AUTHOR=Poulet Geoffroy , Garlan Fanny , Garrigou Sonia , Zonta Eleonora , Benhaim Leonor , Carrillon Marie-Jennifer , Didelot Audrey , Le Corre Delphine , Mulot Claire , Nizard Philippe , Ginot Frederic , Boutonnet-Rodat Audrey , Blons Helene , Bachet Jean-Baptiste , Taïeb Julien , Zaanan Aziz , Geromel Vanna , Pellegrina Laurence , Laurent-Puig Pierre , Wang-Renault Shu-Fang , Taly Valerie
TITLE=Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays
JOURNAL=Frontiers in Oncology
VOLUME=11
YEAR=2021
URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2021.639675
DOI=10.3389/fonc.2021.639675
ISSN=2234-943X
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.