P16 protein Expression in Nasopharyngeal Carcinoma Histologic Subtypes in Kano

Abstract

Background:

Nasopharyngeal carcinoma (NPC) is the most common head and neck malignancy with the highest incidence seen in South China, Southeast Asia, Northeast India and parts of North Africa. P16 (p16INK4a), a cyclin-dependent kinase, is essential for the regulation of cell proliferation and plays a key role in the activation of the apoptotic pathway. The objective of this study is to determine the prevalence and clinicopathological associations of p16 expression in NPC subtypes in Kano.

Methodology:

This was a retrospective study covering five years (2015 to 2019) of all NPC cases diagnosed at the Department of Histopathology, Aminu Kano Teaching Hospital, Kano (AKTH). The collected data included patients’ demographic details, clinical information, histological diagnosis, and p16 Immunohistochemical status.

Results:

NPC cases showed a bimodal age distribution, with a mean age of 40 years. The majority of patients were male, giving a male-to-female ratio of about 4.2:1. P16 positivity was observed in more than a third of the cases, more frequent in males than in females. Non-keratinizing nasopharyngeal carcinoma was the most common subtype and showed more p16-positivity.

Conclusion:

This study identifies p16 expression in a subset of nasopharyngeal carcinoma cases, with a higher frequency among males and a predominance in non-keratinizing nasopharyngeal carcinoma. These findings contribute to the understanding of p16 as a potential biomarker in NPC biology. While the prognostic implications remain uncertain, further molecular subtyping and prospective outcome studies are needed to clarify the role of p16 in risk stratification and therapeutic decision-making.

Introduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the nasopharyngeal epithelium, distinguished by marked geographic and racial variation in incidence. [1] In 2022, the International Agency for Research on Cancer reported 120,416 new cases worldwide, representing 0.6% of all cancers, alongside 73,476 deaths, with a clear male predominance. [2] Although NPC is rare in Western populations (<1 per 100,000), it is highly endemic in Southeast Asia and Southern China. NPC is hyper-endemic, where the lifetime risk may reach 1 in 40. [3,4] The age-incidence curve in these regions is typically monomodal, peaking between the fourth and sixth decades of life. [1] In contrast, African populations, particularly in Nigeria and the Maghreb, display a unique bimodal age distribution. [5,6] While the primary peak aligns with global trends, a significant secondary peak occurs among adolescents aged 11–18 years. [1-9] This early-onset phenotype suggests a distinct genetic susceptibility or earlier intense environmental exposure compared to the Asian model.

Nasopharyngeal carcinoma (NPC) is strongly associated with chronic Epstein–Barr virus (EBV) infection, which serves as a key biomarker for early detection and risk stratification. [10] While most cases are EBV-related, EBV-negative NPC highlights the role of cofactors such as HIV, HPV, and environmental exposures, including cigarette smoke and formaldehyde. Genetic susceptibility, particularly certain HLA haplotypes and immune-regulatory gene polymorphisms, further influences risk. [10,11]

P16, also known as cyclin-dependent kinase inhibitor 2A, is a tumor suppressor protein that hinders cell cycle progression by inhibiting the formation of cyclin-dependent kinase complexes. Its mechanism of action as a CDK inhibitor involves binding to and inactivating the cyclin D1-cyclin-dependent kinase 4/6 complex. These proteins phosphorylate the retinoblastoma (Rb) protein, leading to the unbinding of the E2F transcription factor. This inhibitory action of p16 on CDI- CDK4/6 Complex prevents the transcription of vital cell-cycle regulatory proteins and results in cell-cycle arrest. [12]

There are three mechanisms by which the p16 gene is silenced in neoplastic transformation, and these include loss of heterozygosity, methylation of its promoter gene sequence and point mutation. Its over-expression has been associated with improved progression-free survival (PFS). [13]

Nasopharyngeal carcinoma (NPC) is a major head and neck malignancy with marked geographic variation. Although p16 has been implicated in its pathogenesis, research on its role remains scarce, especially in Nigeria. This gap is particularly evident in Northern Nigeria, where documented NPC cases are few and data on p16 expression are extremely limited compared to the southern regions. Given p16’s importance as a tumor suppressor and potential biomarker, there is a pressing need to evaluate its immunohistochemical expression in NPC. Such studies would help fill existing epidemiological gaps, inform diagnostic and risk-stratification strategies, and contribute to global understanding of p16-NPC association in non-endemic African settings.

Materials and Methods

This was a retrospective study spanning five years, from January 2015 to December 2019, of all cases of nasopharyngeal carcinomas diagnosed in the Department of Histopathology at Aminu Kano Teaching Hospital (AKTH). All available cases within the study period that fulfilled the inclusion criteria were included, totaling 78 cases.

Inclusion Criteria

These records included requisition forms, histopathology reports, tissue blocks, and haematoxylin and eosin (H&E)-stained slides. The data retrieved included patient biodata, clinical information, histological diagnosis, and P16 Immunohistochemistry positivity.

Exclusion Criteria

Exclusion criteria included records with inadequate clinical information, missing archival slides and tissue blocks, and damaged tissue blocks.

