The PIK3CA Assay n°1 (Fig. 1a), combines a drop-off system (Fig. 1b) for the detection of the 542–546 hotspot mutations (Drop-Off542–546) using a HEX-labelled drop-off probe covering the 542–546 hotspot and a Cy5-labelled reference probe located on the same amplicon, and simultaneous detection of N345K, C420R, H1047L and H1047R mutations, covering 90% of the pathogenic mutations identified for BC tumor samples in the COSMIC database (Supplementary Table S4). Here, PIK3CA is linked to breast cancer.