& articles



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. Read More

Abstract : Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the
quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS… Read More

Abstract : Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCRTM, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows threecolor multiplexing, which entails a different approach to data analysis. In the present publication, we
present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M… Read More



  • April 2017, “Monitoring EGFR mutations in lung cancer patients using 3-color Crystal Digital PCR” by Magali Droniou; Director of Applications at Stilla Technologies
    At the qPCR dPCR & NGS Congress, the 8th international Gene Quantification Event (3 – 7 of April 2017), Freising-Weihenstephan, Germany
  • October 2016, “Monitoring EGFR mutations in lung cancer patients using 3-color Crystal Digital PCR” by Rémi Dangla; CEO at Stilla Technologies
    At the 4th qPCR & Digital PCR Congress (20 – 21 of October 2016); London, UK
  • July 2016, “Digital PCR: Possibilities & Opportunities: Monitoring EGFR mutations in lung cancer patients using 3-color Crystal Digital PCR” by Rémi Dangla; CEO at Stilla Technologies
    At the 2nd q PCR & Digital PCR Congress (12-13 of July 2016); Philadelphia, USA