Past, Present, and Future Diagnostic Methods for the Early Noninvasive Detection of Oral Premalignant Lesions: A State of the Art and Systematic Review

https://doi.org/10.1177/01455613241245204 Ear, Nose & Throat Journal 1 –21 © The Author(s) 2024 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/01455613241245204 journals.sagepub.com/home/ear Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Review Introduction White lesions of the oral mucosa are commonly encountered in daily practice by dentists, otolaryngologists, and maxillofacial surgeons.1,2 Oral leukoplakia is rarely symptomatic, and the importance of screening and early diagnosis is derived from its frequent association with oral cavity squamous cell carcinoma.3,4 Oral leukoplakia constitutes 85% of all potential malignant disorders occurring in the oral cavity, with a described prevalence of 2.89% to 3.6%, a higher incidence among men.5-7 The etiology of oral leukoplakia is multifactorial.8-12 Consumption of tobacco, whether through smoking or chewing, appears to be the sole direct risk factor implicated in the induction of oral leukoplakia.13-17 The most commonly described locations are represented by mandibular alveolus (25%-40%), buccal mucosa (22%-46%), palate (27%), tongue (26%), and floor of the mouth (19.3%).18-21 Negative prognostic risk factors include being of the female gender, advanced age, having a size greater than 200 mm², and having a Candida albicans infection. HPV, or human papillomavirus, plays a significant role in developing oral leukoplakia.22 Particularly, high-risk strains like HPV-16 and HPV-18 are associated with an increased likelihood of developing oral leukoplakia and its progression to cancer.22 Also, different genes involved in DNA damage response and repair pathways 1245204EARXXX10.1177/01455613241245204Ear, Nose & Throat JournalKhong et al review-article2024 Past, Present, and Future Diagnostic Methods for the Early Noninvasive Detection of Oral Premalignant Lesions: A State of the Art and Systematic Review Brendan Khong, MD1, Salvatore Ferlito, MD, PhD2, Stuart Quek, MD3, Gianluca Conte, MD4, Angelo Ingrassia, MD2, Jerome Rene Lechien, MD, PhD5, Carlos Chiesa-Estomba, MD, PhD6, Miguel Mayo, MD, PhD7, Antonino Maniaci, MD, PhD8, Thomas Radulesco, MD, PhD9, Justin Michel, MD, PhD9, Nicolas Fakhry, MD, PhD9, and Riccardo Polosa, MD, PhD10,11 Abstract Objectives: To provide an in-depth analysis of noninvasive methods for the early diagnosis of oral premalignant lesions, focusing on novel biomarkers and optical technologies, and to discuss their potential in improving the prognosis of patients with oral oncological diseases. Methods: This state-of-the-art review examines various noninvasive diagnostic techniques, including the utilization of salivary microRNAs and optical technologies such as Raman spectroscopy, elastic scattering spectroscopy, diffuse reflectance spectroscopy, narrow-band imaging, autofluorescence imaging, toluidine blue staining, and microendoscopy. Results: Several noninvasive techniques have shown varying degrees of effectiveness in detecting oral cancer. Autofluorescence imaging exhibited sensitivities up to 100% but had variable specificity. toluidine blue staining reported sensitivity between 77% and 100% for high-risk lesions or cancer, with specificity around 45% to 67%. Spectroscopy techniques achieved 72% to 100% sensitivities and specificities of 75% to 98%. Microendoscopy presented a sensitivity of 84% to 95% and a specificity of 91% to 95%. Conclusion: The review highlights the strengths and limitations of each noninvasive diagnostic method and their recent advancements. Although promising results have been demonstrated, there is a need for further development of reliable strategies for early detection and intervention in oral oncology. Keywords oral leukoplakia, diagnosis, noninvasive test, noninvasive biomarkers, oral cancer, saliva testing 2 Ear, Nose & Throat Journal 00(0) have been reported as candidates for cancer susceptibility.23,24 Oral leukoplakia can present carcinoma in situ in 7% to 7.6% of cases,25-27 with nonhomogenous leukoplakia possessing greater malignancy probability (20%-25%).28-31 Visual examination represents the first cost-effective approach, compromised by its inherent subjectivity and the heavy reliance on the clinician’s experience. Toluidine blue staining is employed to supplement visual examination, due to selectively stain areas of dysplasia or malignancy.32,33 However, diagnostic accuracy is sometimes