European Journal of Cardiovascular Medicine

Oral squamous cell carcinoma (OSCC) represents more than 90% of all oral malignancies and ranks among the most prevalent cancers globally, particularly in developing countries (1). It is often preceded by potentially malignant disorders (PMDs), with oral epithelial dysplasia (OED) being the most significant histopathological indicator of malignant transformation risk (2). The early identification and monitoring of dysplastic lesions are crucial to improving the prognosis and survival rates in affected individuals.

Among the various molecular markers studied for their role in carcinogenesis, p16^INK4a^ has gained prominence due to its function as a tumor suppressor gene. p16 regulates the cell cycle by inhibiting cyclin-dependent kinases, thereby controlling the G1-S phase transition (3). Aberrant expression of p16 has been linked to disruptions in the retinoblastoma (Rb) pathway, a common feature in several cancers, including OSCC (4).

Interestingly, overexpression of p16 in oral lesions has been associated not only with oncogenic human papillomavirus (HPV) infection but also with cellular senescence and dysregulation of tumor suppressor pathways in HPV-negative cases (5,6). Therefore, p16 immunohistochemistry is increasingly being explored for its diagnostic and prognostic implications in both dysplastic and malignant oral lesions.

This study aims to evaluate and compare the immunohistochemical expression of p16 in different grades of OED and OSCC to determine its potential utility as a biomarker in assessing disease progression.

This cross-sectional, observational study was conducted on formalin-fixed, paraffin-embedded tissue samples obtained from archived cases diagnosed histopathologically as oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). A total of 60 cases were selected and categorized into two groups: Group I included 30 cases of OED (10 mild, 10 moderate, and 10 severe), while Group II comprised 30 cases of OSCC (10 well-differentiated, 10 moderately differentiated, and 10 poorly differentiated).

All selected tissue blocks were sectioned at 4 µm thickness and mounted on poly-L-lysine–coated slides. Hematoxylin and eosin staining was performed initially to confirm histopathological grading. Subsequently, immunohistochemical staining was carried out using monoclonal antibodies against p16 (clone-specific, ready-to-use). The staining procedure included deparaffinization, rehydration, antigen retrieval in citrate buffer (pH 6.0), and incubation with the primary antibody, followed by detection using a polymer-based detection system and DAB chromogen. Slides were counterstained with hematoxylin, dehydrated, and mounted.

Evaluation of p16 expression was performed under a light microscope by two independent observers. Positivity was determined based on both nuclear and cytoplasmic staining. A semi-quantitative scoring system was used, considering both the intensity of staining (graded 0–3) and the percentage of positive cells (graded 0–4). The final immunoreactivity score (IRS) was calculated by multiplying the intensity and percentage scores, yielding a value between 0 and 12.

Data were compiled and analyzed statistically using SPSS software version 22.0. Mean values and standard deviations were calculated, and the association between p16 expression and histological grades was evaluated using the Chi-square test and one-way ANOVA. A p-value less than 0.05 was considered statistically significant.

A total of 60 cases were analyzed for p16 expression using immunohistochemistry. These included 30 cases of oral epithelial dysplasia (OED) and 30 cases of oral squamous cell carcinoma (OSCC), equally distributed across histological grades.

p16 Expression in Oral Epithelial Dysplasia

Among the OED cases, p16 positivity increased with the severity of dysplasia. Mild dysplasia showed weak expression in 4 cases (40%), moderate dysplasia showed moderate positivity in 6 cases (60%), while strong expression was seen in 8 cases (80%) of severe dysplasia. The mean immunoreactivity score (IRS) values for mild, moderate, and severe dysplasia were 2.3 ± 1.2, 4.5 ± 1.4, and 6.8 ± 1.7 respectively, showing a significant upward trend (p < 0.05) (Table 1).

Table 1. p16 Expression in Different Grades of Oral Epithelial Dysplasia

p16 Expression in Oral Squamous Cell Carcinoma

In the OSCC group, an increasing trend of p16 expression was observed with advancing histological grade. Well-differentiated OSCC showed positive staining in 5 cases (50%), moderately differentiated in 7 cases (70%), and poorly differentiated in 9 cases (90%). The mean IRS for well, moderately, and poorly differentiated OSCC was 3.2 ± 1.5, 5.7 ± 1.6, and 8.1 ± 1.9 respectively, indicating a statistically significant correlation (p < 0.01) (Table 2).

