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Oncotarget. 2018 Dec 21;9(100):37393-37406. doi: 10.18632/oncotarget.26446. eCollection 2018 Dec 21.

EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR.

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Stilla Technologies, 1 Mail du Professeur Georges Mathé, Villejuif, France.
Plateforme de Génomique, Module de Biopathologie Moléculaire et Centre de Ressources Biologiques, AMMICa, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, France.
Ecole Polytechnique, Route de Saclay, Palaiseau, France.
Département d'Oncologie Médicale, Gustave Roussy, Villejuif, France.
Département de Biologie et Pathologie Médicales, Institut Gustave Roussy, Villejuif, France.
Contributed equally



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.


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.


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 wild-type 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.


This 6-color Crystal digital PCR assay could represent a robust solution using digital PCR for the monitoring of EGFR mutations.


EGFR; NSCLC; digital PCR; monitoring; multiplexing

Conflict of interest statement

CONFLICTS OF INTEREST Jordan Madic, Julien Lopez, Barbara André and Magali Droniou are employees of Stilla Technologies. Benjamin Besse's research is funded in part by Abbvie, Amgen, AstraZeneca, Biogen, Blueprint Medicines, BMS, Celgene, Eli Lily, GSK, Ignyta, IPSEN, Merck KGaA, MSD, Nektar, Onxeo, Pfizer, Pharma Mar, Sanofi, Spectrum Pharmaceuticals, Takeda and Tiziana Pharma.

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