compromised by potential false positives and negatives. As a more sensitive approach, brush biopsy and cytology are often utilized.34,35 While minimally invasive, their sensitivity and specificity can vary, and more severe or deeper dysplastic changes may not be captured. Although tissue biopsy remains the definitive diagnostic method with high diagnostic accuracy, the procedure can present risks and potential discomfort for the patient. In the face of emerging technologies, optical imaging techniques such as Raman spectroscopy (RS) and narrow-band imaging (NBI) are gaining attention.31-34 These innovative, noninvasive techniques can pinpoint subtle structural and biochemical tissue changes in real time. However, their successful implementation requires specialized equipment and expertise for accurate interpretation. To evaluate and analyze the different diagnostic indications, the advantages, and limitations of each process, we conducted a comprehensive review of the literature on the diagnostic procedures involved in the early detection of oral leukoplakia. Methods Study Design This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, the Cochrane Handbook for Systematic Reviews of Interventions, and the PICOTS framework (Population, Intervention, Comparison, Outcomes, Timing, and Setting). Search Strategy The authors performed a comprehensive literature search in the following electronic databases: PubMed/Medline, Embase, Web of Science, Google Scholar, and the Cochrane Library. The search strategy included the combination of keywords and MeSH terms related to “oral leukoplakia,” “oral precancerous lesions,” “early diagnosis,” “early detection,” “neoplastic lesions,” “oral cavity,” “diagnostic techniques and procedures,” and “biomarkers.” The search was limited to English-language publications with no publication date restrictions. Inclusion Criteria Studies were considered eligible for inclusion if they met the following criteria: 1. Study design: Cross-sectional studies, case-control studies, retrospective cohort studies, prospective cohort studies, primary science articles, and epidemiological studies. 2. Population: Patients with oral leukoplakia. 3. Intervention: Procedures or techniques used for the early diagnosis of oral leukoplakia. 4. Outcomes: Diagnostic accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and other relevant measures. Exclusion Criteria Studies were excluded if they: 1. Were not published in English. 2. Were case reports, case series, reviews, commentaries, editorials, or letters to the editor. 1 Ashford and St Peter’s Hospitals NHS Trust, Chertsey, UK 2 Department of Medical and Surgical Sciences and Advanced Technologies “GF Ingrassia” ENT Section, University of Catania, Catania, Sicilia, Italy 3 Bedfordshire Hospitals NHS Foundation Trust, Bedfordshire, UK 4 Department of General Surgery and Medical-Surgical Specialties, University of Catania, Catania, Sicilia, Italy 5 Department of Human Anatomy and Experimental Oncology, Faculty of Medicine, UMONS Research Institute for Health Sciences and Technology, University of Mons (UMons), Mons, Belgium 6 Department of Otorhinolaryngology—Head and Neck Surgery, Hospital Universitario Donostia, San Sebastian, Spain 7 Department of Otorhinolaryngology—Head and Neck Surgery, University Hospital Complex of A Coruña, A Coruña, Spain 8 Faculty of Medicine and Surgery, “Kore” University of Enna, Enna, Italy 9 Department of Otorhinolaryngology—Head and Neck Surgery, APHM, Aix Marseille University, La Conception University Hospital, Marseille, France 10 Center of Excellence for the Acceleration of HArm Reduction (CoEHAR), University of Catania, Catania, Sicilia, Italy 11Department of Clinical and Experimental Medicine, University of Catania, Catania, Sicilia, Italy Received 15 October 2023; revised March 10 2024; revised manuscript accepted 15 March 2024 Corresponding Author: Antonino Maniaci, MD, PhD, Faculty of Medicine and Surgery, “Kore” University of Enna, via Santa Sofia 78, Enna 95124, Italy. Email: [email protected] Khong et al 3 3. Focused on diagnosing other oral lesions or conditions without a specific focus on oral leukoplakia. 