Résumé
We investigated size distributions of circulating cell-free DNA (ccfDNA) to determine its value as a potential theranostic tool. CcfDNA quantification by Q-PCR was optimal when amplifying target lengths between 60 and 100 bp. CcfDNA sizes were previously believed to be greater than the length of the DNA per nucleosome (~180 bp). Q-PCR analyses of plasma samples from xenografted mice and from colorectal cancer (CRC) patients showed that tumor derived ccfDNA exhibited specific profiles of ccfDNA sizes with significantly higher ccfDNA fragmentation than was observed in samples from healthy individuals. Atomic Force Microscopy analysis confirmed these observations and, for the first time, enables visual representation of ccfDNA molecules. CcfDNA from plasma of CRC patients exhibited a median size of 135 bp and 88% of ccfDNAs are less than 180 bp in size. Based on these observations, we developed a refined single-step Allele Specific Q-PCR based method (Intplex) with improved specificity and sensitivity for enabling single-copy detection of variant alleles to a sensitivity of ≥ 0.005% mutant to WT ratio. We performed a blinded prospective study to compare KRAS and BRAF mutation status data by analysis of tumor tissue of CRC patients by standard methods and of plasma DNA (n=79). CcfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the six tested KRAS point mutations, the method exhibited 100% specificity and 87% sensitivity with a concordance value of 96%. Mutation load expressed as the proportion of mutant alleles in ccfDNA, was highly variable (0.03% to 69%, median, 10.9%; n=128) and was determined in the plasma of every patient at stage I, II, III and IV (100% success rate). Tumor-derived ccfDNA did not correspond to "a tiny fraction" as it has been previously stated and it is likely not derived mainly from stroma cells. CcfDNA analysis could replace advantageously tumor-section analysis for determining KRAS and BRAF mutation status and expand the scope of personalized medicine for cancer patients.
Citation Format: Florent Mouliere, Dalong Pang, Safia El Messaoudi, Denis Pezet, Muriel Mathonnet, Marc Ychou, Anatoly Dritschilo, Alain R. Thierry. Detection of circulating cell-free DNA is enhanced by targeting short fragments: high concordance of the KRAS and BRAF mutation status from plasma analysis, with tumor-tissue analysis in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-78. doi:10.1158/1538-7445.AM2013-LB-78