Oral potentially malignant disorders (OPMDs) are a heterogeneous group of oral mucosal diseases that are characterized by a heightened risk of oral squamous cell carcinoma (OSCC).[1] The disorders involve oral leukoplakia, oral erythroplakia, oral submucous fibrosis, proliferative verrucous leukoplakia, and oral lichen planus.[2] They have a wide spectrum of clinical and histopathological features, and predicting malignant transformation is a significant problem in oral pathology.[3] Oral cancer continues to be a significant disease around the world, with an estimated 389,846 new cases of lip and oral cavity cancer in 2022.[4] In addition,

a recent systematic review and meta-analysis estimated the prevalence of histopathologically confirmed OPMDs at about 4.67% globally, with a relatively higher prevalence of 5.44% in Asia.[3] These statistics highlight the need for the early detection and effective risk assessment of potentially malignant oral lesions, especially in high-risk populations who have high exposure to tobacco, betel quid, and other carcinogenic substances.

Histopathological examination, especially evaluation of epithelial dysplasia, is the primary method to assess OPMDs' malignant potential.[5] These traditional grading systems, however, have some drawbacks in terms of interobserver variability and that they are not as precise in predicting the biological behaviour of individual lesions.[6] Importantly, some lesions with minimal dysplasia may develop into malignancy, while some lesions with significant dysplasia may persist for a long time.[2] The malignant transformation rates of oral leukoplakia, oral submucous fibrosis, and proliferative verrucous leukoplakia were reported to be 6.64%, 6.00%, and 43.87%, respectively, in a recent review.[7] This diversity indicates the necessity of further molecular and immunohistochemical markers to support the traditional histopathological evaluation and aid in identifying high-risk lesions.

Ki-67 is a nuclear proliferation-associated protein that is expressed during the active phase of the cell cycle and is generally used to assess cellular proliferative activity.[8] Its expression pattern in normal oral epithelium is primarily found in the basal and parabasal layers, and its expression pattern and spreading into the suprabasal epithelial compartments may suggest abnormal proliferative activity and progressive epithelial dysregulation.[9] This has led to a great interest in the use of Ki-67 immunohistochemistry as a marker of epithelial dysplasia and malignant progression.[10] In an immunohistochemical study of 61 cases of oral leukoplakia, Miguel et al. (2023) showed a significant increase in the expression of Ki-67 in high-risk epithelial dysplasia compared to low-risk epithelial dysplasia (p=0.013).[11]

In contrast, p53 is a tumour suppressor protein that regulates progression through the cell cycle, DNA repair, cellular senescence, and apoptosis in response to genomic damage.[12] Genetically damaged epithelial cells may survive and acquire further molecular defects due to changes in the TP53 pathway.[13] Therefore, p53 overexpression is believed to be a possible early molecular change in oral carcinogenesis, and immunohistochemical detection of p53 is regarded as a potential marker of early changes involved in the oral carcinogenesis process.[14] In a systematic review and meta-analysis of 24 studies and 1,210 OPMD patients, Ramos-García et al. (2022) found that there is a 1.88 increase in malignant transformation in patients with OPMD when p53 was overexpressed (95% CI: 1.39–2.56).[15]

Although more and more evidence has emerged indicating that Ki-67 and p53 have individual diagnostic and prognostic significance, their relative ability to predict malignant transformation in OPMDs remains of clinical significance. Increased levels of the Ki-67 biomarker reflect increased cellular proliferation, while p53 abnormalities reflect disruptions of important tumor-suppressive mechanisms. The evaluation of both markers may thus give a more comprehensive picture of the molecular alterations associated with oral epithelial carcinogenesis than either marker alone. In addition, their relative expression pattern has the potential to help identify lesions with higher malignant potential, which cannot be well separated by traditional histopathological grading. Thus, the present study aimed to compare the expression of Ki-67 and p53 in OPDs and to assess their respective correlation with histopathological grades and malignant transformation risk with the ultimate goal of identifying the more informative immunohistochemical marker for better risk stratification and early clinical management

A comparative cross-sectional study was conducted at the Department of Oral Pathology, Fatima Jinnah Dental College, Karachi, Pakistan. The study period was six months, from 1st November, 2025 to 30th April, 2026. Patients with clinically suspicious oral potentially malignant disorders were assessed, and histopathological examination and immunohistochemical analysis of eligible tissue specimens were performed.