4. Did not provide sufficient data to assess the diagnostic accuracy or other relevant outcome measures. Study Selection and Data Extraction Two independent reviewers screened the titles and abstracts of the identified articles. Full-text articles were obtained for those that appeared to meet the inclusion criteria or when there was uncertainty. Disagreements between reviewers were resolved through discussion or by involving a third reviewer. Data extraction was performed using a standardized data collection form. The extracted data included study design, population characteristics, diagnostic procedures, outcomes, and follow-up. In addition, the risk of bias and quality of the included studies was assessed using appropriate tools, such as the Quality Assessment of Diagnostic Accuracy Studies and the Newcastle-Ottawa Scale. Data Synthesis A narrative synthesis of the findings was conducted, summarizing the included studies’ main features, diagnostic procedures, treatment modalities, outcomes, and followup. The primary objective of this review was to evaluate the diagnostic accuracy of various methods for the early detection of oral leukoplakia. The secondary objective was to compare different diagnostic techniques and identify potential biomarkers that could aid in the early diagnosis of oral leukoplakia. Due to the expected heterogeneity in study designs and diagnostic methods, a formal statistical analysis was not performed. Instead, a qualitative synthesis of the findings was presented, highlighting the strengths and limitations of the included studies. Results Study Design and Patient Inclusion After assessment for eligibility, 16 articles were included for quantitative analysis. The systematic protocol is summarized in Figure 1. The sample sizes varied significantly across the studies, ranging from a minimum of 18 to a maximum of 184 subjects. The studies included patients with various oral conditions, from benign inflammatory lesions to potentially malignant disorders and oral squamous cell carcinoma (OSCC). Methodologies and Outcomes Reported Several different diagnostic methods were used across the studies. These included salivary microRNA, methylene blue staining, Rose Bengal (RB) staining, blue toluidine staining, Lugol’s iodine staining, RS, elastic scattering spectroscopy (ESS), diffuse reflectance (DR) spectroscopy (DRS), autofluorescence, NBI, high-resolution microendoscopy (HRME), and photodynamic diagnosis. The sensitivity of the diagnostic methods ranged from 64.3% to nearly 100%. The specificity of the methods ranged from around 60% to 100%. Several studies reported instances of false positives and false negatives. The diagnoses made included normal tissues, dysplasia, potentially malignant disorders (PMDs), and OSCC. Some studies reported on the ability of the diagnostic method to restrict the margins of premalignant lesions or differentiate between different types of oral lesions. The risk of bias for each included study, according to the Joanna Briggs Institute tool, is summarized in Figure 2. Oral Leukoplakia—Diagnosis Early detection of oral premalignant lesions (ie, leukoplakia) is essential to reduce the high morbidity and mortality rate associated with ensuing oral cancer.36,37 The different noninvasive test approaches available to screen lesions of the oral mucosa are summarized in Figure 3. During oral examination, it is important to identify the following by visual inspection and palpation: •• Location •• Size •• If the lesion is raised •• Presence of ulcers •• Borders of the lesion—whether it is well-defined or irregular Patients who are suspected to have premalignant disease will undergo incisional biopsy for histological examination to confirm the diagnosis. However, oral leukoplakia remains a diagnostically challenging lesion that is a potential hurdle for clinicians.38 It was reported that the 5 year survival rates have not improved despite advancements in treatment.39 Given the aggressive nature of this condition, the high rates of malignant transformation, and its propensity for early lymphatic spread, early diagnosis is critical in limiting treatment morbidity and maximizing oncologic control. Noninvasive Tests The new noninvasive test that this article discusses can be used to aid the identification and diagnosis of oral leukoplakia. Different diagnostic methods for leukoplakia evaluation are summarized in Table I. 4 Ear, Nose & Throat Journal 00(0) Recordsidentified from*: Databases (n =2892) Registers (n =0) Records removedbefore screening: Duplicate records removed (n =19) Records removedfor other reasons (erratum,retracted publication) (n =18) Fulltext screened. (n =2855) Records excluded (n =0) Reportssought for retrieval (n =2855) Reportsnot retrieved (n =1852) Review (n = 826) Case reports(n = 708) Letter,Editorial,Comment, (n=221) Article notinEnglish (n =97) Reportsassessed for eligibility (n =1003) Reportsexcluded (n= 993): 1) Partial or incomplete data 2) Notadequate patient selection Studies included in review for: Qualitative analysis (n =16) Identification of studiesvia databases and registers Identification Screening Included • Figure 1. Flow-diagram describing systematic protocol according to PRISMA guidelines. