European Journal of Cardiovascular Medicine

Oral cancer accounts for approximately 5% of all cancers worldwide, whereas, it represents approximately 40% of all cancer cases in India. It is estimated that nearly 60,000 new cases of oral cancer are reported annually in India, with over five deaths occurring every hour due to the disease, indicating its highly fatal nature [1]. Although tobacco and alcohol remain the primary risk factors, there is growing evidence that human papillomavirus (HPV) may also function as a co-carcinogen, contributing to the development of oral cancer [2]. Initial small biopsy followed by radical surgery, and radiotherapy for advanced stage is the traditional method of management of oral and oropharyngeal cancers. Onco-surgery facilities are unavailable for many patients and significant number present in advanced inoperable stage of disease explain the higher mortality and emphasizing the importance of early detection. Early detection is the most effective strategy for reducing the morbidity and mortality associated with oral and oropharyngeal cancers. Visual inspection alone is insufficient for distinguishing benign lesions from premalignant and early malignant lesions, necessitating further evaluation of persistent oral lesions of unknown etiology [3]. The use of brush cytology is a cost-effective and risk-free method for evaluating oral lesions, particularly in resource-limited countries such as India. It has the potential to bridge the diagnostic gap that currently hinders the early detection of oral cancers. Brush cytology is a useful and non-invasive method for identifying dysplasia and early carcinoma in asymptomatic individuals or those with mild symptoms that do not warrant urgent biopsy [2].

This study aimed to evaluate the efficacy of brush cytology in detecting oral premalignant and malignant lesions in correlation with histopathology and immunohistochemical p16 expression

The study was approved by the Institutional ethical committee (IEC/RMC/2022/805).

Participants:

This prospective study was conducted over 20 months from November 2022 to June 2024 in the departments of ENT, Dental, and Pathology at Government general hospital, Rangaraya Medical college, Kakinada, India. The study included 65 cases of clinically suspected premalignant and malignant oral and oropharyngeal lesions that were evaluated using brush cytology, followed by histopathology and immunohistochemistry.

Inclusion Criteria:

Patients with clinically suspected premalignant and malignant lesions of the oral cavity and oropharynx who underwent oral brush cytology followed by biopsy were included in this study. Patients who provided informed consent were included.

Exclusion Criteria:

Patients with confirmed malignancies, those undergoing treatment, those with infective and non-neoplastic lesions, those with inadequate samples, and those who declined to provide informed consent were excluded.

Patient preparation

The procedure and benefits were explained to the patients, and consent was obtained prior to brush cytology. A thorough history was taken, including present complaints, duration, and presence of any adverse habits, followed by oral cavity examination using a head torch, focusing on the lesion size, site, tenderness, and consistency. After a detailed explanation of the procedure, the patients were instructed to gargle with chlorhexidine mouthwash for one minute.

Exfoliative cytology procedure

A sterile disposable nylon pediatric toothbrush was used for sampling. Repeated brushing in one direction at moderate pressure until pinpoint bleeding occurs allows cells from the full thickness of the epithelium to be extracted. The exfoliated material was transferred onto two or three clean glass slides, and smears were prepared using a spreader. The smears were immediately fixed with 95% isopropyl alcohol for staining with hematoxylin and eosin or Papanicolaou stain.

Cytology Analysis:

The stained cytology smears were analyzed and reported by two independent pathologists.

Histopathology Analysis:

Punch biopsy specimens obtained from the same patient with the biopsy requisition form were fixed in 10% formalin. The fixed specimens were then subjected to grossing, tissue processing, and sectioning. Microscopic evaluation of hematoxylin and eosin-stained sections of paraffin blocks was performed.

Immunohistochemical Analysis

Immunohistochemical staining was performed on paraffin-embedded tissue sections to detect the expression of p16 protein. Tissue sections were deparaffinized and rehydrated, and the antigens were retrieved. Primary antibodies against  p16 were used, followed by suitable secondary antibodies. Diaminobenzidine (DAB) was used as the chromogen, and hematoxylin was used as a counterstain. Two expert pathologists, blinded to the clinical data, independently assessed the intensity and extent of the staining.

Data Analysis:

Data analysis was performed using SPSS version 28. Numerical data are presented as frequencies and percentages. Comparisons between groups of categorical data were performed using the chi-square test.

A total of 65 patients with suspected premalignant and malignant oral lesions were subjected to brush cytology. The patient age ranged from 20 to 100 years, with a peak in the fifth decade. The male-to-female ratio was 1.5:1. The results are presented in Table-1. 

