In this study, we measured the kinase activity profiles of 32 pre-treatment tumour biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels in prediction of treatment success against HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis, based on targeted mass spectrometry measurement of kinase activation loops, allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. Furthermore, we found eleven kinases differentially regulated between treatment outcome groups.

Targeted MS analysis of phosphorylated kinase T-loops as previously published by Schmidlin et al (Schmidlin, T., Debets, D. O., van Gelder, C. A., Stecker, K. E., Rontogianni, S., van den Eshof, B. L., ... & Altelaar, M. (2019). High-throughput assessment of kinome-wide activation states. Cell systems, 9(4), 366-374.) here we applied this method to 32 treatment-naive HER2+ breast cancer needle biopsies to predict treatment outcome against HER2-targeted therapy. In short, samples were lysed by freeze-thaw, pestle and mortar and sonication. 200ug of each biopsy was digested ON using trypsin. Peptides were desalted and heavy labelled standards were spiked-in prior to phospho-peptide enrichment. Samples were analysed using an Ultimate 3000 RSLCnano system coupled to a TSQ Altis Triple Quadrupole.

Patients were enrolled in the TRAIN2-study (van Ramshorst, M. S., van der Voort, A., van Werkhoven, E. D., Mandjes, I. A., Kemper, I., Dezentjé, V. O., ... & Dutch Breast Cancer Research Group. (2018). Neoadjuvant chemotherapy with or without anthracyclines in the presence of dual HER2 blockade for HER2-positive breast cancer (TRAIN-2): a multicentre, open-label, randomised, phase 3 trial. The Lancet Oncology, 19(12), 1630-1640.). 14G needle biopsies of treatment naive HER2+ breast cancer patients were taken. After this, patients received multiple treatment cycles and treatment outcome was determined at surgery.