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Salivary biomarkers. The technique involves the collection of saliva samples from patients, which is a noninvasive, easy-to-perform, and stress-free procedure. Once the saliva sample is collected, it is processed to extract the microRNA. This extraction can be done using various methods, including commercial kits. The levels of specific microRNAs associated with oral leukoplakia are measured after the extraction. This is often done using quantitative Khong et al 5 1. Were thegroups comparable otherthanthe presence of diseaseincases or theabsence of diseaseincontrols? 2. Were casesand controls matchedappropriately? 3. Were thesamecriteriausedfor identification of casesand controls? 4. Wasexposure measured in astandard, valid and reliableway? 5. Wasexposuremeasuredinthe same wayfor casesand controls? 6. Were confounding factorsidentified? 7. Were strategies to deal with confoundingfactors stated? 8. Were outcomes assessed in astandard, validand reliableway forcases and controls? 9. Wasthe exposure period of interest long enough to be meaningful? 10.Was appropriatestatistical analysis used? C.-J.Chang. et al., 2005 Ya-Wei Chen et al., 2006 A. Sharwani et al., 2006 Ge-fei Du et al., 2007 ManjuMStephen et al., 2013 KevinGuzeetal., 2014 F. Zahran et al., 2015 ShereenFatimaetal., 2016 QuangTet al., 2016 BoscoloNataF.etal., 2021 NChainani-Wu et al., 2015 Onofre JB et al.,2001 Elvers Detal.,2015 Koch FP et al., 2011 Chenxi Li et al., 2022 Moro et al., 2010 Figure 2. Risk of bias summary author’s judgments for each included study, assessed by the JBI. Critical appraisal checklist for case-control studies. JBI, Joanna Briggs Institute. 6 Ear, Nose & Throat Journal 00(0) Figure 3. Noninvasive test subclasses and main features. real-time polymerase chain reaction (qRT-PCR), a technique that precisely measures the amount of a specific RNA. The levels of these microRNAs are then compared to a control group or established thresholds to determine whether they are elevated or reduced. In 2015, there was a study that investigated the use of 3 salivary microRNAs (miRNA-21, miRNA-184, and miRNA-145) as markers for oral cancers.40 This study isolated RNA from saliva samples using the microRNA Isolation Kit (Qiagen), and miRNA expression analysis was performed using qRTPCR (Applied Biosystems). This study showed a highly significant increase in salivary miRNA-21 and miRNA184 in OSCC and PMDs. The miRNA-184 was found to discriminate between OSCC and PMDs with dysplasia. It has also provided good diagnostic value, with a specificity of 75% and sensitivity of 80%.6 The usage of salivary interleukin-6 (IL-6) has also been investigated. A study involving 40 patients showed that IL-6 levels were elevated in leukoplakia with coexisting periodontitis and periodontitis patients compared to healthy controls. Within the group of patients with leukoplakia, IL-6 levels also correlated with the severity of dysplasia.41 Several comparative studies have collectively advanced the understanding of other salivary biomarkers in the detection and monitoring of oral diseases with potential malignant transformations.42-47 Agha-Hosseini and Mirzaii-Dizgah42 identified increased salivary p53 in patients with plaque-like Oral Lichen Planus (OLP), suggesting a higher malignancy risk compared to erosive OLP, while Jacob et al43 found elevated salivary total sialic acid (TSA) levels in oral precancer and OSCC, indicative of disease progression. Complementing these findings, Varun et al44 reported that salivary Her2/neu levels were significantly higher in OSCC than in PMDs and controls, underscoring its potential as a localized biomarker for malignancy. The study by Jancsik et al45 reinforced the concept that saliva testing could be an effective and reliable method for the early detection of OSCC, particularly in high-risk populations such as those with diabetes. A salivary proteomic analysis was conducted to identify potential biomarkers for OSCC, utilizing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight (MALDI TOF/ TOF) mass spectrometry, the researchers found elevated levels of annexin