Table 1: Demographic distribution in present study

All patients were assessed for the presence of risk factors, and 39 out of 65 patients were identified as smokers.

Table-2: Location of the lesion

The most common sites of the lesions were the lateral border of the tongue and hard palate, followed by the buccal mucosa (Table -2). 

Table-3: Clinical appearance of lesions

The lesions exhibited variable clinical appearances, as depicted in Table 3, ranging from ulcero-proliferative lesions, which were the most common type, to leukoplakia, proliferative lesions, and ulcers. Ten patients out of 65 were presented with associated cervical lymphadenopathy. 

Table-4: Cytological diagnosis of lesions

All 65 patients underwent cytological examination using brush cytology. The lesions were categorized as normal, reactive, atypical-probably reactive/low-grade squamous intraepithelial lesion (LSIL), atypical-probably high-grade, high-grade squamous intraepithelial lesion, squamous cell carcinoma, and inadequate smear. The results are shown in Table 4, where the majority were diagnosed with squamous cell carcinoma, followed by normal smears without any abnormality. 

Table -5: Histopathology diagnosis

Among the 65 patients assessed by cytopathology, 50 patients underwent histopathological examination. The diagnoses included 36 cases of squamous cell carcinoma, and the remaining cases comprised non-neoplastic lesions, dysplasia, and other conditions. The details are presented in Table 5. 

Table -6: Comparison of histopathology and brush cytology findings

A comparative analysis was conducted on 50 cases between cytology and histopathology and results are depicted in Table 6. All cases categorized as atypical probably reactive/LSIL (2 cases), atypical probably high grade (3 cases), HSIL (5 cases), and squamous cell carcinoma (23 cases) in cytology were confirmed as malignant on histopathology. Of the seven cases reported as normal smears on cytology, two were non-neoplastic, three exhibited dysplasia, and three were found to be malignant upon histopathological evaluation. Among the ten cases identified as reactive on cytology, four were non-neoplastic, four showed dysplasia, and two were malignant on histopathology. 

Table -7: Brush cytology in correlation to histopathology diagnosis

Of the total 50 cases of brush cytology cases followed by histopathology, 33 were true positive, 12 were true negative, 05 cases were false negative and no false positives. Based on this sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were calculated.

Table-8: Diagnostic accuracy

Table-9: Chi-square test

*a-67 cells (95.7%) had an expected count of less than 5, with the minimum expected count being 0.03. Consequently, the null hypothesis is rejected in favor of the alternative hypothesis, indicating a high level of significance. This suggests a significant difference between cytopathology and histopathology

Table 10: P16 immunohistochemistry

Table 11: Oropharyngeal squamous cell carcinomas

The patient age ranged between  53-70 years and include both males and females. The site of lesion, histology type and grade, lymph node involvement and p16 staining was analyzed.

All patients had smoking as a risk factor. Immunohistochemistry with p16 staining was positive in two cases, one case with 25% positivity was considered as negative and another case with more than 70% staining was considered as positive.

IMAGES

Oral cancer continues to be a major public health challenge worldwide. According to global estimates, approximately 377,713 new cases and 177,757 deaths due to oral cancer were reported in 2020 [4]. Early diagnosis is a critical determinant of prognosis and survival; however, conventional diagnostic approaches such as visual examination and biopsy are associated with limitations including invasiveness, patient discomfort, and sampling errors.

In recent years, oral brush cytology has gained attention as a valuable, minimally invasive screening tool for the early detection of oral malignancies. This technique involves the collection of exfoliated epithelial cells from the oral mucosa using a brush or spatula, followed by cytological evaluation for dysplastic or malignant changes. Compared to surgical biopsy, brush cytology offers several advantages such as ease of use, minimal discomfort, ability to sample larger mucosal areas, and suitability for repeated examinations. Its utility has been demonstrated particularly in screening for oral squamous cell carcinoma (OSCC) and potentially malignant disorders, especially in high-risk individuals or in clinical situations where biopsy may be contraindicated or poorly tolerated, such as in patients with Fanconi anemia [5,6].

The present study comprised 65 cases, of which 24 (37%) were clinically diagnosed as premalignant lesions and 41 (63%) as clinically malignant lesions. All cases were subjected to cytological evaluation, while 50 cases underwent histopathological examination. Among the 15 cases not evaluated histopathologically, 13 were clinically premalignant and 2 were clinically malignant. One patient, a person living with HIV/AIDS (PLWHA), was lost to follow-up, while the remaining cases were managed conservatively, as they were clinically suspected to be traumatic or frictional ulcers that did not warrant biopsy.

