Mechanisms Underlying Negative p53 Immunocytochemistry in Oral Cytology: TP53 mRNA Expression and Sampling...

Background: Reliable molecular biomarkers that complement cytomorphological assessment for early identification of high-risk oral epithelial lesions remain limited. Although p53 immunocytochemistry (ICC) is used as a surrogate marker of TP53 abnormalities, p53 protein expression does not always reflect TP53 transcriptional activity. This study investigated the relationships between p53 ICC, TP53 mRNA expression, and p53 immunohistochemical (IHC) staining patterns in the categories of oral high-grade squamous intraepithelial lesions (OHSILs) and oral squamous cell carcinoma (SCC). Methods: We classified 504 liquid-based cytology (LBC) specimens according to the Bethesda System for Reporting Oral Cytology: negative for an intraepithelial lesion or malignancy (NILM) (n = 394), oral low-grade squamous intraepithelial lesion (OLSIL) (n = 72), OHSIL (n = 18), and SCC (n = 20). p53 ICC was performed on cytological specimens, TP53 mRNA expression was quantified by quantitative real-time PCR using residual LBC samples, and corresponding biopsy specimens were evaluated for p53 IHC staining patterns. Associations between p53 ICC and clinicopathological variables were also analyzed. Results: The p53 labeling index significantly increased with cytological severity, and TP53 mRNA expression was significantly higher in OHSIL and SCC than in NILM and OLSIL. In OHSIL and SCC, lesions showing a null-type p53 IHC staining pattern showed significantly lower TP53 mRNA expression than lesions with non-null staining patterns. No significant associations between p53 ICC and clinicopathological variables were observed in OHSIL. In contrast, p53 ICC positivity in SCC was significantly associated with tumor size, depth of invasion, and p53 IHC staining patterns. Conclusions: Negative p53 ICC may result from sampling limitations or null-type TP53 expression. TP53 mRNA analysis provides complementary molecular information that distinguishes false-negative p53 ICC caused by sampling limitations from biologically reduced TP53 expression. Integrating cytomorphology, p53 ICC, histopathology, and TP53 mRNA analysis may improve diagnostic accuracy and risk stratification of high-risk oral epithelial lesions.