A8, peroxiredoxin-2, and tyrosine kinase in the saliva of diabetic individuals, proteins previously associated with cancer and OSCC in saliva. In the study led by Punyani and Sathawane,47 the focus was on evaluating the salivary levels of IL-8 in patients with oral precancer and OSCC to understand its potential as a biomarker. The research revealed that salivary IL-8 concentrations were significantly higher in OSCC patients compared to both the precancer group and healthy 7 Table 1. Systematic Table for Different Diagnostic Methods for Leukoplakia Assessment. Authors, year Study design Sample size Type of tests Results Sensitivity/specificity Advantages Disadvantages Zahran etal, 2015 Prospective controlled 100 Salivary microRNA Highly significant increase in salivary miRNA-21 and miRNA-184 in OSCC and PMDs. A 4-fold increase in miRNA-21 was associated with a specificity and sensitivity of 65%. A decrease of 0.6 in miRNA-145 resulted in a specificity of 70% and a sensitivity of 60%. In addition, a 3-fold rise in miRNA-184 was linked to a specificity of 75% and a sensitivity of 80%. miRNA-184 was found to discriminate between OSCC and PMDs with DP. Not high levels of specificity and sensitivity in this method. (75%-80%, respectively) Chen etal, 2006 Prospective uncontrolled 58 Methylene blue staining Histological examination identified 16 cases of squamous cell carcinomas and 13 precancerous lesions, which included a spectrum of DPs featuring varying concentrations of atypical cells in the basal and parabasal layers. In addition, 29 benign lesions were diagnosed, which encompassed conditions such as epithelial hyperplasia, hyperkeratosis, and lichen planus. It achieved a sensitivity of 90%, the specificity was 69%, reflecting its ability to correctly recognize those without the condition. The positive predictive value stood at 74%, the NPV was 87%. Low toxicity and cheaper than toluidine blue High number of false negatives and false positives. Du etal, 2007 Prospective uncontrolled 132 RB staining RB staining appeared to be a promising technique for identifying dysplastic changes within oral leukoplakia, lichen planus, and leukokeratosis. In the context of this study, the staining method successfully identified 5 out of 6 cases of DP or OSCC prior to confirmation by histological examination. The sensitivity and specificity to detect epithelial DP and OSCC are 93.9% and 73.7%, respectively. The positive and negative likelihood ratios are 3.570 and 0.082, respectively. RB staining seems promising to detect DP in OLKia, lichen planus, and leukokeratosis. High rates of false positives and false negatives. Fatima etal, 2016 Retrospective uncontrolled 100 Lugol’s iodine staining Effective in demarcating the precancerous lesions’ margins, identifying the precancerous lesion’s correct size and extent. The sensitivity and specificity were 100% for surgical margins of dysplastic tissue lesions. Good availability, ease of use, costeffectiveness, and widespread use by clinical in identifying intraepithelial neoplasia of the oral cavity. The specificity of Lugol’s iodine stain ranged from 60%-84.2%. (continued) 8 Table 1. (continued) Authors, year Study design Sample size Type of tests Results Sensitivity/specificity Advantages Disadvantages Guze and Pawluk, 2014 Prospective controlled 18 Raman spectroscopy The differential spectra derived from premalignant and malignant lesions exhibited clear distinctions from those of normal, benign tissues. The predictive accuracy for premalignant and malignant lesions was extremely high, with a sensitivity of 100% and a specificity of 77%. Noninvasive, convenient, and relatively inexpensive technology. During data acquisition, the operator can view the laser light, thus allowing illumination of the lesion site to ensure accurate positioning. The detection needs a sensitive and highly optimized instrumentation. Fluorescence of impurities or of the sample itself can hide the Raman spectrum. Sharwani etal, 2006 Prospective uncontrolled 25 ESS Out of the 11 biopsies that were histologically confirmed as dysplastic, ESS correctly classified 8 of these cases as dysplastic. Conversely, ESS mistakenly identified 3 dysplastic sites as normal, marking them as false negatives. Regarding the normal tissues, 4 biopsies were found to be normal histologically. ESS correctly identified 3 of these as normal, which are true negatives, while it incorrectly classified 1 biopsy as dysplastic, a false negative. On obtaining 2 sets of spectra and applying