Paradoxical Expression of R-10G-reactive Antigen in Human Testicular Embryonal Carcinoma

Abstract

Thus far, several monoclonal antibodies directed against cell-surface carbohydrate antigens have been generated. Among them, R-10G reportedly reacts selectively with human embryonic stem and induced pluripotent stem cells, but not with embryonal carcinoma (EC) cells. However, EC cells derived from patients' EC tumors may exhibit varying levels of R-10G-reactive antigen expression. Thus, we asked whether human EC tissues or germ cell tumor (GCT) tissues other than EC express R-10G-reactive antigen. To do so, we quantitatively analyzed R-10G-reactive antigen expression in 83 testicular GCT surgical specimens containing a total of 125 various GCT components. Accordingly, in all EC components examined, the EC cell plasma membrane was immunolabeled with R-10G, while most seminoma components were R-10G-negative. In non-seminomatous GCT (NSGCT) other than EC (non-EC NSGCT), R-10G-reactive antigen expression was variable, but signal distribution was focal, and the average intensity was weaker than that seen in EC. The percentages of R-10G-positive cells in these three groups varied with high statistical significance (p<0.001 for all combinations). These findings indicate that the R-10G-reactive antigen is preferentially expressed in human testicular EC tissues and, thus, could be used as a diagnostic marker for this malignancy.

Keywords: immunohistochemistry; keratan sulfate; testis.

Figure 1.

(A) Analysis of positive and negative controls for R-10G immunostaining using porcine corneal tissue rich in keratan sulfate glycosaminoglycans. Untreated (positive control) or keratanase-II-treated (negative control) sections were stained with R-10G. Signals were visualized with 3,3’-diaminobenzidine (DAB) (brown), and tissues were counterstained with hematoxylin. (B) Expression of R-10G-reactive antigen in tissues of indicated testicular GCTs, including EC, yolk sac tumor, choriocarcinoma, and seminoma. Tissue sections containing GCT components were stained with H&E (left column) or immunostained for CD30 (second column from left) or R-10G (right two columns). Boxed areas in column 3 are enlarged in adjacent images in column 4. Signals were visualized with DAB (brown), and tissues were counterstained with hematoxylin. Representative cases are shown. In the lower right panel, arrows indicate seminoma cells, arrowheads indicate lymphocytes, and asterisks indicate fibrous septa. Bar A = 200 µm. Bar B = 50 µm for columns 1–3; 12.5 µm for column 4. Abbreviations: GCT, germ cell tumor; EC, embryonal carcinoma; H&E, hematoxylin and eosin.

Figure 2.

(A) Double immunofluorescence of testicular embryonal carcinoma (EC) (top row), yolk sac tumor plus EC (middle row), and seminoma plus EC (bottom row) using respective diagnostic markers (left column; red) and R-10G (middle column; green). Yellow signals in merged images (right column) indicate antigen colocalization. Tissues were stained with 4’,6-diamidino-2-phenylindole (DAPI) to mark nuclei. Arrows in GPC3 “Merged” indicate weak colocalization signals. (B) Expression patterns of glycosaminoglycans other than R-10G-reactive keratan sulfate in EC tissues. Serial EC sections were immunostained with R-10G, CS-56 (which recognizes chondroitin sulfate), or 10E4 (which recognizes heparan sulfate), as indicated. Signals were visualized with 3,3’-diaminobenzidine (DAB) (brown), and tissues were counterstained with hematoxylin. Arrows indicate vessel walls of the tumor stroma, and arrowheads indicate EC tumor cells. Bars A and B = 50 µm. GPC3, glypican 3.

Figure 3.

Expression of R-10G-reactive antigen in testicular teratoma. Tissue sections were stained with H&E (left column) or immunostained for CD30 or R-10G, as indicated. Images in right panels are enlarged from boxes in panels in third column from left. Signals were visualized with 3,3’-diaminobenzidine (DAB) (brown), and tissues were counterstained with hematoxylin. Shown are sections representing three germ layers (neural tube for ectoderm, gastrointestinal gland for endoderm, and cartilage for mesoderm). Bar = 50 µm for columns 1–3; 12.5 µm for column 4. Abbreviation: H&E, hematoxylin and eosin.

Figure 4.

Scatter plot showing the percentage of CD30-positive (A) and R-10G-positive (B) cells in EC, non-seminomatous germ cell tumor other than EC (non-EC NSGCT) and seminoma. The percentage of CD30-positive or R-10G-positive cells was determined by dividing the number of immunolabeled cells by the total number of tumor cells. Median and interquartile ranges are overlaid on plots for each histological type. The dotted line at 5% indicates the threshold between positive and negative cases. Abbreviation: EC, embryonal carcinoma; NSGCT, non-seminomatous germ cell tumor. ***p<0.001.