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  • GSK3 br We and others have


    We and others have shown that cases classified as MES subtype have the lowest rates of complete resection and have the poorest prognosis
    amongst HGSOC [5–8]. In our own cohort we observed that MES sub-type was an independent predictor of overall survival (OS) in HGSOC (34.2 vs. 44.6 months; p = 0.009, compared to non-MES subtype) [7]. We also reported that MES subtype is associated with higher rates of miliary and upper abdominal disease patterns suggesting a more in-vasive and metastatic phenotype. Clinically, these disease patterns are associated with dense stromal reaction and commonly contain areas of clinical fibrosis [7]. These results support the oft-quoted statement regarding HGSOC that the underlying biology of the disease impacts both phenotype (spread pattern) and outcomes.
    Classification systems based on global transcriptional profiling have typically been considered to represent the expression specifically from cancer cells. However, whole-tumor samples contain both cancer GSK3 and stroma, with stroma contributing a median of 50% of the OC tumor [9]. In the Tothill study, 40% of tumors within the C1 (MES) sub-group had a high percentage (defined as N50%) stromal component [3]. In TCGA analysis, the stromal content of samples was set at b30% but was not further described [4]. Relevant to the contribution from stroma to these gene signatures is the observation that specific genes (i.e., ACTA2, FAP, POSTN, ZEB1, and COL11A1) associated with worse outcomes in cancer are generally thought to arise from stroma not epi-thelial cells. By using IHC staining Tothill et al. observed that ACTA2 ex-pression was strong in areas of stromal desmoplasia surrounding tumor, and suggested that this stromal contribution to the genetic signature was important [3]. Calon et al. reported that FAP showed higher expres-sion in stroma and POSTN was exclusively expressed in stroma in colo-rectal tumors [10]. Isella et al. also reported that ZEB1 showed stronger staining in stroma compared with cancer cells in colorectal cancer [11]. Liu et al. found that COL11A1 was associated with stroma in HGSOC [6]. Many of these same genes are also significantly overexpressed in MES subtype of HGSOC [3–6,12,13]. It was also reported that stromal activa-tion provides a favorable tumor microenvironment and stimulates OC metastasis [5,8,15]. Defining the cells of origin has significant implica-tions for our understanding of events related to metastasis and clinical behavior of the cancer. Taken together, these observations support the concept that expression of MES subtype gene signatures is derived from both cancer and stromal populations and commonly from stromal compartment.
    In this study, our primary hypothesis is that MES subtype gene sig-natures partially arise from cancer-associated stroma. We performed IHC to study the origin of several MES subtype gene signatures in tumor samples. An important strength of our study is the inclusion of both primary tumor and metastatic sites. This approach allowed us to ask if the stromal reaction was different at metastatic implantation sites or varied across tissue types.
    2. Methods
    2.1. Patients and slides
    We studied 15 patients diagnosed with HGSOC between 2010 and 2013. Institutional Review Board approval was obtained for all studies and a written informed consent was obtained from all patients for use of data and biospecimens for research. Molecular subtype analysis categorized these 15 patients into MES subtype. A total of 45 formalin-fixed paraffin-embedded (FFPE) blocks (1 primary and 2 metastatic sites per patient) from primary surgery were selected to make slides.
    2.2. Gene signatures
    From the literature we selected 8 genes/proteins associated with the MES subtype. Table 2 describes the genes selected for this study and evidence from the literature of expression pattern in MES subtype in comparison with other molecular subtypes. 
    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 antibodies 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).