Expression and clinical significance of PD‑1 in hepatocellular carcinoma tissues detected by a novel mouse anti-human PD‑1 monoclonal antibody

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignancies and causes of death worldwide. Research investigating novel therapeutic strategies for the treatment of HCC is urgently required. Monoclonal antibodies (mAbs) that target the programmed cell death‑1 (PD‑1/PDCD1)/programmed death-ligand 1 (PD-L1) immune checkpoint have demonstrated substantial clinical benefit for a variety of solid tumors; however, these mAbs have not been well studied in HCC. In the present study, Sp2/0-Ag14 myeloma cells and spleen cells derived from BALB/c mice immunized with the recombinant human PD‑1/PDCD1 protein were fused for the production of novel antibodies. The 9E11 mAb, which exhibited the highest specificity for PD‑1 in HCC tissues in western blot and immunohistochemical staining analyses, was used to investigate the clinical significance of PD‑1 expression in HCC tissues from 77 cases, which were collected and examined histologically. Overexpression of PD‑1 was identified in peritumoral tissues, primarily in the liver portal region. Importantly, by analyzing the clinical data from 77 HCC patients, the expression of PD‑1 was observed to be significantly correlated with larger tumor size (>5 cm) and poorly differentiated tumors. In addition, PD‑1 expression was moderately correlated with venous thrombosis, but not correlated with patient sex or age, liver cirrhosis, hepatitis B, tumor, node and metastasis (TNM) stage or tumor location. The results of the present study suggest that high-level PD‑1 expression may be an important factor associated with the immune checkpoint pathway in HCC. The results suggest that PD‑1 serves an important role in tumor immune evasion and may be a valuable immunodiagnostic marker. In addition, PD‑1 may serve as a therapeutic target for patients presenting with poorly differentiated HCC, thus indicating the potential application of a PD‑1 inhibitor for the treatment of HCC patients.

Figure 1

Specificity of mouse anti-human PD-1 mAbs by western blot analysis. The recombinant PD-1 protein (40 kDa) was subjected to SDS-PAGE and blotted with the 1C4, 1H8, 2C4, 2H7, 3E5, 5B2, 5H7, 7C9, 7E5, 7G12, 9A5, 9B4, 9E11 and B1C4 mAb clones. PD-1, programmed cell death-1; mAb, monoclonal antibody.

Figure 2

Specificity of mouse anti-human PD-1 mAbs by immunohistochemical staining. (A) Representative immunohistochemical staining images of hepatocellular carcinoma tissue sections (magnification, ×400). (B) Mean optical densities of the positively stained specimens. ^**P<0.001 vs. UMAB199. PD-1, programmed cell death-1.

Figure 3

Location of PD-1⁺ T lymphocytes in hepatocellular carcinoma tissues. Representative microscope images showing the (A) tumor interior, (B) nodular margin of the tumor, (C) tumor-normal adjacent tissue junction and the (D) liver tissue surrounding the tumor. Black arrows indicate tumor tissues, and red arrows indicate peritumoral tissues (magnification, ×40). PD-1, programmed cell death-1.

Figure 4

Location of PD-1⁺ T lymphocytes in different hepatocellular carcinoma tissue regions as detected by immunohistochemical staining. Representative microscope images of the (A) nodular margin of the tumor, (B) liver tissue surrounding the tumor, (C) tumor-normal adjacent tissue junction and (D) tumor interior (magnification, ×100). PD-1, programmed cell death-1.

Figure 5

Expression of PD-1 in hepatocellular carcinoma tissues and the clinicopathological characteristics of patients based on the number of PD-1⁺ lymphocytes per field of view. The number of PD-1⁺ T lymphocytes in patients grouped by (A) degree of tumor differentiation, (B) tumor size and (C) the presence of vascular tumor emboli. The data were collected at ×200 magnification by two different investigators. Each dot represents the number of PD-1⁺ lymphocytes per field of view in one tissue section from each patient. Black bars indicate the mean ± standard deviation. ^**P<0.01, as indicated. PD-1, programmed cell death-1; ns, not significant.