Robust dPCR Hyper-Plexing Approach by Color Combination for Detection of 17 ESR1 Mutations in Breast Cancer Patient Samples.

Digital PCR has seen increasing adoption in recent years since it overcomes most of the main technical limitations of real-time PCR. However, the level of multiplexing remains limited when facing the need to develop approaches that can detect multiple targets in a single reaction with a high sensitivity to obtain a more complete genetic characterization of samples. This need concerns most molecular biology applications, but it is especially valuable in fields such as oncology, for which it is often necessary to detect numerous specific mutations, particularly those involving hotspots, in order to adapt a treatment, monitor the efficacy of a drug, and detect resistance mutations. This is the case of the ESR1 gene in breast cancer, where numerous mutations can be responsible for mechanisms of resistance to the backbone therapy. In this sense, the implementation of color combination using Crystal Digital PCR® allows high levels of multiplexing to be achieved, while maintaining simplicity and robustness of analysis. This approach has been successfully applied to the detection of 17 ESR1 mutations in a single assay.