A slight male predominance was observed in the present study, consistent with findings reported in previous studies [7–9]. The higher prevalence among males has traditionally been attributed to increased exposure to risk factors such as tobacco and alcohol consumption. However, a notable observation in the present study was the relatively higher incidence among females, which may be explained by the regional practice of reverse smoking. Cancers of the lip and oral cavity are among the most common malignancies in males and rank fourth among females. The age of patients in this study ranged from 20 to 100 years, with the majority belonging to the 41–70 year age group, findings that are in agreement with earlier reports [9,10]. The increasing incidence with advancing age suggests the cumulative effect of genetic alterations and prolonged exposure to carcinogenic risk factors [11].

Smoking emerged as the most frequently reported risk factor in the present study, corroborating observations by Mehrotra et al. [3] and Jairajpuri et al. [8]. The most commonly affected anatomical sites were the tongue and hard palate, similar to findings reported by Kokubun et al. [10]. The buccal mucosa was the next most frequently involved site, consistent with earlier Indian studies [3,8]. In India, the buccal mucosa is widely reported as the predominant site for oral cancer, largely due to the habitual use of smokeless tobacco and betel nut products, which are retained in the oral cavity for prolonged periods, leading to chronic irritation, inflammation, and genetic alterations. The increased involvement of the hard palate observed in the present study may be attributed to the regional prevalence of reverse smoking, particularly in the Srikakulam and Kakinada districts of Andhra Pradesh.

Histopathological evaluation revealed that all non-neoplastic lesions were cytologically reported as normal or reactive. Among the seven cases of histologically confirmed dysplasia, all were interpreted as reactive on cytology. Of the 37 histopathologically confirmed malignancies—including 35 squamous cell carcinomas, one verrucous carcinoma, and one adenoid cystic carcinoma—23 were correctly identified as malignant on cytology, while 10 were categorized as dysplasia.

The diagnostic performance of brush cytology in the present study demonstrated a sensitivity of 75%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 41.7%, with an overall diagnostic accuracy of 78%. The absence of false-positive cases supports the high specificity of the technique, while the presence of 10 false-negative cases contributed to reduced sensitivity and negative predictive value. These findings are comparable to those reported by Babshet et al. [7], who utilized brush cytology without computer-assisted analysis. Higher sensitivity values have been reported in studies incorporating cytomorphometric or computer-assisted analysis, such as those by Raj et al. [12] and Haque et al. [9], highlighting the potential benefits of adjunctive analytical techniques. Comparable diagnostic values have also been reported by Trakroo et al. [11] and Driemel et al. [13] using conventional cytological assessment.

Immunohistochemical evaluation revealed p16 positivity in one of six oropharyngeal squamous cell carcinomas, with positivity defined as moderate to strong diffuse nuclear and cytoplasmic staining in at least 70% of tumor cells. The overall p16 positivity rate in the present study was 16.6%, with all positive cases occurring in the 50–70 year age group. Smoking was identified as a common risk factor among these patients. The relatively low p16 positivity may be attributed to the predominance of keratinizing squamous cell carcinomas and the limited sample size. Additionally, sociocultural factors and lower prevalence of HPV-associated risk behaviors may explain the reduced incidence of HPV-related tumors. These findings are consistent with observations reported by Bhosale et al. [14] and Raphael et al. [15].

The current study revealed that early diagnosis of premalignant and malignant lesions in the oral and oropharyngeal regions is required and can be accomplished using a noninvasive, painless, and simple approach such as toothbrush cytology. Brush cytology is very effective for individuals who are concerned about the lesion but are unwilling to undergo a biopsy. Brush cytology has the advantages of being noninvasive, painless, cost-effective, and requires little technical ability. It has good specificity and positive predictive value. The disadvantages include low sensitivity and negative predictive value (NPV). Brush cytology also has low rates of technical and procedural errors. Brush cytology can be utilized as a first-line inquiry for the early diagnosis of malignancies in rural settings; however, it cannot completely replace biopsy. p16 IHC is important for understanding the HPV relationship, particularly in metropolitan regions with diverse sexual habits. However, p16 IHC was not necessary for histopathological diagnosis. Further research is needed to demonstrate the effectiveness of brush cytology for oral and oropharyngeal lesions. At the cytological level, it is also critical to implement novel techniques, such as liquid-based cytology, computer-assisted analysis, AgNOR analysis, and p16 antibody detection. If p16 IHC positivity is detected, additional testing can be performed using the HPV PCR DNA detection method.

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