Significance of P53-Binding Protein 1 as a Novel Molecular Histological Marker for Hypopharyngeal Squamous Neoplasms

Abstract

The DNA damage response protein p53-binding protein 1 (53BP1) accumulates and forms foci at double-strand DNA breaks, indicating the extent of DNA instability. However, the potential role of 53BP1 as a molecular biomarker for hypopharyngeal squamous cell carcinoma (HPSCC) diagnosis remains unknown. Here, we evaluated the potential of immunofluorescence-based analysis of 53BP1 expression to differentiate the histology of hypopharyngeal neoplasms. A total of 125 lesions from 39 surgically or endoscopically resected specimens from patients with HPSCC was histologically evaluated. 53BP1 expression in the nucleus was examined using immunofluorescence. The number of 53BP1 nuclear foci increased with the progression from non-tumorous to low-grade dysplasia, high-grade dysplasia, and squamous cell carcinoma. Unstable 53BP1 expression served as an independent factor for distinguishing lesions that required intervention. Colocalization of 53BP1 foci in proliferating cells, as assessed by Ki67, was increased in tumors ≥ 1000 µm in depth compared to those <1000 µm in depth at the tumor surface. Hence, the expression patterns of nuclear 53BP1 foci were associated with the progression of hypopharyngeal neoplasms. These findings suggest that 53BP1 could serve as an ancillary marker to support histological diagnosis and predict the factors that influence prognosis in patients with HPSCC.

Keywords: DNA double-strand break; genomic instability; hypopharyngeal squamous cell carcinoma; p53-binding protein 1.

Figure 1

Representative Hematoxylin–Eosin staining of squamous epithelium employed in our study (original magnification: ×40): (a) non-tumor, (b) low-grade dysplasia, (c) high-grade dysplasia, and (d) squamous cell carcinoma. Scale bar = 50 µm. (e) Tumor thickness indicates the distance from the tumor surface to its deepest point.

Figure 2

Representative images of nuclear expression patterns of 53BP1. Stable (a) zero, (b) one, or (c) two foci; (d) unstable; (≥3 nuclear foci); (e) large foci (≥1 μm in diameter); (f) colocalization of 53BP1 foci and Ki67. 53BP1 (green), Ki67 (red), and DAPI (blue).

Figure 3

Expression patterns of 53BP1 nuclear foci during the progression of squamous neoplasms in the hypopharynx. (a) Representative image of double-label immunofluorescence staining of 53BP1 and Ki-67 in the hypopharynx. LD, low-grade dysplasia; HD: high-grade dysplasia; SCC: squamous cell carcinoma. 53BP1 (green), Ki67 (red), and DAPI (blue). Original magnification: ×400. (b) Quantification of stepwise increase in the following nuclear expression patterns: unstable expression, large foci (LF), and colocalization of 53BP1/Ki67 during carcinogenesis. p < 0.0001, Jonckheere–Terpstra test. Data were calculated as a percentage (%) of the total number of epithelial cells and are presented as mean ± standard error of the mean (SEM). LD, low-grade dysplasia; HD: high-grade dysplasia; SCC: squamous cell carcinoma. LF: large nuclear foci, 53BP1/Ki67: colocalization of 53BP1 and Ki67.

Figure 4

Association between 53BP1 expression pattern and T factor of TNM classification. LF: large nuclear foci; 53BP1/Ki67: colocalization of 53BP1 and Ki67. Data are expressed as mean ± SEM; NS, not significant.

Figure 5

Expression pattern of 53BP1 nuclear foci according to tumor depth. (a) Representative images of 53BP1 (green) and Ki67 (red) staining in tumors with a thickness <1000 µm and ≥1000 µm at the surface of squamous cell carcinoma (SCC) visualized by 53BP1 (green) and Ki67 (red) staining with nuclei stained with DAPI (blue). Original magnification: ×400. (b) Quantification of unstable, large foci (LF), and colocalization of 53BP1 and Ki67 (53BP1/Ki67) at the surface of HPSCC with a tumor thickness <1000 µm and ≥1000 µm. Data are expressed as mean ± SEM; NS, not significant; * p < 0.05.

Figure 6

Comparison of 53BP1 expression patterns between the tumor surface and invasive front. LF: large nuclear foci; 53BP1/Ki67: colocalization of 53BP1 and Ki67. Data were calculated as a percentage (%) of the total number of epithelial cells and are presented as mean ± SEM; NS, not significant, * p < 0.05.

Figure 7

Expression pattern of 53BP1 nuclear foci according to lymphatic invasion. (a) Quantification of 53BP1 expression at the tumor surface in the presence or absence of lymphatic invasion (Ly+/Ly−). Data are expressed as mean ± SEM. (b) Quantification of 53BP1 and Ki67 at the tumor surface in the presence or absence of vascular invasion (v+/v−). LF: large nuclear foci; 53BP1/Ki67: colocalization of 53BP1 and Ki67. Data are expressed as mean ± SEM; NS, not significant.

Figure 8

Expression pattern of 53BP1 nuclear foci according to venous invasion. (a) Quantification of 53BP1 expression patterns at the tumor invasion area, in the presence or absence of lymphatic invasion (Ly+/Ly−). LF: large nuclear foci; 53BP1/Ki67: colocalization of 53BP1 and Ki67. Data are expressed as mean ± SEM. (b) Quantification of 53BP1 and Ki67 at the tumor invasion area in the presence or absence of vascular invasion (v+/v−). Data are expressed as mean ± SEM; NS, not significant; * p < 0.05, ** p < 0.001.