a robust pathology-specific modification that is not impacted by preanalytical methods, 23 has already found clinical application and is FDA approved in colorectal cancer. 27 What makes it especially valuable is that research has shown that cfDNA methylation can be detected even when ctDNA makes up less than 10% of the total cfDNA fraction.…”

“… 13 , 18 , 23 , 27 Of these, the most promise as cancer biomarkers has been shown by cfDNA methylation and mutation analysis. 21 , 28 , 29 cfDNA’s non-genetic properties also vary between different biological states. The quantity of cfDNA can reflect tumor burden and progression, while differences in the fragmentation profile can discriminate cancer patients from healthy individuals, making cfDNA integrity a promising cancer diagnostic and prognostic biomarker in its own right.…”

“…The quantity of cfDNA can reflect tumor burden and progression, while differences in the fragmentation profile can discriminate cancer patients from healthy individuals, making cfDNA integrity a promising cancer diagnostic and prognostic biomarker in its own right. 18 , 29 – 31 …”