linear discriminant analysis, the authors achieved a sensitivity of 72% and a specificity of 75% for identifying DP in oral tissues. ESS has the advantage of being fast, reliable, and cost-effective and potentially offers a noninvasive diagnosis in situ and in real time. When comparing spectroscopy to histopathology, the accuracy for normal tissues was 91.6% (22/24) compared to 97% (33/34) for abnormal tissues. When examining DP, these figures fell at 64.3% (9/14) and carcinoma, 50% (5/10). Stephen etal, 2013 Prospective controlled 55 active vs 23 controls DR spectroscopy vs biopsy The median pixel value of the R545/R575 image ratio was 0.87 (IQR = 0.82-0.94) for normal/ clinically healthy tissue, while it was 1.35 (IQR = 1.13-1.67) and 2.44 (IQR = 1.78-3.80) for premalignant and malignant lesions, respectively. The AUC showed differentiating malignant from normal/clinically healthy yielded an AUC of 0.99 (95% CI: 0.99-1.00), premalignant from normal/clinically healthy an AUC of 0.94 (95% CI: 0.86-1.00), malignant from premalignant an AUC of 0.84 (95% CI: 0.73-0.95), and premalignant and malignant from normal/clinically healthy an AUC of 0.97 (95% CI: 0.94-1.00). High sensitivity and specificity in differentiating between malignant and normal tissues, with both values at 97%. When identifying premalignant vs normal tissues, sensitivity remains high at 95%, while specificity is slightly lower at 92%. In distinguishing between premalignant and malignant lesions, with lower sensitivity and specificity of 76% and 80%, respectively. Finally, in detecting both malignant and premalignant lesions vs normal tissues, the method shows strong sensitivity at 92% and specificity at 95% The imaging method has the advantage of noninvasively scanning the entire lesion and its surrounding areas in real time and categorizing oral lesions into normal/clinically healthy, premalignant, and malignant tissue. Furthermore, it efficiently delineates the boundaries of neoplastic changes and locates the site with the most malignant potential for a biopsy, thereby avoiding unnecessary repeated biopsies and delays in diagnosis. Relatively good diagnostic accuracy while comparing it to the gold standard histopathology. (continued) 9 Table 1. (continued) Authors, year Study design Sample size Type of tests Results Sensitivity/specificity Advantages Disadvantages Nata etal, 2021 Prospective uncontrolled 160 NBI The difference between NBI and HD WL sensitivity was statistically significant (P <.001). The NBI diagnostic advantage was 62.5%, highest in the hypopharynx (P = .05), and was not influenced by previous RT or CT (P = .49). Index tumor site statistically related with recurrence site (P < .001), but not with the risk of developing recurrence (P = .81). Among the patients, 30 lesions from 21 individuals were biopsied. NBI identified 26 lesions as positive, of which 24 were confirmed as true positives on histological examination, while 2 were false positives. No significant correlation was found between the initial tumor site and recurrence risk. However, there was a significant association between the original tumor site and the site of recurrence, with the pattern of recurrence varying based on the initial tumor location. The use of NBI with flexible videoendoscope was better tolerated by the patient and allowed closer inspection of the laryngeal and hypopharyngeal subsites with the tip of the endoscope, with in-depth visualization of mucosal and submucosal vascular patterns and without the need for local spray anesthesia in the majority of patients. A learning curve characterizes NBI, and it is an operator-dependent investigation. NBI uses light at specific wavelengths to enhance the visibility of superficial blood vessels. The interpretation of NBI images can be subjective and requires considerable expertise. Quang etal, 2016 Prospective uncontrolled 177 HRME The tablet-interfaced HRME demonstrated comparable imaging performance at a lower cost than first-generation laptop-interfaced HRME systems. In a post hoc quantitative analysis, the algorithm identified neoplasia with The sensitivity and specificity of 95% and 91% in the validation set compared with 84% and 95% achieved in the original study. HRME uses a lowcost, fiber-optic fluorescence microscope to image the cellular morphology of the surface epithelium. The cost of goods to build an HRME system is less than $5000 Although promising, widespread use of the HRME is limited by the need for a bulky laptop to control the system