A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care

Part of the Methods in Molecular Biology book series (MIMB, volume 1392)


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.

Key words

Circulating DNA Cell-free DNA Plasma Mutation Q-PCR Cancer KRAS BRAF Sensitivity Diagnostic 


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institut de Recherche en Cancérologie de Montpellier (IRCM)MontpellierFrance
  2. 2.INSERM, U1194MontpellierFrance
  3. 3.Université de MontpellierMontpellierFrance
  4. 4.Institut Régional du Cancer de MontpellierMontpellierFrance
  5. 5.DiaDx SARLMontpellierFrance

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