Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer.
Development of a Q-PCR-based assay for the high-performance analysis of circulating cell-free DNA (ccfDNA) requires good knowledge of its structure and size. In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q-PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele-Specific Blocker (ASB) Q-PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi-marker method, termed Intplex, enables sensitive and specific non-invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory.
Institut de Recherche en Cancérologie de Montpellier, U896 Inserm, Montpellier, France.
Florent
F
Mouliere
Institut de Recherche en Cancérologie de Montpellier, U896 Inserm, Montpellier, France.
Safia
S
El Messaoudi
Department of Radiation Oncology, Lombardi Cancer Center, Georgetown University, Washington DC, USA.
Dalong
D
Pang
Department of Radiation Oncology, Lombardi Cancer Center, Georgetown University, Washington DC, USA.
Anatoly
A
Dritschilo
Institut de Recherche en Cancérologie de Montpellier, U896 Inserm, Montpellier, France. Electronic address: alain.thierry@inserm.fr.
Alain R
AR
Thierry
D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D003106:Colon / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000097:blood* / Q000235:genetics* / Q000473:pathology; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics / Q000648:ultrastructure*; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D057285:Precision Medicine / Q000379:methods; D012007:Rectum / Q000378:metabolism / Q000473:pathology
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
Circulating cell-free DNA; Colorectal cancer; Multi-marker analysis; qPCR