and the need for training to interpret acquired images. Chang etal, 2005 Prospective uncontrolled 20 Photodynamic diagnosis Of the patients studied, 25% showed hyperkeratosis, 45% exhibited squamous hyperplasia, and 30% had SCC. The sensitivity was approximately 92% and 94%, and the specificity was about 96% and 98% in the macroscopic and microscopic studies, respectively. Light-induced fluorescence detection using topical Photofrin provides a sensitive, noninvasive technique for the early identification of malignant neoplasms in the oral cavity. The lesions must be scanned point by point with the tip of the collection fiber to assess the oral tissue mucosa. This method is time-consuming, especially for examining large areas of lesions. (continued) 16 Ear, Nose & Throat Journal 00(0) sensitivity. However, the sensitivity of CLE has decreased to 80% while specificity increased to 100%, indicating an improvement in excluding nonmalignant lesions.71 Confocal reflectance microscopy utilizes laser light at a near-infrared wavelength (830 nm), penetrating the tissue of interest and illuminating a single point. A study that compared confocal reflectance microscopy and histopathology with hematoxylin and eosin staining showed that this is a promising method of diagnosis, with a sensitivity of 96.3%, specificity of 92.3%, positive predictive value of 93%, and negative predictive value of 96%.72 The HRME is a diagnostic tool that leverages a coherent fiber bundle to capture high-resolution fluorescence images of the tissue in contact with the device’s distal tip. In this setup, a camera plays the crucial role of seizing high-quality digital images, which are then transferred to a computer for further analysis.73 The HRME provides a noninvasive method to visualize tissue architecture and changes in cellular behavior, potentially aiding in the early detection and diagnosis of conditions like oral leukoplakia. However, this technique requires specialized equipment and trained personnel, and the effectiveness of HRME can be influenced by factors such as the specific location of the tissue being examined and the patient’s individual characteristics. Fluorescence lifetime imaging (FLIM) is a promising diagnostic technique that analyzes a tissue area of about 1.6 cm × 1.6 cm. It operates by measuring both tissue autofluorescence and the decay of fluorescence over time. This unique approach allows for the potential estimation of the contributions of specific fluorophores like, Nicotinamide Adenine Dinucleotide (NADH), Flavin Adenine Dinucleotide (FAD), and collagen, which are molecules that emit fluorescence when excited by light. The differential presence of these fluorophores can provide valuable information about tissue health and potential abnormalities. Particularly useful in differentiating dysplastic lesions from benign inflammatory lesions,74 FLIM offers a noninvasive method to identify precancerous or cancerous changes. However, the technique requires specialized equipment and expertise in data interpretation, and its effectiveness can be affected by factors such as the patient’s individual characteristics and the specific location of the tissue being examined. Multiphoton microscopy represents a fluorescence imaging technique that provides cross-sectional images of tissues at different depths, reaching up to 1 mm.75 This approach allows for the assessment of cellular invasion beyond the basement membrane, a key characteristic of invasive diseases. By offering a detailed look at tissue architecture and cellular behavior in their native environment, multiphoton microscopy can contribute to the early detection and diagnosis of conditions like oral leukoplakia. In a study conducted by Matsui et al, this technique demonstrated a high sensitivity of 96% and a specificity of 84%,76 indicating its potential as a precise diagnostic tool. However, like all diagnostic techniques, multiphoton microscopy comes with its own set of requirements, including the need for specialized equipment and trained personnel for accurate operation and interpretation of results. Light-based systems. Light-based detection systems such as chemiluminescence and photodynamic diagnosis have been developed to aid in diagnosing oral leukoplakia at an early stage. By utilizing the structural abnormalities in oral leukoplakia, healthy and cancer cells emit different wavelengths of light. There are 2 main types of light-based systems, namely chemiluminescence and photodynamic diagnosis. There are multiple