iMALDI as a tool to improve patient stratification for targeted cancer therapies




Popp, Robert

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The PI3K/AKT/mTOR signaling pathway is commonly dysregulated in cancer. The goal of this thesis project was to assess the hypothesis of a strong correlation between PI3K/AKT/mTOR pathway activity and the response to targeted therapies, by using a protein quantitation technique called immuno-matrix assisted laser desorption/ionization (iMALDI). The use of iMALDI as a clinical tool was demonstrated by automating an established iMALDI assay for quantifying plasma renin activity. The results from the automated method gave high correlation coefficients of ≥0.98 with a clinical LC-MS/MS method and could be performed significantly faster than with manual sample preparation. The 7.5-fold faster analysis compared to LC-MS/MS, reduction in human error, and higher throughput, demonstrated the suitability of this assay for clinical use. The automated iMALDI platform was then adapted for use with cancer cell lines and tissue analysis, targeting the kinases AKT1 and AKT2 as surrogate proteins for signaling pathway activity. Using minute amounts (10 µg/capture), AKT1 and AKT2 expression and phosphorylation stoichiometry (PS) were successfully quantified via their C-terminal tryptic peptides, which encompassed key phosphorylation sites. After assay optimization, the assays were analytically validated for linear range, accuracy, and interferences. In addition, PS cut-off values based on measurement errors were established for confident PS quantitation. The functionality of the assay was demonstrated with cell lines, and flash-frozen and FFPE tissue lysates, with, on average, lower AKT1/AKT2 measurements obtained from FFPE samples. The developed assays were sensitive and precise enough to detect differences between matched normal and adjacent tumor tissues. To answer the hypothesis, patient-derived xenograft (PDX) mouse-model tumors treated with Herceptin, Everolimus, a combination of both (E+H), or with no treatment, were assessed for molecular patterns linked to tumor response. One mouse from the E+H group showed a partial response, with elevated total and phosphorylated AKT1/AKT2. Unfortunately, overlapping values between treatment groups were obtained in this study, and the large within-group spread and the low number of biological replicates made it difficult to confirm a definite correlation between PI3K/AKT/mTOR pathway activity and response to treatment. A follow-up study with additional protein targets, a larger number of samples, and serial biopsies will be required to determine if there is, in fact, a correlation between PI3K/AKT/mTOR pathway activity and response to treatment.



Cancer, Proteomics, MALDI-TOF, Protein analysis, Patient stratification