All the cases diagnosed as Chs (primary tumours, recurrences and/or metastasis and xenotransplanted Chs) from the files of our Department were collected. Only cases with paraffin blocks available were selected. All the clinical data of the patients were reviewed. (Total: 32 cases). A reclassification of all the cases according to the new criteria for Chs diagnosis (WHO) was performed.
Assembly of TMA
Six TMAs were performed and all the cases and biopsies were distributed into the following groups: a) only paraffin block available from primary and/or metastatic tumours (3 TMA), b) paraffin block available from primary and/or metastatic tumours as well as from the corresponding Nude mice xenotransplant (2 TMA), c) only paraffin block available from xenotransplanted Chs (1 TMA). A hematoxylin and eosin stained (H/E) section from each primary tumour and xenograft was prepared and areas of representative non-necrotic neoplasm circled on coverslip. The TMAs were assembled using a manual tissue arrayer (Beecher Instruments, Sun Prairie, WI). Normally, two cores (1 mm in thickness) of each biopsy were performed; nevertheless, more than 2 cores were made if the biopsy revealed a different pattern. All TMAs included two cores of normal kidney or liver as control tissues. Following TMA construction, H/E stained section of the TMA recipient block was prepared and reviewed to confirm the presence of intact neoplasm. Several sections of 5 μ were prepared in order to perform H/E stain as well as different IHC staining.
IHC analysis was performed using anti-CD99 antibody (clone 12E7, DakoCytomation) at a 1:50 dilution, anti-S100 polyclonal antibody (DakoCytomation) at a 1:200 dilution, anti-SOX-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution, anti-survivin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1: 50 dilution, anti-p16 antibody (clone F12, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution, anti-p53 antibody (clone DO7, Novocastra) at 1:50 dilution, pan-CK (AE1/AE3) antibody (DakoCytomation) at 1:50 dilution, anti-Ki-67 antibody (MIB-1, DakoCytomation) at 1:50 dilution, anti-Caveolin (CAV) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution, anti-Bcl-2 antibody (clone 124, Novocastra) at 1:50 dilution. Antigen retrieval was performed by pressure cooker boiling for 3 minutes in 10 mmol/L of citrate buffer (pH 6.0). The LSAB method (DakoCytomation) was used, followed by revelation with 3,3'-diaminobenzidine. Cytoplasmic and/or membrane staining was considered positive for CD99, S100, CK, CAV and Bcl-2 antibodies, nuclear staining was considered positive for SOX-9, Survivin, p53, p16 and Ki-67 antibodies. Sections were examined and immunoreactivity was defined as follow: negative, fewer than 5% of tumour cells stained; poorly positive (+), 5% to 10% of tumours cells stained; moderately positive (++), 10% to 50% of tumours cells stained and strongly positive (+++), more than 50% of the tumours cells were stained. All sections were evaluated independently by 3 pathologists (IM, SN and ALLB). The agreement of staining intensity scoring by all was recorded, and in cases of disagreement, intensity and score was determined by consensus.
Male nude mice, were purchase from IFFA-CREDO (Lyon, France), kept under specific pathogen-free conditions throughout the experiment, and provided with vinyl isolates plus sterilized food, water, cage and bedding. The specimens for xenotransplant were obtained at surgery (OT) and placed in a culture medium (RPMI 1640) plus antibiotic at 37 ℃ until transplantation, usually 6 hours after surgery. Fragments of non-necrotic tumour, about 3 to 5 mm in size, were transplanted into the subcutaneous tissue in the backs of two nude mice. The new tumour transfers were made by following the same procedure as in the initial xenotransplant and always under highly sterile conditions. Material from different passages was obtained in order to perform all TMAs. Additional material was obtained for electron microscopy, culture, and frozen sections.