Abstract B21: Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue
Authors: Sobsey Constance A. , Popp Robert , Ibrahim Sahar , Froehlich Bjoern C , Aguilar-Mahecha Adriana , Basik Mark , Batist Gerald , Borchers Christoph 
Published: December 2020

Abstract
Introduction: Many clinical trials for inhibitors targeting the PI3K pathway now include genetic screening of tumors to try to select probable treatment responders. Genetic testing identifies some relevant mutations, but this approach does not address many mechanisms of PI3K pathway regulation. Epigenetic changes and post-translational modifications may increase pathway activity without any detectable DNA mutation. Where mutations are detected, mutated proteins may not be expressed or may be quickly degraded. Direct measurement of oncogenic proteins may therefore offer more useful molecular profiles for characterizing cancer-driving alterations and matching patients with appropriate treatments.
Purpose: The purpose of this study was to develop and implement protein quantitation assays to accurately assess PI3K pathway activity in tumors, as a means of matching patients with targeted cancer treatments.
Methods: Protein extracted from tumor tissue lysate is subjected to proteolytic digestion to generate proteotypic peptides used for protein quantitation. Synthetic stable isotope-labeled peptides are added to the digest as an internal standard, after which the target peptides are immuno-enriched with antibodies coupled to magnetic beads. MS analysis is then performed by either eluting peptides for measurement with liquid chromatography multiple reaction monitoring tandem MS (LC-MRM-MS/MS) or by spotting beads onto a plate for matrix-assisted laser desorption MS (MALDI-MS). External calibration curves are used for accurate quantitation.
Results: We developed and implemented MS-based methods to determine the concentration of PI3K pathway proteins in tumor samples. The developed methods enable precise and reproducible quantitation of Akt1, Akt2, PI3K P110α, and PTEN. We were able to distinguish and quantify specific isoforms, including phospho-Akt1 (Ser473), phospho-Akt2 (Ser474), and mutant-Akt (E17K). Endogenous protein levels were quantified in cell lines, fresh-frozen tumor samples, and formalin-fixed, paraffin embedded tumor tissue. 10 μg of protein extract per analyte was sufficient for quantitation. Subsets of assays could be multiplexed through simultaneous or sequential enrichment without compromising assay performance.
Novel Aspect: The multiplexing of immuno-MALDI assays through sequential enrichment and the immuno-MRM-MS assays for PTEN and mutant Akt represent new developments. Recent adoption of monoclonal antibodies for Akt1 will facilitate this assay's translation to a clinical setting.
Conclusions: This panel of quantitative proteomics assays enables comprehensive PI3K pathway assessment in tumor tissues. Previously collected genetically screened tumor samples were obtained with patient consent (as approved by the Jewish General Hospital Research Ethics Committee). Samples will be analyzed to assess PI3K pathway activity and determine its correlation to clinical features and response to PI3K-targeted treatments.
Note: This abstract was not presented at the conference.
Citation Format: Constance A. Sobsey, Robert Popp, Sahar Ibrahim, Bjoern C Froehlich, Adriana Aguilar-Mahecha, Mark Basik, Gerald Batist, Christoph Borchers. Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B21.