Formalin-fixed, paraffin-embedded tumour specimens from patients who met the inclusion criteria were used for tissue microarray construction. We marked 1 representative tumour area on the haematoxylin and eosin-stained tissue sections. Next, using a tissue arrayer (Tissue Microprocessor KIN-2; Azumaya, Japan), we arrayed a cylindrical 3-mm tissue core from the corresponding paraffin block into a recipient block. In total, there were 444 available cases with adequate cores for immunohistochemical analysis.
The immunohistochemical analysis was performed to evaluate TAMs in the primary tumour. We obtained 4-µm-thick sections from the tissue microarray blocks and then stained them with anti-CD68 antibody (clone KP1, Dako; 1:50) using a BenchMark ULTRA automated immunohistochemical slide staining system (Ventana Medical Systems, Inc.). For immunohistochemically stained tissue sections with immune markers, the 3 tumour areas with the highest density of immune cell infiltration (hot spots) were photographed using an Olympus BX53 microscope equipped with a DP22 digital camera (Olympus Corporation, Japan) using a 20× objective. CD68+ TAMs were counted on each of the 3 photographs using the manual counting method. The average count of 3 areas was considered as the number of CD68+ TAMs for each patient.
The immunohistochemical analysis was performed to evaluate TAMs in the primary tumour. We obtained 4-µm-thick sections from the tissue microarray blocks and then stained them with anti-CD68 antibody (clone KP1, Dako; 1:50) using a BenchMark ULTRA automated immunohistochemical slide staining system (Ventana Medical Systems, Inc.). For immunohistochemically stained tissue sections with immune markers, the 3 tumour areas with the highest density of immune cell infiltration (hot spots) were photographed using an Olympus BX53 microscope equipped with a DP22 digital camera (Olympus Corporation, Japan) using a 20× objective. CD68+ TAMs were counted on each of the 3 photographs using the manual counting method. The average count of 3 areas was considered as the number of CD68+ TAMs for each patient.