A sample size was determined using the OpenEpi online sample size calculator for a single population proportion. The calculation was based on the previous study by Khan et al. (2022), which reported p53 immunohistochemical positivity in 73.3% (44/60) of patients with oral potentially malignant disorders with a confidence level of 95%, an Absolute precision (d) of 10%, and a Z value of1.96.[16] In total, 83 patients presenting with oral potentially malignant disorders were included in the study.

Non-probability consecutive sampling was used. The study period was divided into two months, during which all the patients who visited the study department meeting the study inclusion criteria were consecutively recruited until the desired sample size was obtained. This method allowed eligible patients to be included without selection of cases according to their clinical features or histopathological data.

Patients aged 18 years or older, of either gender, presenting with clinically suspected oral potentially malignant disorders were included. The clinical conditions studied were: oral leukoplakia, oral erythroplakia, oral submucous fibrosis and other OPMDs recognised clinically. Patients with histopathologically confirmed OPMDs and with sufficient FFPE tissue blocks for immunohistochemical evaluation were selected. Eligible lesions were both dysplastic and non-dysplastic. Patients who had been diagnosed with oral squamous cell carcinoma, those who had undergone chemotherapy or radiotherapy for head and neck cancers, or those who had undergone surgery for the lesion were excluded. Specimens that were too poorly preserved and had inadequate epithelial tissue or extensive necrosis or severe tissue artefacts were excluded. Patients with incomplete clinical and histopathological records and those with recurrent lesions were not included.

After obtaining approval from the institutional ethical review committee, eligible patients were enrolled following written informed consent. Demographic and clinical data were collected with a structured data collection proforma. Age, gender, medical history, tobacco or betel quid use, duration of the oral lesion, anatomic location, clinical diagnosis, and the type of OPMD were recorded.

Detailed clinical examination of the oral cavity was carried out, and incisional/excisional biopsy was taken as clinically indicated. The tissues were fixed in 10% neutral buffered formalin, processed by conventional histopathological techniques, and embedded in paraffin wax. About 3-4 μm thick sections were cut and stained with haematoxylin and eosin (H&E) to assess the histopathology. The specimens were reviewed under the microscope to ensure the diagnosis and assess the presence and grade of epithelial dysplasia. Dysplasia was graded as absent, mild, moderate, or severe based on pre-established histopathology criteria.

In addition, other paraffin-embedded tissue sections were placed on positively charged slides for immunohistochemical evaluation. The sections were deparaffinised in xylene and rehydrated in a descending series of alcohols. Appropriate antigen retrieval buffer was used to retrieve the antigen, and endogenous peroxidase activity was blocked. Sections were then incubated with validated antibody dilutions and incubations in accordance with manufacturers' instructions with primary antibodies against Ki-67 and p53. Washed and then an appropriate secondary antibody and detection system was applied. The chromogen was Diaminobenzidine (DAB), and the sections were counterstained with haematoxylin. Each staining batch included appropriate positive and negative controls to ensure the reliability of the immunohistochemical procedure.

Ki-67 expression was assessed by the detection of brown nuclear staining in epithelial cells. A minimum of 500 cells in representative microscopic fields were counted, and the percentage of cells with Ki-67-positive nuclei was determined as the Ki-67 labelling index. Positive cells were also identified and counted in the basal, parabasal, and suprabasal layers to evaluate expression. In the case of p53, brown staining of nuclei was interpreted as a positive result, and the proportion of immunoreactive epithelial cells was noted. Staining intensity was assessed as weak, moderate, or strong. Cells that were positive and the staining intensity were documented separately to enable comparison of the two biomarkers. The results of the immunohistochemical stains were then compared with the histopathological grades of epithelial dysplasia. All slides were independently examined by two experienced oral pathologists who were blinded to the clinical details of the patients. Disagreements for interpretation were settled by consensus building and joint microscopic examination. This was done in order to reduce the observer-related variability and to standardize the immunohistochemical evaluation.

