title: A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.
abstract: Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.
authors: Institut de Recherche en Cancérologie de Montpellier (IRCM), Campus Val d'Aurelle, 208 rue des Apothicaires, Montpellier, Cedex 5, 34298, France. alain.thierry@inserm.fr.|INSERM, U1194, Montpellier, 34298, France. alain.thierry@inserm.fr.|Université de Montpellier, Montpellier, 34090, France. alain.thierry@inserm.fr.|Institut Régional du Cancer de Montpellier, Montpellier, 34298, France. alain.thierry@inserm.fr.|DiaDx SARL, Montpellier, 34298, France. alain.thierry@inserm.fr.
authors: Alain R
authors: AR
authors: Thierry
mesh_terms: D014408:Biomarkers, Tumor; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D019468:Disease Management; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
keywords: BRAF; Cancer; Cell-free DNA; Circulating DNA; Diagnostic; KRAS; Mutation; Plasma; Q-PCR; Sensitivity