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  • br Five micrometer thick paraffin


    Five micrometer-thick paraffin sections were mounted on Superfrost Adhesion Slides and dried at room temperature for over-night. The sections were deparaffinized in xylene and hydrated and rehydrated in graded solutions of ethanol. The endogenous peroxidase was blocked with Peroxidazed 1 (Biocare Medical, Pacheco, CA, USA) for 5 mins. Antigen retrieval procedure was performed by using water bath heating in pH = 8.0, 0.001 M EDTA buffer or pH = 6.0, 0.01 M cit-rate buffer (Newcomer Supply, Middleton, WI, USA). Sections were in-cubated with DAKO Protein block serum-free ready-to-use solution (Agilent, Santa Clara, CA, USA) for 1 h at room temperature to reduce the nonspecific binding. Primary Pyocyanin were diluted with DAKO Antibody diluent (Agilent, Santa Clara, CA, USA) and incubated at 4 °C for overnight. After washing, the sections were then incubated with Sig-nal Stain Boost IHC Detection Solution (Cell signaling, Danvers, MA, USA) at room temperature for 30 mins. The slides were developed with Betazoid Diaminobenzidine (DAB) Chromogen (Agilent, Santa Clara, CA, USA), counterstained with haematoxylin, dehydrated in ethanol and xylene, and finally mounted. The stained slides then scanned with Desktop Scanner (Objective Imaging, Kansasville, WI, USA). Primary antibodies ACTA2 (ab5694), COL5A1 (ab7046), FAP (ab207178), POSTN (ab79946), and VCAN (ab177480) purchased from Abcam (Cambridge, UK); COL11A1 (PA5-36227) and p-SMAD2 (44-244G) from Thermo Fisher Scientific (Waltham, MA, USA); and ZEB1 (HPA027524) from Sigma-Aldrich (St. Louis, MO, USA).
    Two researchers independently, in a blinded manner, evaluated the intensity and distribution of positive staining of each slide. Slides and scores were reviewed together using a dual-headed scope to reach a final consensus score for any specimens that were discordant. A stan-dard 4-point scale scoring system as 0 (absent), 1 (weak), 2 (moderate), and 3 (strong) was used to score intensity to study origin (stroma vs. cancer) and gene expression alteration (primary vs. metastatic sites). In expression alteration analysis, the average of 2 scores of metastatic sites was compared with the score of primary site.
    2.5. Statistical analysis
    The analysis of protein expression was performed using GraphPad Prism 7 software and statistical differences were evaluated using t-test.
    3. Results
    3.1. Patient demographics
    Patients' characteristics are shown in Table 1. All patients had MES subtype of HGSOC, and underwent cytoreductive surgery. The vast ma-jority of patients had either high or intermediate complexity surgery. All patients had b0.5 cm of gross residual disease at the conclusion of sur-gery with 40% having complete gross resection. Most patients were ei-ther platinum sensitive (47%) or resistant (47%) showing disease progression within 6 months of completion of chemotherapy.
    3.2. Gene signatures
    Based on existing literature, an initial candidate list selected proteins that are recognized to be highly expressed in MES vs. non-MES sub-types. We then narrowed this candidate list to focus on those with established antibodies that further performed well in testing in FFPE samples to arrive at the 8 proteins selected. Published reports of micro-array analyses show correlation with overexpression and prognosis for many of these genes (Table 2). These proteins include alpha-smooth muscle (ACTA2), collagen type V alpha 1 (COL5A1), collagen type XI
    Table 1
    Patient demographics.
    (40%) metastatic sites, compared with absent expression in only 2/15
    this likely reflects small sample size.
    Tumor stage
    In contrast, COL11A1 demonstrated increased expression in the
    Surgical complex
    tionship was reversed when considering expression from cancer cells
    instead of stroma: COL11A1 expression was reduced in metastatic vs.