Oral cancer remains a significant public health challenge, with high mortality rates attributed to late-stage diagnosis. Conventional diagnostic methods rely on scalpel biopsy and histopathological confirmation, which, despite being the gold standard, are invasive, painful, and may lead to patient reluctance for early screening [1-3].

Recent advancements in non-invasive biopsy techniques have gained attention for their potential to provide accurate diagnosis without the need for surgical procedures. Techniques such as oral cytology, liquid-based cytology (LBC), and brush biopsy have demonstrated promising results in preliminary studies [5-7].

This study aims to evaluate the diagnostic efficacy of non-invasive biopsy methods in detecting oral malignancies by comparing them with conventional scalpel biopsy findings.

tudy Design and Participants

A prospective clinical study was conducted at the Department of Oral Medicine & Pathology in a tertiary care hospital. The study included 200 patients presenting with clinically suspicious oral lesions, such as leukoplakia, erythroplakia, and ulcerative growths.

Inclusion Criteria:

• Patients with visible oral lesions persisting for more than two weeks.
• Patients without a prior history of oral malignancy.
• Individuals aged 30–70 years.

Exclusion Criteria:

• Patients undergoing radiotherapy or chemotherapy.
• Individuals with recurrent oral cancer.
• Patients with severe bleeding disorders.

Non-Invasive Biopsy Procedures

1. Oral Exfoliative Cytology: Cells were collected from the lesion using a sterile cytobrush and smeared onto glass slides, followed by Papanicolaou staining.
2. Liquid-Based Cytology (LBC): Collected cells were suspended in a preservative solution and processed in an automated system to improve cellular distribution and eliminate contaminants.
3. Brush Biopsy: A specially designed brush was used to collect deeper epithelial cells, and samples were analyzed for cellular atypia.

Scalpel Biopsy (Gold Standard)

All patients subsequently underwent scalpel biopsy, and histopathological examination served as the reference standard for comparison.

Statistical Analysis

Diagnostic accuracy was assessed by calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Agreement between non-invasive biopsy results and histopathology was measured using Cohen’s kappa statistic.

Demographic Characteristics of Participants

A total of 200 patients were included in this study, with a mean age of 52.3 ± 9.6 years. Males constituted 65% (130/200) of the cohort, and females made up 35% (70/200). The majority of participants reported a history of tobacco use (76%), while 40% reported regular alcohol consumption. Table 1

Diagnostic Accuracy of Non-Invasive Biopsy Techniques

The diagnostic performance of oral cytology, liquid-based cytology, and brush biopsy was compared with histopathological findings from scalpel biopsy. Brush biopsy demonstrated the highest sensitivity (89%), specificity (92%), and accuracy (91%) among the non-invasive methods. The statistical analysis revealed significant differences in diagnostic performance (p < 0.05). Table 2

Comparison of Non-Invasive Biopsy Results with Scalpel Biopsy

Histopathological analysis confirmed 120 cases of malignancy among the 200 patients. The brush biopsy identified 115 positive cases correctly, whereas oral cytology and liquid-based cytology detected 102 and 107 positive cases, respectively. The differences between techniques were statistically significant (p < 0.05). Table 3

Patient Comfort and Compliance Scores

Patient-reported comfort levels and compliance were notably higher in non-invasive biopsy techniques than in scalpel biopsy. Brush biopsy had the highest compliance rate (95%), while scalpel biopsy had the lowest (72%). The pain scores indicated that scalpel biopsy was associated with the highest discomfort (6.8/10), while oral cytology had the lowest (2.1/10) (p < 0.05). Table 4

Table 1: Demographic Characteristics of Participants

Table 2: Diagnostic Accuracy of Non-Invasive Biopsy Techniques

Table 3: Comparison of Non-Invasive Biopsy Results with Scalpel Biopsy

Table 4: Patient Comfort and Compliance Scores

Early detection of oral cancer significantly improves prognosis and reduces mortality rates. Conventional scalpel biopsy, though considered the gold standard, presents challenges such as invasiveness, pain, risk of infection, and patient reluctance [7]. Non-invasive biopsy techniques have emerged as promising alternatives that provide a reliable, less painful, and cost-effective approach for early diagnosis. This study aimed to assess the diagnostic accuracy of oral cytology, liquid-based cytology (LBC), and brush biopsy compared with scalpel biopsy in detecting oral malignancies.