Table 2. p16 Expression in Different Grades of Oral Squamous Cell Carcinoma

Comparative Analysis

When comparing the p16 expression between OED and OSCC groups, a progressive increase in IRS values was observed with higher grades of dysplasia and carcinoma (p < 0.01). This suggests a potential link between increased p16 expression and malignant transformation (Table 3).

Table 3. Comparative Mean IRS between OED and OSCC

As shown in Tables 1–3, there was a consistent trend of increased p16 expression associated with histopathological severity in both OED and OSCC groups.

The present study evaluated the immunohistochemical expression of p16 in various grades of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), revealing a progressive increase in p16 expression with advancing histological grade in both groups. These findings suggest that p16 may serve as a useful biomarker in predicting malignant transformation and tumor aggressiveness.

p16^INK4a^ is a tumor suppressor protein encoded by the CDKN2A gene, which plays a key role in regulating the cell cycle by inhibiting cyclin-dependent kinases 4 and 6 (1). Loss of function or abnormal overexpression of p16 is frequently observed in malignancies, including those of the oral cavity (2,3). In our study, mild dysplasia showed relatively low p16 expression, while severe dysplasia and poorly differentiated OSCC demonstrated stronger positivity, indicating a correlation between p16 expression and the degree of cellular atypia.

These results are in agreement with studies by Gonzalez-Moles et al. and Hall et al., which reported increased p16 immunoreactivity in high-grade dysplastic lesions and poorly differentiated carcinomas (4,5). Similarly, other studies have found that p16 expression may serve as an early molecular event in oral carcinogenesis and may precede morphological changes detectable by conventional histopathology (6,7).

The increasing trend of p16 expression in OSCC, especially in poorly differentiated tumors, may reflect a compensatory mechanism to counteract unchecked cell proliferation due to retinoblastoma (Rb) pathway disruption (8). This paradoxical overexpression of p16 has also been observed in HPV-negative oral cancers, highlighting the complexity of its role in tumor biology (9,10). In contrast, some studies have reported reduced p16 expression in OSCC, possibly due to promoter hypermethylation or gene deletion, indicating that p16 dysregulation can occur through multiple pathways (11,12).

It is noteworthy that p16 has been widely used as a surrogate marker for high-risk HPV in oropharyngeal squamous cell carcinoma (13). However, its diagnostic role in HPV-independent oral cancers remains controversial and is often context-dependent (14). While this study did not evaluate HPV status, the increasing expression of p16 across non-HPV-related lesions reinforces its relevance as a marker of tumor progression rather than HPV association alone.

Furthermore, the immunoreactivity score (IRS) used in this study provided a reliable semi-quantitative method for evaluating p16 staining, allowing for better correlation with histopathological grading. Similar scoring methods have been employed effectively in previous research to standardize immunohistochemical interpretations (15).

Despite these findings, the study has limitations, including the relatively small sample size and lack of correlation with clinical outcomes or HPV status. Future studies incorporating larger cohorts, HPV DNA analysis, and follow-up data are needed to validate the prognostic significance of p16 in oral lesions.

In conclusion, the study supports the role of p16 as a potential biomarker in assessing the progression from OED to OSCC. Its increased expression in higher grades of dysplasia and carcinoma underscores its utility in risk assessment and early intervention strategies.