Histopathological Diagnosis

The histopathologic diagnoses were made from old slides and classified according to the 2017/2022 WHO classification of nasopharyngeal carcinomas. [14] In cases where slides were missing, faded, or broken, new sections were cut from archived formalin-fixed paraffin-embedded (FFPE) tissue blocks and stained with haematoxylin and eosin (H&E).

Immunohistochemistry Procedure

The DB-Biotech immunohistochemistry staining protocol was used to assess P16 expression in tumour cells. Abcam Monoclonal p16 antibody (clone E6H4) was utilised, while the Abcam Expose Mouse and Rabbit Specific HRP/DAB 27 (ab80436) detection kit was employed to visualise the signals from the cells.

Sections were cut at 3 microns from tissue blocks and placed on 2 negatively charged APES-coated slides. These sections were then deparaffinized in xylene and rehydrated in decreasing concentrations of alcohol. The 2 slides for each case were then processed for immunohistochemistry staining using the P16 antibody. Antigen retrieval was done by immersion in 10mM citrate buffer followed by heating in a pressure cooker for 30 minutes.

Endogenous peroxidase activity was blocked by treating with hydrogen peroxide for 10 minutes before applying primary antibodies for 45 minutes. After staining with primary antibodies, the signal was then amplified with a secondary antibody-linked horse radish peroxidase polymer, which was then visualised by applying a chromogenic substrate (DAB). Counterstaining was performed using hematoxylin, followed by dehydration and cover slipping. All steps were carried out at room temperature, and both negative controls (pancreas) and positive controls (High-grade Squamous Intra-epithelial Lesion) were included alongside the test samples to validate the staining.

Immunohistochemistry Interpretation

P16 protein expression was observed under a light microscope by an independent and experienced pathologist, and the staining intensity was graded using the German semi-quantitative scoring system by considering the staining intensity and extent as adopted in previous studies. [12] Here, each case was given a score based on the intensity of nuclear and cytoplasmic staining as:

Intensity	Score	Extent (score)
No staining	0	0: 0% stained cells
Weak intensity	1	1: 1 — 10 stained cells
Moderate intensity	2	2: 11 — 50 stained cells
Strong intensity	3	3: 51 — 80 stained cells
	4	4: >80-stained cells

The final immunoreactivity score was determined by multiplying the intensity score by the extent of positivity score of stained cells, giving a minimum score of 0 and a maximum score of 12. The cut-off point was set at 4. Expression index 0-3 was classified as negative (or “loss of P16 gene expression”), while 412 was classified as positive (with at least moderate staining in 11-50% tumor cells).

Data Analysis

The data obtained were analysed using SPSS version 23, and the results were presented in the form of photomicrographs, figures, and tables. Quantitative data were summarised and shown as means/medians, ranges, and frequency distribution tables, while qualitative data were summarised and presented using frequencies and percentages. Statistical comparisons between groups were done using the Chi-square test as appropriate. The histopathological data included histological subtypes in relation to age and sex, age and histologic subtypes, and sex and histologic subtypes. This set of data was correlated with immunohistochemistry staining (P16) data. Statistical significance was set at a 95% confidence interval with a p-value ≤ 0.05.

Ethical Consideration

Ethical approval was obtained from the Ethics and Scientific Committee of Aminu Kano Teaching Hospital, Kano, with approval number: NHREC/28/01/2020/AKTH/EC/2857.

Results

A total of 83 NPC cases were diagnosed during the five-year study period (2015 to 2019). Five (6%) cases were excluded for not meeting the inclusion criteria due to missing tissue blocks or exhausted tissues. Therefore, only 78 cases were analysed.

Age distribution: The age of the patients ranges from 12 to 81 years, with a mean of 40.8±13.5 years and a median of 35 years. The most common age group was 31 to 40 years (30.2%), while the least common was 10 to 19 years (7.7%). Nasopharyngeal carcinoma was more frequent in patients under 50 years (70%) than in older patients (30%).

Sex distribution: Males constituted 63 (77%) of the 78 cases, and females made up the remaining 15 (23%), resulting in a male-to-female ratio of 4.2:1. The mean age for males was 41.9 ± 13.5 years, while for females it was 36.3 ± 13.5 years. The peak age among males was in the 31–40-year age group, whereas for females it was in the 21–30-year age group. Among males, NPC cases also showed a bimodal age distribution, with a higher peak in the 3rd decade and a lower peak in the 5th decade (Figure 1).

Figure 1.

Age and Sex Distribution of Nasopharyngeal Carcinoma in Kano

P16 Expression: Twenty-one 21 (27%) of the 78 NPC cases were P16-positive, while the remaining 57 tumours (73%) were negative. Among males, 17 (27%) of the 63 cases were P16-positive, whereas only 4(27%) of the 15 female cases were p16-positive. P16 overexpression was most frequent among cases of non-keratinizing nasopharyngeal carcinoma, accounting for 18 (86%) of the 21 positive cases. (Table 1).

