BMI-1 expression increases in oral leukoplakias and correlates with cell proliferation

Abstract Oral leukoplakia (OL) is a white lesion of an indeterminate risk not related to any excluded (other) known diseases or disorders that carry no increased risk for cancer. Many biological markers have been used in an attempt to predict malignant transformation; however, no reliable markers have been established so far. Objective To evaluate cell proliferation and immortalization in OL, comparing non-dysplastic (Non-dys OL) and dysplastic OL (Dys OL). Methodology This is a cross-sectional observational study. Paraffin-embedded tissue blocks of 28 specimens of Non-dys OL, 33 of Dys OL, 9 of normal oral mucosa (NOM), 17 of inflammatory hyperplasia (IH), and 19 of oral squamous cell carcinomas (OSCC) were stained for Ki-67 and BMI-1 using immunohistochemistry. Results A gradual increase in BMI-1 and K-i67 expression was found in oral carcinogenesis. The immunolabeling for those markers was higher in OSCC when compared with the other groups (Kruskal-Wallis, p<0.05). Ki-67 expression percentage was higher in OL and in IH when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). Increased expression of BMI-1 was also observed in OL when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). No differences were observed in expression of both markers when non-dysplastic and dysplastic leukoplakias were compared. A significant positive correlation between Ki-67 and BMI-1 was found (Spearman correlation coefficient, R=0.26, p=0.01). High-grade epithelial dysplasia was associated with malignant transformation (Chi-squared, p=0.03). Conclusions These findings indicate that BMI-1 expression increases in early oral carcinogenesis and is possibly associated with the occurrence of dysplastic changes. Furthermore, our findings indicate that both Ki-67 and BMI-1 are directly correlated and play a role in initiation and progression of OSCC.


Introduction
Oral leukoplakia (OL) is a lesion with a risk of malignant transformation ranging from 0.13% to 17.5%. 1 Histopathologically, OL is characterized by a variety of epithelial changes, including dysplasia.
The presence of epithelial dysplasia is considered the most important predictive factor for OL prognosis. [1][2][3] Although many biological markers have been explored, no reliable ones have yet been established for predicting malignant transformation of OL, thus necessitating additional studies to increase our knowledge of the biological processes underlying OL and oral carcinogenesis. 4 Ki-67 is a nuclear protein expressed in the G1, S, G2, and M phases of the cell cycle, and its expression reflects the total growth fraction in tissues. 5 Ki-67 expression correlates with the severity of epithelial dysplasia and histological grading of oral squamous cell carcinoma (OSCC). 6,7 Another protein named BMI-1, which mediates gene silencing by regulating chromatin structure, 8,9 plays a central role in cell cycle regulation and cell immortalization, as well as cell senescence and epithelial-mesenchymal transition (EMT). 10,11 BMI-1 is associated with the initiation and progression of various tumors, including oropharyngeal, 10 nasopharyngeal, 11 and prostate 12 cancers. Furthermore, increased BMI-1 expression was found in bronchial premalignant lesions as well as squamous cell carcinoma (SCC), indicating that its expression in neoplastic cells may be an early event in lung carcinogenesis. 13 BMI-1 is also overexpressed in OSCC cells when compared with normal oral mucosa cells and has been presumed to influence proliferation and immortalization of epithelial cells in oral carcinogenesis. 14 This study aimed to evaluate cell proliferation and immortalization in OL by studying Ki-67 and BMI-1 expression in non-dysplastic (Non-dysOL) and dysplastic (DysOL) cases.

Histopathological analysis
All selected samples were fixed in 10% neutral formalin and embedded in paraffin. Five-micrometer H&E (hematoxylin and eosin) stained sections were blindly reviewed by two pathologists (V.C.C. and I.P.K.). The epithelial dysplasia in OL samples was graded according to the criteria and definition proposed by Reibel, et al. 15 (2017) and Kujan, et al. 16 (2006). A consensus was reached in cases that were graded differently by the two pathologists.

Statistical analysis
The Kruskal-Wallis test, followed by Dunn's test, was used for multiple comparisons and the Chisquare test was used for comparisons of scores distribution among the groups. Student t/ANOVA and Mann-Whitney/Kruskal Wallis tests were used to assess the influence of OL characteristics on Ki-67 and BMI-1 expression, respectively. Spearman's correlation analysis was conducted to determine the relationship between BMI-1 and Ki-67 expression.
The SPSS Statistics software, 18.0 Version, was used for statistical analysis, and p value threshold used was 5%. Table 1 shows the demographic and clinical characteristics of the OL and OSCC patients. The cases were divided into two groups, namely "tongue or floor of the mouth" and "other sites", according to the lesion site. 20 The tongue or floor of the mouth
Ki-67 immunolabeling data is shown in Figure 2.   Association of Ki-67 and BMI-1 immunolabeling w i t h O L c l i n i c a l c h a ra c t e r i s t i c s a n d histopathological changes (Table 4) Higher Ki-67 and BMI-1 expression was observed in lesions in the tongue and floor of the mouth, epithelial dysplasia, OSCC development and poor clinical evolution; however, the associations were not statistically significant.   To the best of our knowledge, this study is the first to assess Ki-67 and BMI-1 expression in OL. 19 As expected, the expression of these markers was increased in OL when compared with NOM.

BMI-1 is involved in the transcriptional repression
of Hox genes, thus affecting stem cell self-renewal, embryonic development, and proliferation. 24,25 Our results show that BMI-1 expression increases during carcinogenesis. However, BMI-1 immunolabeling analysis showed no differences between OL and IH.
OL presented a higher and statistical positivity of BMI-1 when compared with NOM. Our results agrees with those of Kang, et al. 14 (2007)

Conclusion
The findings of this study indicate that BMI-1 expression increases in early oral carcinogenesis and that it may be associated with the occurrence of dysplastic changes. Furthermore, our findings indicate that both Ki-67 and BMI-1 are directly correlated and possibly play a role in initiation and progression of OSCC.