Multiplex detection of ten ESR1 mutations and AKT1 E17K in breast cancer using digital PCR

ESR1 mutations are now established as a key mechanism of resistance to endocrine therapy in estrogen-receptor-positive breast cancer (ER​+ ​breast cancer) and their sensitive and specific detection in plasma-cell free DNA (plasma-cfDNA) is crucial to monitor during patient treatment. In the present proof-of-principle study, we evaluated the performance of a novel multiplex assay (12plex) for the detection of ten ESR1 mutations and AKT1 E17K in plasma-cfDNA based on Crystal Digital PCR® (Stilla Technologies, France). We analyzed 35 plasma-cfDNA samples from ER+ ​breast cancer patients and 10 samples from healthy donors and further compared the results with our previously reported ESR1 NAPA assay for D538G, Y537S, Y537C and Y537 N ESR1 mutations. Using this novel 12plex ESR1-AKT 6-color Crystal Digital PCR® assay we detected both AKT1 E17K and ESR1 D538G mutations in 5/35 (14.3%) plasma-cfDNA samples. ESR1 D538G was detected in 4/35 (11.4%) of these plasma-cfDNA samples using the ESR1 NAPA assay. Direct comparison between Crystal Digital PCR™ and the ESR1 NAPA assay revealed a high concordance (97.1%, k = 0.871, p < 0.001) for the detection of D538G mutation. The Stilla 12plex ESR1-AKT 6-color Crystal Digital PCR® assay is multiplex, highly sensitive and robust and can be used in liquid biopsy.