The data were entered and analyzed in IBM SPSS Statistics version 27. Quantitative variables were presented as mean ± SD. Normality was tested by the Shapiro–Wilk test. Categorical variables such as gender, OPMD subtype, histopathological grade, and p53 expression categories were displayed as frequencies and percentages. The relationship between the expression of Ki-67, p53, and the histopathological grades of epithelial dysplasia was evaluated using the chi-square test. The differences in Ki-67 labelling index for each dysplasia grade were analyzed with a one-way analysis of variance (ANOVA) test. Each biomarker was tested for its discriminatory value using receiver operating characteristic curve analysis between the presence of moderate or severe epithelial dysplasia and the absence of dysplasia or the presence of mild dysplasia. Calculations of area under the curve (AUC), sensitivity, and specificity were made, and the optimum cut-off value was determined. Multivariable logistic regression was used to evaluate the independent relationships between biomarker expression and the presence of high-grade dysplasia after adjusting for potentially confounding demographic and clinical characteristics. The level of significance was set at p < 0.05.

The study involved a total of 84 patients with oral potentially malignant disorders. The mean age was 46.8 ± 12.4 years, with 60.7% of the study population being male. The most commonly reported behavioural exposure was tobacco use, followed by smoking and the use of betel quid. The average duration for the oral lesions was 2.4 ± 1.6 years (Table 1).

Oral leukoplakia was the most common disorder, followed by oral submucous fibrosis, oral lichen planus, and oral erythroplakia. Buccal mucosa was the most frequently involved site of the body. The homogeneous lesions and solitary presentation were more common than the non-homogeneous lesions and multiple presentation (Table 2).

Histopathological examination showed that the most frequent lesions were mild epithelial dysplasia, moderate epithelial dysplasia, no dysplasia, and severe dysplasia. Immunohistochemical evaluation revealed varying expression of both markers. Ki-67 demonstrated increased proliferative activity, with the labelling index being higher in lesions with more advanced dysplastic changes. Likewise, the expression of p53 showed different staining intensity in the specimens analysed (Table 3).

Ki-67 and p53 expression showed a significant relationship with the histopathological grades of epithelial dysplasia (p< 0.001). The mean Ki-67 labelling index gradually rose from the lesions without dysplasia to those with severe dysplasia. As with p53 positivity, the proportion of lesions with positive Ki-67 staining increased, with the highest proportion found in severe dysplastic lesions (Table 4).

The correlation analysis showed that there was a positive correlation between Ki-67 expression and histopathological grade, which was greater than that between p53 expression and the severity of dysplasia. The discriminatory performance of Ki-67 was better than that of p53 in the detection of high-grade epithelial dysplasia as demonstrated by ROC curve analysis (AUC 0.89 vs. 0.83). Both biomarkers demonstrated acceptable sensitivity and specificity (Table 5).

High Ki-67 expression and positivity of p53 were independently associated with high-grade epithelial dysplasia in the multivariable logistic regression analysis. High Ki-67 expression was associated to a greater extent than positivity of p53. Age, gender, tobacco consumption, betel quid consumption, and duration of the lesion did not show statistically significant independent association with high-grade dysplasia (Table 6).