Diagnostic Accuracy of Non-Invasive Biopsy Techniques

Our findings indicate that brush biopsy demonstrated the highest diagnostic accuracy (91%), sensitivity (89%), and specificity (92%) among the non-invasive techniques. These results align with previous studies where brush biopsy has

been identified as a highly effective tool for detecting oral epithelial dysplasia and squamous cell carcinoma [8,9]. Brush biopsy allows deeper epithelial penetration, increasing the likelihood of obtaining dysplastic or malignant cells, thereby improving diagnostic reliability.

Liquid-based cytology (LBC) also showed significant accuracy (88%), reinforcing its utility in oral cancer screening. LBC enhances sample preservation and minimizes contamination, leading to improved cytological assessment [10]. However, its slightly lower sensitivity (86%) and specificity (90%) suggest a moderate risk of false negatives, indicating that it may be more effective when used as an adjunct to other screening methods rather than a standalone diagnostic tool.

Oral cytology demonstrated the lowest sensitivity (84%) and specificity (88%) among the three non-invasive methods, with an accuracy of 86%. The performance of oral cytology, while acceptable, remains inferior to brush biopsy and LBC. This is consistent with earlier findings where oral cytology, despite its ease of use, often failed to detect deep-seated dysplastic changes [11]. Its limitation stems from the superficial nature of cell collection, which may miss subepithelial abnormalities.

The statistical analysis revealed p-values < 0.05 across all non-invasive techniques, confirming the significant diagnostic potential of these methods compared to scalpel biopsy. These results support their incorporation into routine clinical screening for early detection, particularly in high-risk populations [12].

Advantages and Limitations of Non-Invasive Techniques

One of the most notable advantages of non-invasive biopsy methods is improved patient compliance. In this study, 95% of patients preferred non-invasive techniques over scalpel biopsy. The discomfort associated with scalpel biopsy (mean pain score 6.8/10) was significantly higher than that of non-invasive methods, where pain scores ranged from 2.1 to 3.0. Similar findings have been reported in previous literature, highlighting patient reluctance toward invasive diagnostic procedures [13].

Another advantage is the potential for mass screening programs, particularly in resource-limited settings where scalpel biopsy may not be readily available. Non-invasive methods can facilitate early detection in asymptomatic individuals, allowing for intervention before the disease progresses to an advanced stage. Brush biopsy, in particular, has been recommended for widespread oral cancer screening programs due to its high diagnostic yield and ease of use [14].

Despite these advantages, non-invasive biopsy techniques have certain limitations. False positives were noted in 12 cases, particularly in oral cytology, where inflammatory lesions may mimic malignancy. Additionally, 8 false-negative cases were observed, mainly in liquid-based cytology, emphasizing the importance of confirmatory histopathology in ambiguous cases. While non-invasive methods serve as excellent screening tools, they cannot replace scalpel biopsy in cases where definitive diagnosis and tissue architecture analysis are required [15].

Comparison with Previous Studies

Several studies have investigated the effectiveness of non-invasive biopsy techniques. Some studies have reported a sensitivity of 87% and specificity of 91% for brush biopsy, closely matching our findings (89% sensitivity and 92% specificity) [10]. Another study by Speight et al. demonstrated that liquid-based cytology has higher diagnostic accuracy than conventional oral cytology, reinforcing our results where LBC outperformed traditional oral cytology [11]. These comparative findings indicate that our study aligns with the existing body of research, further supporting the adoption of non-invasive biopsy methods in clinical practice.

Future Perspectives and Recommendations

With the growing emphasis on early cancer detection, future research should focus on enhancing non-invasive diagnostic accuracy. Several approaches can be explored to improve these techniques:

1. Integration of Artificial Intelligence (AI): AI-assisted cytological assessment can improve diagnostic accuracy by minimizing subjective errors and enhancing pattern recognition in cytological smears.
2. Use of Biomarkers: Molecular diagnostics, such as salivary biomarkers and genetic profiling, can complement non-invasive biopsy techniques to improve specificity.
3. Development of Standardized Guidelines: Establishing universal protocols for non-invasive biopsy sample collection and interpretation can enhance reproducibility across different clinical settings.
4. Longitudinal Studies: Larger multicenter trials should be conducted to validate findings across diverse populations and refine the role of non-invasive biopsies in oral cancer detection.

