90 CHAPTER 4 We here aimed to identify baseline tissue-based molecular biomarkers for prediction of (lack of) treatment benefit to sunitinib in patients with advanced RCC, using MS-based pTyr-phosphoproteomics and global expression proteomics. MATERIALS AND METHODS PATIENT SELECTION From the hospital pathology database, patients with RCC were selected who had undergone tumor nephrectomy or metastasectomy between 2000 and 2013, and thereafter received palliative treatment with sunitinib in the Amsterdam University Medical Centers (Amsterdam UMC), location VUmc. Clinical data were collected retrospectively from the hospital case records. Patients were classified as “sensitive” if they had PFS ≥ 12 weeks and radiological stable disease or objective response, or “primary resistant” if they exhibited radiological progressive disease at first evaluation (PFS < 12 weeks). Since archival tissue was used for the purpose of scientific research, and collected within the context of routine clinical practice procedures, the Dutch Medical Research Involving Human Subjects Act does not apply. Patients treated at Amsterdam UMC had the possibility to opt-out for the use of their data and tissue for research purposes. TUMOR TISSUE COLLECTION AND SAMPLE PROCESSING FOR LC-MS/MS Frozen pre-treatment tumor resection specimens, acquired through standard care procedures and stored at -80 °C, were collected from the hospital biobank. The tumor samples were cut (Leica CM1850) in 10-µm cryosections at -20 °C, transferred to precooled 1.5-ml Eppendorf vials and stored at -80 °C. Lysis was performed using approximately 1 ml 9 M urea buffer per sample, followed by 1 min vortexing (maximum speed), sonication (18-μm amplitude) and centrifugation (15 min, maximum speed). The cleared lysate was aliquotted and stored at −80 °C until further use. The BCA protein assay (ThermoPierce, Rockford, IL) was used to determine protein concentration. Cell lysates were reduced in 4 mMDTT for 20 min at 60 °C, cooled to room temperature, and subsequently alkylated in 10mMiodoacetamide for 15min in the dark. After dilution to 2 M urea using 20 mM HEPES buffer pH 8.0, the lysate was digested with 20 μg Sequencing Grade Modified trypsin/ (Promega, Leiden, The Netherlands) per mg protein by overnight incubation at 22 °C. Digestion was then stopped by adding trifluoroacetic acid (TFA) to a final concentration of 1%. Samples were incubated for 15 min on ice, centrifuged for 5min at 1800 ×g, and transferred to a new tube. Tryptic digests were desalted using 1-ml Oasis HLB cartridges (Waters, Milford, MA). After pre-wetting with acetonitrile (ACN) and equilibration of the column with 0.1% TFA, peptides were loaded. The column was washed using 0.1% TFA before elution into glass vials with 40% ACN/0.1% TFA. Eluates were lyophilized for 48 h and stored at −80 °C until further use.
RkJQdWJsaXNoZXIy MTk4NDMw