165 Discussion and future perspectives lished to date on clinical trials using global phosphoproteomics for targeted therapy selection in patients with cancer. Phosphoproteomics knowledge and facilities are not mainstream and are still confined to centers of expertise, often academic laboratories. At this time, it seems still too early to implement phosphoproteomics analysis for therapy selection in patients with malignancies, due to (i) limited tissue availability, although down-scaling of the pTyr phosphoproteomics protocol now allows for reproducible analysis on only 1 mg of protein input52, which is the yield of a 14G core needle biopsy53, (ii) specific logistic requirements and pre-analytical handling of the tissue to allow this complex analysis, (iii) the time-consuming laborious technique and required expertise of the research staff, (iv) required expertise in interpretation of the results, and (v) the lack of clinical validation and reproducibility. A targeted assay or immunohistochemistry analysis with a selection of differential phosphosites and/or proteins could facilitate the implementation of these signatures as a decision-making tool for treatment selection in clinical practice. Such an assay would prevent unnecessary toxicity and enable alternative (combination) treatment in patients upfront predicted to be resistant to sunitinib. PRE-ANALYTICAL REQUIREMENTS FOR (MULTI)OMICS ANALYSIS ON CLINICAL TISSUE SAMPLES Increasingly complex molecular analyses of tissue samples require a standardized and controlled method of tissue preservation. To facilitate multi-omics analysis on clinical tissue samples, high-quality fresh frozen tissue samples are required. Particularly post-translational modifications in tumor tissue are sensitive to variations in pre-analytical handling54-57. Tumor biopsies collected for research and precision oncology purposes are generally placed in a cryovial by trained staff and immediately immersed in liquid nitrogen (LN2). This process is referred to as snap freezing and currently the golden standard58. Snap freezing is a laborious, potentially hazardous, and not user-friendly procedure. In addition, LN2 is not widely available and the use of sacrificial LN2 is non-sustainable due to its energy-intensive synthesis. To circumvent the limitations of snap freezing in LN2, a new liquid nitrogen-free snap freezer was developed for snap freezing biospecimens, to conserve molecular profiles under standardized and optimized pre-analytical conditions59. In chapter 5, we benchmarked the performance of the electrically powered snap freezer prototype to the golden standard of LN2-quenching with regard to preservation of biology. We used cancer cell line K562 specimens and core needle biopsies from normal human liver resection specimens, snap frozen using either the golden standard or the new snap freezer, to compare mass spectrometry (MS)-based global phosphoproteomic and transcriptomic profiles and RNA integrity. We found that cell line RNA integrity, phosphoproteomic and transtriptomic profiles of snap freezer versus LN2 quenching were highly comparable, the samples could not be distinguished based on the freezing method used, while the positive control sample (that was left at room temperature for 2 hours) clearly formed a separate cluster. Molecular profiles of liver tissue biopsy samples clearly clustered per patient, regardless of the applied freezing 6
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