Formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples were retrospectively collected from the enrolled patients. Representative hematoxylin-eosin-stained tumor sections were reviewed by a pathologist (M.B.) and representative tumor areas were selected for the construction of the tissue microarray (TMA) blocks, as previously described [8 (link)]. Each case was represented by two tissue cores, 1.5 mm in diameter. Various neoplastic, non-neoplastic, and reactive tissues were also included in each TMA block to assist in the orientation and provide internal assay controls. Cases inadequately represented on the TMA sections were re-cut from the original blocks and whole tissue sections were used for immunohistochemical analysis.
Serial 2.5-μm-thick thick tissue sections from the TMA or the original blocks were used for the immunohistochemical staining, performed in the Laboratory of Molecular Oncology of the Hellenic Foundation of Cancer Research/Aristotle University of Thessaloniki, using a Bond MaxTM autostainer machine (Leica Microsystems, Wetzlar, Germany). The primary antibodies employed, retrieval conditions, dilution, and incubation time are presented inTable 1 , as previously described [9 (link)].
Serial 2.5-μm-thick thick tissue sections from the TMA or the original blocks were used for the immunohistochemical staining, performed in the Laboratory of Molecular Oncology of the Hellenic Foundation of Cancer Research/Aristotle University of Thessaloniki, using a Bond MaxTM autostainer machine (Leica Microsystems, Wetzlar, Germany). The primary antibodies employed, retrieval conditions, dilution, and incubation time are presented in