Multi‐marker analysis of circulating cell‐free DNA toward personalized medicine for colorectal cancer. Issue 5 (24th March 2014)

• Record Type: Journal Article
• Authors: Mouliere, Florent; El Messaoudi, Safia; Pang, Dalong; Dritschilo, Anatoly; Thierry, Alain R.
• Is Part Of: Molecular oncology. Volume 8:Issue 5(2014:Jul.)
• Page Start: 927
• Page End: 941
• Publication Date: 2014-03-24
• Subjects: Circulating cell‐free DNA -- Multi‐marker analysis -- qPCR -- Colorectal cancer; Cancer -- Molecular aspects -- Periodicals; 616.994005
• DOI: 10.1016/j.molonc.2014.02.005
• Languages: English
• ISSNs: 1574-7891

Abstract

Abstract : 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. Highlights: We present the first visual determination of ccfDNA short fragments from plasma samples by AFM. AFM analysis confirms that ccfDNA is highly fragmented. This report describes the first integrated ccfDNA multi‐marker Q‐PCR analytical test. ccfDNA concentration, detection of mutated allele, and integrity index are analyzed in this test. This test provides a fast, cost effective, sensitive, and simple analysis of ccfDNA.