Table 1:

Sex distribution and P16 positivity by histologic subtypes of nasopharyngeal carcinoma (NPC)

Histologic Subtype	Male	Female	Total (M: F Ratio)	P16+	P16-
KNPC	20	7	27 (2.8:1)	3	24
NKNPC	43	8	13 (5.3:1)	18	33
BNPC	0	0	0 (0:0)	0	0
Total	63	15	78 (4.2:1)	21	57

Chi-square for sex distribution by subtype: 1.9095, p=0.38.

p-value for p16 pattern by subtype: 0.03.

Age Group and P16 expression: P16 positivity was observed between the age groups of 21-30, but also showed a steady decline in positivity with increasing age. There was no statistically significant difference in the pattern of P16 expression with age (p= 089) ( Table 1)

Morphologic subtype and P16 Expression: Of the 27 keratinising nasopharyngeal carcinomas, 11% were P16+. Of the 51 non-keratinising nasopharyngeal carcinomas, 35% were P16+. None of the cases was basaloid nasopharyngeal carcinoma.

Overall, the P16+ phenotype was more frequent among the NKNPC phenotypes and was least frequent among the KNPC. These differences in phenotypic expression were found to be statistically significant (p = 0.015). Table 1.

Key: KNPC = keratinizing nasopharyngeal carcinoma; NKNPC = Non-keratinizing nasopharyngeal carcinoma; BNPC = Basaloid nasopharyngeal carcinoma.

Discussion

A total of 78 cases of NPC were analysed in the five-year study period (2015 to 2019) in Aminu Kano Teaching Hospital.

Age and Sex: The patients ranged in age from 12 to 81 years, with a mean of 40.8 ± 13.5 years and a median of 35 years. Comparable age patterns have been reported across other Nigerian regions and parts of Africa, including Ibadan, Sokoto, Jos, Maiduguri, and Nairobi, Kenya, where cases span 11 to 85 years and mean ages fall between 40 and 42. [15 – 19]

Our study demonstrated a clear male predominance in nasopharyngeal carcinoma (M: F ratio = 4.2:1), which is higher than figures reported from Northern Nigeria (2.1:1) but closely aligns with data from the South (3.5:1). This disparity may reflect regional differences in risk factors or genetic predisposition across Nigeria. [15-17,19,21] Notably, males reached peak incidence about a decade earlier than females, suggesting sex-specific risk patterns. A bimodal age distribution was also observed, with peaks in the third and fifth decades, consistent with reports from Ibadan, Lagos, Ghana, and Ethiopia, thereby reinforcing the reproducibility of this pattern across African populations.

Histologic Subtypes: In our study, non-keratinising nasopharyngeal carcinoma was the predominant histologic subtype (65%), followed by keratinizing and non-keratinizing nasopharyngeal carcinomas (35%). None of the cases was diagnosed as basaloid nasopharyngeal carcinoma. This pattern is comparable to findings from Ilorin, Nigeria, where Alabi et al. reported 71% [21] and Switzerland, where Sidler et al. documented 70%. [22] Several international studies identify non-keratinizing nasopharyngeal carcinoma as the most frequent subtype (>90%), [15-17,22,23]

Age Group and p16 expression: In this study, p16 positivity was most frequent in the 21–30-year age group and declined steadily with increasing age. Overexpression was therefore more common in younger patients than in older ones, suggesting the potential for better outcomes among younger individuals. However, no statistically significant relationship was observed between age and the expression patterns of p16 (p = 0.89).

Morphologic subtype and P16 Phenotypes: Of the 78 NPC cases evaluated in the index study, only 21(27%) were p16-positive. This is lower than 68% reported by park et al [24], the 35% frequency reported by Hwang et al [25] in Taiwan and Jiang et al [13] in Texas, USA, but higher than the 11% reported by Shimizu et al [26] in Japan. Furthermore, this study found that p16-overexpression was more frequent among cases of non-keratinising nasopharyngeal carcinoma, accounting for 18 (86%) of the 21 positive cases, and this was much higher than the 18% reported in Japan by Shibosawa et al. [27]. The high number seen in this study could be explained by the predominance of the non-keratinising variant.

Conclusion

This study demonstrates that p16 expression is present in a subset of nasopharyngeal carcinoma cases in Kano, with variation across histological subtypes. However, given the absence of outcome data and molecular characterization, the clinical and prognostic significance of p16 in this population remains uncertain. Further studies incorporating EBV status, HPV analysis, and survival outcomes are required.

Limitations of the Study

The study has several limitations. First, the relatively small sample size and single-centre design may restrict the generalizability of the findings to wider Nigerian or African populations. In addition, patients’ outcomes such as survival and treatment response were not evaluated, limiting the ability to establish the prognostic significance of p16 expression. The cross-sectional nature of the study also prevents conclusions about causality or temporal changes in biomarker expression. Furthermore, reliance on immunohistochemistry without complementary molecular techniques such as polymerase chain reaction (PCR) may have constrained the depth of characterization. Regional representation was uneven, with limited data from Northern Nigeria. Finally, environmental and lifestyle risk factors, including smoking and occupational exposures, were not assessed, which restricts the interpretation of co-factor influence.