Jordan Madic1,*, Cécile Jovelet2,*, Julien Lopez1, Barbara André1, Jean Fatien3, Isabelle Miran2, Aurélie Honoré2, Laura Lezquita5, Benjamin Besse5, Ludovic Lacroix2,4,* and Magali Droniou1,*

Published: December 21, 2018

Journal : OncoTarget

Background:

Detection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an
extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated.

Methods: We developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and
evaluated this assay on 82 tumor and plasma samples from NSLC patients.

Results: Limits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled
detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wildtype DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease.

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