We further validated the approach in breast cancer cells with mutational activation of PIK3CA, where tandem mass spectrometry detected and quantitatively measured the abundance of a helical domain mutant (E545K) of PIK3CA connected to PI3K activation.
Using a variety of physiologically relevant model systems with defined natural or knock-in PIK3CA mutations and/or PI3K hyperactivation, we show that PIK3CA-E545K mutations (found in ∼20% of PIK3CA-mutant breast cancers), but not PIK3CA-H1047R mutations (found in 55% of PIK3CA-mutant breast cancers), preferentially activate AKT1.
Formalin-fixed paraffin-embedded tumour specimens from 118 HER2-overexpressing breast cancer patients treated with radical local therapy and trastuzumab in adjuvant setting were used for the assessment of: (1) PIK3CA gene mutations (p.H1047R and p.E545K) by qPCR, and (2) expression of Ki-67, EGFR, MUC4, HER3 and PTEN by immunohistochemistry.