Table 1. Demographic and behavioural characteristics of study participants (n = 84)

Table 2. Clinical characteristics and distribution of oral potentially malignant disorders (n = 84)

Table 3. Histopathological grading and immunohistochemical expression of Ki-67 and p53 (n = 84)

Table 4. Association of Ki-67 and p53 expression with histopathological grades of epithelial dysplasia

Table 5. Correlation and diagnostic performance of Ki-67 and p53 for identifying high-grade epithelial dysplasia

Table 6. Multivariable logistic regression analysis of factors associated with high-grade epithelial dysplasia

In this study, 84 patients with oral potentially malignant disorders (OPMDs) were comparatively assessed according to immunohistochemical expression of Ki-67 and p53. Both biomarkers showed that there was a significant correlation between the grade of epithelial dysplasia and the expression level, with higher expression noted in moderate and severe dysplasia. The findings are consistent with the hypothesis of a role for cellular proliferation and modification in tumour-suppressor pathways in the genesis of oral epithelial carcinogenesis. Increase in Ki-67 expression with increasing dysplasia grades agrees with that of Miguel et al. (2023), who assessed 61 cases of oral leukoplakia and found significantly more Ki-67 expression in high-risk compared to low-risk epithelial dysplasia (p = 0.013).[17] Likewise, de Villalaín et al. (2023), in a longitudinal study of 60 patients with oral leukoplakia, found that higher dysplasia grade was correlated with higher expression of Ki-67 and tongue cancer.[18] In the present study, these observations support the findings of proliferation changes.

In the current study, there was a progressive increase in the mean Ki-67 labelling index from 10.2% in lesions with no dysplasia to 45.8% in severe dysplasia. This is a pattern that is indicative of the higher the degree of histopathology the higher the epithelial proliferative activity. Similar results were observed by Kamala et al. (2024), who studied the expression of Ki-67 and p53 in 60 patients with oral submucous fibrosis as well as normal oral mucosa and an oral cancer control group.[19] They found that the expression of Ki-67 was considerably higher in OSF than in the normal oral mucosa; however, it was low when compared with oral cancer.[8]

Deshpande et al. (2025) also showed that the level of Ki-67 expression was markedly higher in OSF than in non-dysplastic lesions. In addition, Satir and Mercantas (2026), in their systematic review of 50 studies, found that Ki-67 can be helpful in differentiating normal oral mucosa from epithelial dysplasia, but this is limited by the differences in staining methods and scoring systems used for the interpretation of the results in a standardized clinical interpretation.[19] The present study also proved that higher expression of p53 was associated with increasing levels of epithelial dysplasia, indicating that changes in the pathways of p53 may occur in the course of oral epithelial abnormalities.[20]

Khan et al. (2022) examined p53 expression in tissues and serum p53 antibodies in OPMDs and oral squamous cell carcinoma (OSCC) patients and found similar results. They identified that p53 changes may be of interest in oral cancer. Similarly, Ramos-García et al. (2022) conducted a systematic review and meta-analysis of 24 studies (1210 patients) which showed that p53 overexpression was linked to a higher risk of malignant transformation in OPMDs (RR = 1.88; 95% CI: 1.39–2.56). Kamala et al. (2023) also studied the expression of p53 in OSF and its potential as an immunohistochemical marker of epithelial changes.[19] Altogether, these results are similar to the upregulated p53 expression we observed in the present study.

A correlation between p53 expression and the severity of dysplasia was also investigated by Sawada et al. (2022), who showed that p53 immunohistochemical staining patterns can identify the TP53 mutation status in OED.[21]  Chang and Wang (2024) conducted a retrospective study of 65 cases of oral epithelial dysplasia (OED) where they found that abnormal p53 staining patterns were more closely related to severe dysplasia, while increased upward distribution of Ki-67 positive cells was more common in moderate and severe lesions of OED.[22] The results presented here are in line with the expression profile found in the present study, as well as with the simultaneous evaluation of both biomarkers.