1. Balcere A, Sperga M, Čēma I, Lauskis G, Zolovs M, Rone Kupfere M, Krūmiņa A. Expression of p53, p63, p16, Ki67, Cyclin D, Bcl-2, and CD31 markers in actinic keratosis, in situ squamous cell carcinoma and normal sun-exposed skin of elderly patients. J Clin Med. 2023;12(23):7291. doi:10.3390/jcm12237291. PMID: 38068343.
2. Hodges A, Smoller BR. Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin. Mod Pathol. 2002;15(11):1121–5. doi:10.1097/01.MP.0000032536.48264.D1. PMID: 12429789.
3. Einspahr JG, Xu MJ, Warneke J, Saboda K, Ranger-Moore J, Bozzo P, et al. Reproducibility and expression of skin biomarkers in sun-damaged skin and actinic keratoses. Cancer Epidemiol Biomarkers Prev. 2006;15(10):1841–8. doi:10.1158/1055-9965.EPI-06-0378. PMID: 17021352.
4. Nindl I, Gottschling M, Krawtchenko N, Lehmann MD, Röwert-Huber J, Eberle J, et al. Low prevalence of p53, p16(INK4a) and Ha-ras tumour-specific mutations in low-graded actinic keratosis. Br J Dermatol. 2007;156 Suppl 3:34–9. doi:10.1111/j.1365-2133.2007.07857.x. PMID: 17488404.
5. Talghini S, Halimi M, Baybordi H. Expression of P27, Ki67 and P53 in squamous cell carcinoma, actinic keratosis and Bowen disease. Pak J Biol Sci. 2009;12(12):929–33. doi:10.3923/pjbs.2009.929.933. PMID: 19777788.
6. Brasanac D, Boricic I, Todorovic V, Tomanovic N, Radojevic S. Cyclin A and beta-catenin expression in actinic keratosis, Bowen's disease and invasive squamous cell carcinoma of the skin. Br J Dermatol. 2005;153(6):1166–75. doi:10.1111/j.1365-2133.2005.06898.x. PMID: 16307653.
7. Wrone DA, Yoo S, Chipps LK, Moy RL. The expression of p63 in actinic keratoses, seborrheic keratosis, and cutaneous squamous cell carcinomas. Dermatol Surg. 2004;30(10):1299–302. doi:10.1111/j.1524-4725.2004.30403.x. PMID: 15458526.
8. Sakiz D, Turkmenoglu TT, Kabukcuoglu F. The expression of p63 and p53 in keratoacanthoma and intraepidermal and invasive neoplasms of the skin. Pathol Res Pract. 2009;205(9):589–94. doi:10.1016/j.prp.2009.01.010. PMID: 19577853.
9. Neto PD, Alchorne M, Michalany N, Abreu M, Borra R. Reduced P53 staining in actinic keratosis is associated with squamous cell carcinoma: a preliminary study. Indian J Dermatol. 2013;58(4):325. doi:10.4103/0019-5154.113935. PMID: 23919015.
10. Mai KT, Alhalouly T, Landry D, Stinson WA, Perkins DG, Yazdi HM. Pagetoid variant of actinic keratosis with or without squamous cell carcinoma of sun-exposed skin: a lesion simulating extramammary Paget's disease. Histopathology. 2002;41(4):331–6. doi:10.1046/j.1365-2559.2002.01523.x. PMID: 12383215.
11. Kanellou P, Zaravinos A, Zioga M, Stratigos A, Baritaki S, Soufla G, et al. Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis. Cancer Lett. 2008;264(1):145–61. doi:10.1016/j.canlet.2008.01.042. PMID: 18331779.
12. Conscience I, Jovenin N, Coissard C, Lorenzato M, Durlach A, Grange F, et al. P16 is overexpressed in cutaneous carcinomas located on sun-exposed areas. Eur J Dermatol. 2006;16(5):518–22. PMID: 17101472.
13. Cockerell CJ. Histopathology of incipient intraepidermal squamous cell carcinoma ("actinic keratosis"). J Am Acad Dermatol. 2000;42(1 Pt 2):11–7. doi:10.1067/mjd.2000.103344. PMID: 10607351.
14. Nasiri S, Azhari V, Bidari-Zerehpoosh F, Asadi-Kani Z, Talebi A. The diagnostic value of p63, p16, and p53 immunohistochemistry in distinguishing seborrheic keratosis, actinic keratosis, and Bowen's disease. Dermatol Ther. 2021;34(2):e14817. doi:10.1111/dth.14817. PMID: 33497503.
15. Bito T, Ueda M, Ahmed NU, Nagano T, Ichihashi M. Cyclin D and retinoblastoma gene product expression in actinic keratosis and cutaneous squamous cell carcinoma in relation to p53 expression. J Cutan Pathol. 1995;22(5):427–34. doi:10.1111/j.1600-0560.1995.tb0