The antibodies used in the immunohistochemistry included antibodies for PSA (mouse monoclonal, clone ER-RP8, 1:100 dilution), P504S (rabbit monoclonal, clone 13H4, 1:200 dilution), P501S (mouse monoclonal, clone 10E3, 1:100 dilution), PSMA (mouse monoclonal, clone 3E6, 1:100 dilution), pancytokeratin (mouse monoclonal, clone AE1/AE3, 1:50 dilution), LMW-CK (mouse monoclonal, clone CAM5.2, 1:10 dilution), and HMW-CK (mouse monoclonal, clone 34βE12, 1:200 dilution). In these antibodies, CK CAM5.2 was supplied by Becton Dickinson Immunocytometry Systems (San Jose, CA, USA), and all of the others were supplied by Dako (Glostrup, Denmark). In antigen retrieval, a heat-induced procedure was performed using an autoclave at 121°C for 10 min, in a 0.01 mol/L citrate buffer, pH 6, for PSA and CK 34βE12, and in Target Retrieval Solution, pH 9 (Dako), for P504S, P501S and PSMA. A trypsin digestion was performed for CK AE1/AE3 and CK CAM5.2. The staining was carried out using the streptavidin–biotin–peroxidase complex technique with a Histofine kit (Nichirei, Tokyo, Japan) following the manufacturer's instructions. The sections were made to react with the chromogen, 3,3′-diaminobenzidine-tetrahydrochloride (Merck, Darmstadt, Germany), and were then counter-stained with hematoxylin. A specimen with the primary antibodies omitted was used as the negative control.
Immunohistochemistry was assessed on prostate secretory cells of either the carcinoma glands or non-neoplastic glands. The staining intensity in immunohistochemistry was categorized on the following scale based on positive cell rate (Fig. 1): category 1, negative or minimally positive (<10%) cells were observed (Fig. 1c); category 2, focal positive (≥10% and <90%) cells were observed (Fig. 1d,e,g); and category 3, diffuse positive (≥90%) cells were observed (Fig. 1c,f,g).
Figure 1. Histological features of prostatectomy specimens after hormone therapy. (a) Small glands and clusters of residual carcinoma are densely distributed in the left-lower part (arrowheads) and expanded round glands with basal cell hyperplasia of non-neoplastic prostate glands are situated in the right upper part (arrows). (b) Residual carcinoma cells were arranged in single cells, cords, and small glands (arrowheads), and the non-neoplastic glands had enlarged diameter (arrow). (c) The carcinoma was category 3 (arrowhead) and the non-neoplastic glands was category 1 (arrow) based on prostate-specific antigen (PSA) staining. (d) The carcinoma was category 2 (arrowheads) based on P504S staining. (e) The carcinoma with the feature of perineural invasion was category 2 based on P501S staining (arrowhead). The cytoplasm of mast cell is also reactive for P501S (white arrow). (f) Both the carcinoma (arrowhead) and the non-neoplastic glands (arrows) were category 3 based on prostate-specific membrane antigen (PSMA) staining. (g) The secretory cells of carcinoma were category 2 (arrowhead) and those of non-neoplastic glands were category 3 (arrow) based on low-molecular-weight cytokeratin (LMW-CK) CAM5.2 staining. Some of the basal cell layers of the non-neoplastic glands were also stained with LMW-CK CAM5.2 (white arrowhead). (h) Basal cell layers stained with high-molecular-weight (HMW)-CK 34βE12 are absent in the carcinoma (left side in the panel) and are present with hyperplasia in the non-neoplastic glands (arrow). (a,c,d,f–h) are from one patient; (b,e) are from another.
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On preliminary immunohistochemistry it was easy and verifiable with reproducible results to differentiate the staining intensity between negative/minimally positive cells and almost all positive cells, with the former and the later assessed as category 1 or 3, respectively. But it was difficult to distinguish weak staining intensity from intermediate staining and determine the exact percentage of positive cells without interobserver variation. For practical purposes, therefore, ‘weak’ and ‘intermediate’ results were lumped together in category 2.