3. PIK3CA Mutations and Their DetectionSomatic point mutations and gene amplifications are the two main genetic alterations that can alter PIK3CA functions [15,16].Wu et al.  found a PIK3CA gene copy number gain in 8.7% BC over 92 cases evaluated in their study, an interesting finding since PIK3CA gene amplification has been described as a mechanism of resistance to selective PIK3CA inhibitor in HER2+, PIK3CA mutant breast cancer cell line, KPL-4 .About 80% of PIK3CA somatic mutations are hot-spot mutations, the most common being single nucleotide changes resulting in aminoacidic substitutions: E542K and E545K in the exon 9, H1047R and H1047L in the exon 20. These mutations are oncogenic, determining a gain of function that promotes the constitutive signaling pathway activation, no longer dependent on growth factor stimulation. Of note, PIK3CA mutations have been found in ductal carcinoma in situ (DCIS), suggesting that it is an early event in the BC tumorigenesis .Exon 9 mutations involve the p110 helical domain, avoiding its inhibitory interaction with the p85 nSH2 domain, mimicking the activation by RTK phosphopeptides. These mutants depend on Ras binding for their transformation .By contrast, exon 20 mutations at the hotspot 1047 do not need Ras activation but depend on p85 binding. These mutations occur in the kinase domain causing a conformational change of the activation loop, thus favoring the lipid membrane binding and the PIP2 substrate recognition  [Figure 1].The most three frequent mutations E542K, E545K and H1047R are all responsible for enzymatic activation of the PI3Kα; however, in mice models, PIK3CA H1047R has shown to be a stronger inducer of breast cancer, in terms of both latency and frequency, maybe explained by the higher activation of the downstream AKT signaling .PI3K pathway is also involved in the cell-signaling of the family of Human epidermal growth factor receptors (HER/erbB), including the HER2 that is overexpressed in HER2+ BC subtype [22,23]. In cell membrane, the monomer HER2 is inactive. Following the binding of the ligand, HER2 hetero- or homo-dimerizes to become active, stimulating the downstream PI3K/AKT and the mitogen-activated protein kinase (MAPK) pathway, inducing cell growth and survival.The aforementioned mutations in PIK3CA gene or partial/complete loss of PTEN, mediating mTOR activation, may lead to HER2-independent constitutive activation of the PI3K/AKT pathway that has been associated with HER2-therapy resistance [24,25]. In HR+/HER2+ BC, PI3K is involved in both ER and HER2 pathway signaling, and crosstalk among these pathways is responsible for the occurrence of endocrine- and HER2-resistance .There are three main molecular biology techniques available for the detection of PIK3CA mutations: Sanger sequencing, real-time PCR (RT-PCR) and next-generation sequencing (NGS).Sanger sequencing represents a reliable method that can detect unknown mutations and gene fusions, although limited by a lower sensitivity when compared to NGS. RT-PCR is a commonly employed technique to detect mutations in known genomic regions; however, using primers for specific mutant sequences, only a restricted number of already well-characterized alterations can be assessed. On the other hand, NGS is a high throughput technique for the analysis of tumor DNA samples with higher sensibility than Sanger sequencing, so possibly representing the “ideal technique”, but its adoption is limited because of the complexity of the procedure and, importantly, its costs [27,28].The method used in the SOLAR-1 trial is the Therascreen © PIK3CA RGQ PCR kit, a real time qualitative PCR test that can identify 11 mutations, in particular exon 7 p.C420R; exon 9 p.E542K, p.E545A, p.E545D, p.E545G, p.E545K, p.Q546E and p.Q546R; exon 20 p.H1047L, p.H1047R and p.H1047Y. It analyses both genomic DNA extracted from formalin-fixed, paraffin-embedded tumor tissue or circulating tumor DNA (ctDNA) from plasma derived from liquid biopsies. Along with the approval of alpelisib for the treatment of PIK3CA mutated-mBC, the FDA approved the Therascreen © PIK3CA RGQ PCR kit as the companion diagnostic (CDx) for the detection of PIK3CA mutations .Martines-Saez et al. selected a dataset of 6338 BC tumor samples analyzed by targeted or whole exome sequencing. Exon 20 p.H1047R and exon 9 p.E545K and p.E542K represented approximately 63% of all the PIK3CA mutations found. Nevertheless, other PIK3CA mutations have been found in a non- negligible frequency, such as the exon 4 p.N345K. Since these mutations are not detected in the Therascreen © kit, the SOLAR1 trial did not investigate the efficacy of alpelisib against these variants and this remains unknown .This issue should be considered when choosing a platform for mutational status detection, especially because wide-spectrum NGS assays can detect more alterations beyond those included in the Therascreen. In fact, in the U.S., along with the Therascreen © kit, FoundationOne® CDx and FoundationOne® Liquid CDx are approved as companion diagnostics too.Regarding samples, PIK3CA mutational status can vary among primary tumor and metastases, as for the HR and the HER2 expression . The discordance has been highlighted in different studies, not only in terms of gain or loss of the mutation in the PIK3CA gene but also in terms of increasing or decreasing levels of mutation [32,33,34,35], possibly influencing response to PI3Ki and underlining the importance of molecular characterization of metastatic sites. Moreover, if specimens cannot be obtained (for example due to the specific metastatic site), ctDNA can be used, selecting patients whose tumor shedding is expected to be sufficient to avoid false negative (e.g., cases with adequate tumor burden or with progressive disease) . Among 68 patients included in a study by Dumbrava et al. , an agreement rate of 78% in the detection of PIK3CA mutations has been demonstrated between ctDNA samples and tumor tissue analysis. The concordance even increases up to 91% if only patients with progressing tumors are considered .