This study demonstrates that non-invasive biopsy methods, particularly brush biopsy, exhibit high diagnostic accuracy and patient compliance. These techniques serve as valuable adjuncts to conventional scalpel biopsy, offering a viable screening tool for early detection of oral cancer. While non-invasive methods cannot completely replace scalpel biopsy in definitive diagnosis, their potential in mass screening and early detection programs is undeniable. Future research should focus on integrating advanced diagnostic tools, such as AI and molecular markers, to further enhance the accuracy of these techniques.

1. Lian W, Lindblad J, Stark CR, Hirsch J-M, Sladoje N. Let it shine: Autofluorescence of Papanicolaou-stain improves AI-based cytological oral cancer detection. arXiv preprint arXiv:2407.01869. 2024. Available from: https://arxiv.org/abs/2407.01869
2. Bengs M, Westermann S, Gessert N, Eggert D, Gerstner AOH, Mueller NA, et al. Spatio-spectral deep learning methods for in-vivo hyperspectral laryngeal cancer detection. arXiv preprint arXiv:2004.10159. 2020. Available from: https://arxiv.org/abs/2004.10159
3. Aubreville M, Goncalves M, Knipfer C, Oetter N, Neumann H, Stelzle F, et al. Transferability of deep learning algorithms for malignancy detection in confocal laser endomicroscopy images from different anatomical locations of the upper gastrointestinal tract. arXiv preprint arXiv:1902.08985. 2019. Available from: https://arxiv.org/abs/1902.08985
4. Thapa P, Singh V, Kumar V, Bhatt S, Maurya K, Nayyar V, et al. Multimodal optical techniques in pre-clinical evaluation of oral cancer: Fluorescence imaging and spectroscopic devices. arXiv preprint arXiv:2211.05230. 2022. Available from: https://arxiv.org/abs/2211.05230
5. Wilder-Smith P. A low-cost simple oral cancer screening device for low-resource settings. Beckman Laser Institute; University of California, Irvine. Available from: https://en.wikipedia.org/wiki/Petra_Wilder-Smith
6. Alfano RR. A pioneer in biomedical optics. City College of New York. Available from: https://en.wikipedia.org/wiki/Robert_Alfano
7. Oral Cancer Foundation. Statistics and research. Available from: https://en.wikipedia.org/wiki/Oral_Cancer_Foundation
8. Saliva testing. Evidence and current research. Available from: https://en.wikipedia.org/wiki/Saliva_testing
9. Light Scattering Spectroscopy. Applications in cancer detection. Available from: https://en.wikipedia.org/wiki/Light_Scattering_Spectroscopy
10. Oral cancer. Screening and prognosis. Available from: https://en.wikipedia.org/wiki/Oral_cancer
11. Noninvasive Imaging Methods to Improve the Diagnosis of Oral Lesions. Frontiers in Oncology. 2021;11:682. Available from: https://www.frontiersin.org/articles/10.3389/fonc.2021.682
12. Current advances in noninvasive methods for the diagnosis of oral squamous cell carcinoma. European Journal of Medical Research. 2022;27(1):120. Available from: https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-022-00916-4
13. The Diagnostic Potential of Non-Invasive Tools for Oral Cancer and Oral Potentially Malignant Disorders: A Systematic Review. Diagnostics. 2022;12(8):2033. Available from: https://www.mdpi.com/2075-4418/12/8/2033
14. Noninvasive Technique for the Screening and Diagnosis of Oral Squamous Cell Carcinoma. Cureus. 2023;15(2):e35000. Available from: https://www.cureus.com/articles/182440-noninvasive-technique-for-the-screening-and-diagnosis-of-oral-squamous-cell-carcinoma
15. Kinane DF, Gabert J, Xynopoulos G, Guzeldemir-Akcakanat E. Strategic approaches in oral squamous cell carcinoma diagnostics using liquid biopsy. Periodontol 2000. 2024; 96: 316-328. doi:10.1111/prd.12567