One of the noteworthy findings of the present study was the relatively poor diagnostic value of p53 compared to Ki-67 in the diagnosis of high-grade epithelial dysplasia. In the current study, Ki-67 was able to better discriminate between tumor type and healthy tissue (AUC of 0.89 versus 0.83 for p53). This finding is partially corroborated by de Villalaín et al. (2023), who found that Ki-67 is an independent marker that is linked to the development of oral cancer in patients with leukoplakia.[13] In an immunohistochemical analysis of 70 cases of OPMD, however, Takasaki et al. (2026) found that those lesions that became malignant had significantly higher p53 staining (p = 0.013) but not Ki-67 staining (p = 0.161). They conclude that their findings suggest that p53 could be more helpful in terms of prognosis in some clinical situations.[23]

In a similar study of 64 patients with oral epithelial dysplasia, Poh et al. (2026) determined that abnormal p53 immunohistochemical patterns were significant for shorter progression-free survival.[24] These differences suggest that the relative performance of Ki-67 and p53 might be subtype-dependent, might be influenced by staining techniques, scoring criteria, and by the outcome (histological severity or eventual malignant progression). The results also suggest a complementary role for these biomarkers, rather than one that can be replaced by the others. Ki-67 is mainly associated with proliferation, while the abnormal staining of p53 may suggest abnormalities in tumour suppressor pathways. The study by Kamala et al. (2024) showed that both markers increased when compared with normal oral mucosa in OSF.[19]

Similarly, Deshpande et al. (2025) found that Ki-67 is also very significant in the differentiation of dysplastic lesions from non-dysplastic lesions of the oral potentially malignant lesions.[25] The systematic review by Satir and Mercantas (2026) did highlight, however, that the expression alone of Ki-67 was not consistent in confirming the progression and recurrence of OPMDs.[20] Hence, the immunohistochemical results could be used in conjunction with clinical evaluation and the usual histopathological grading to obtain more comprehensive information about the severity of the lesion. Multivariable logistic regression in the present study showed that high Ki-67 expression and p53 positivity were independently associated with high-grade epithelial dysplasia, while age, gender, tobacco consumption, betel quid consumption, and duration of the lesion were not statistically significant.

The interpretation of these findings should be viewed with caution, as there is possible underpowering of the statistical test to detect associations with clinical risk factors due to the relatively small sample size. In addition, the study by Kamala et al. (2024) and the longitudinal study by de Villalaín et al. (2023) suggest that the expression of biomarkers could be associated with the risk of progression, but the results from these two studies cannot be directly compared as a cross-sectional population.[18, 19] Therefore, the present findings lend credence to the possibility of using Ki-67 and p53 as auxiliary markers to identify high-grade dysplasia, but not to predict malignant transformation. The present study confirms that both Ki-67 and p53 are linked to OPMD histopathological severity, and that Ki-67 has comparatively good discriminatory power in the analysis.

However, several limitations should be considered. The cross-sectional design does not allow the evaluation of the true incidence of malignant transformation in the follow-up periods. The relatively small sample size may also affect the expression of the biomarkers, as may the diversity of OPMD subtypes. Also, there are variations in the use of different antibodies and different staining procedures and criteria for antibody positivity between studies, which makes comparisons difficult. Longitudinal studies with larger patient cohorts and standardized immunohistochemical scoring will allow the investigation of whether these markers are independent prognostic markers and if the combination of these markers could add risk stratification.

LIMITATION

The following are some limitations of this study. First, there was no ability to evaluate the actual malignant transformation during long-term follow-up, as the cross-sectional design did not permit it. Second, the small number of patients and the mixed types of OPMD may have affected the expression patterns of these biomarkers. Third, differences in the intensity of immunohistochemical staining, the choice of antibodies, and the interpretation of their staining have influenced the evaluation of the expression of Ki-67 and p53. Furthermore, the molecular confirmation of TP53 alterations was not available in all samples, which hindered the interpretation of p53 immunoreactivity. Longitudinal studies, with larger populations and standardized immunohistochemical protocols, are recommended in order to confirm the prognostic value of these biomarkers.

Ki-67 and p53 demonstrated significant associations with the histopathological grades of oral potentially malignant disorders. Ki-67 had a relatively good discriminatory ability compared to p53 in the diagnosis of high-grade epithelial dysplasia. The results indicate that both of these markers can be useful adjuncts to routine histopathology, and that Ki-67 may be more clinically informative. Their capability of predicting actual malignant transformation, however, needs to be confirmed by larger longitudinal studies.

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