chemiluminescent devices that are available on the market. These devices use a light-based detection system to detect the different wavelengths reflected due to changes in cancer cell morphology. The main devices on the market are ViziLite (Zila Pharmaceuticals, Phoenix, AZ, United States), ViziLite Plus (Zila Pharmaceuticals, Phoenix, AZ, United States), and Microlux/DL (Microlux DL - AdDent, Inc., Danbury, CT, United States). The oral cavity is first rinsed with acetic acid before being examined under chemiluminescent illumination. This allows the user to differentiate between normal and hyperkeratinized epithelium. Dysplastic or hyperplastic tissue has increased nuclear content that reflects light and hence appears white when viewed at low-energy wavelengths. Conversely, the normal epithelium appears dark.77 Studies have shown that ViziLite has a high sensitivity of 77.3% to 100% but a low specificity of 0% to 55.56% in detecting oral carcinoma.78-80 However, it has been shown to detect leukoplakia more than other forms of oral lesions.75 A newer version of ViziLite called ViziLite Plus combines both ViziLite and TB and has been shown to improve the specificity of 75.5% to 78% but a decreased sensitivity rate. The principles of Microlux/DL are similar to ViziLite. It uses a battery-powered LED light that emits blue light. According to Ibrahim et al, the specificity and sensitivity of identifying oral lesions were 100% and 32%, respectively, meaning that it could locate possible lesions but could not differentiate the types of lesions.80 Photodynamic diagnosis involves treating cells with a photoactivated compound that accumulates more in cells with malignant potential when exposed to photoirradiation. A common compound used is 5-aminolevulinic acid (ALA), which induces the fluorescence of protoporphyrin IX in cancerous and precancerous cells. The procedure involves rinsing the oral cavity with a 0.4% ALA solution followed by exposure to a specific light wavelength of 405 nm. This technique boasts a high sensitivity, as studies Khong et al 17 have reported a range between 80% and 99%.81 However, its specificity can be compromised in patients with a history of radiotherapy, as indicated by some studies.82 Despite this limitation, using ALA and photodynamic diagnosis can provide valuable information for the early detection and diagnosis of conditions like oral leukoplakia. It is worth noting, however, that the effectiveness of this technique can be influenced by factors such as the patient’s individual characteristics and the specific location of the tissue being examined. Discussion Although several diagnostic methods for the early detection of the oral cavity neoplastic lesions have been demonstrated, their use currently remains controversial and debated.20 We aimed to critically describe the noninvasive detection techniques for premalignant oral cavity lesions concerning each method’s sensitivity and specificity. Dentists, ENTs, or maxillofacial surgeons are the specialists who are the first to deal with premalignant lesions of the oral cavity. Identification of such lesions should occur as early as possible.25 Early diagnosis of premalignant lesions of the oral cavity is essential. It is based on oral screening. The latter could avoid delayed referrals, thus reducing mortality in the SCC.19 It has been reported that SCC can develop from oral potentially malignant disorders, and its diagnosis is an important preventive step with a major impact on patient survival and future quality of life.21 However, visual inspection has several limitations, such as the inability to distinguish high-risk benign lesions from other diseases and morbid conditions of the oral mucosa. Tissue biopsy is an invasive, time-consuming, painful, operator-dependent method frequently not readily accepted by patients.24 Despite this, oral biopsy remains the gold standard method today. Typically, nonearly detection of a premalignant lesion leads to an advanced stage at diagnosis.82-84 However, several follow-ups have shown that the risk of malignant transformation can persist for over 10 years. For this reason, long-term follow-up with regular checkups by the oral surgeon, maxillofacial, or ENT specialist is required. The early diagnosis of premalignant lesions of the oral cavity can make use of noninvasive, easy-to-use, and effective methods.21 Salivary diagnostics is a method that has spread in recent years.39 Saliva has a very complex composition, including enzymes, antibodies, hormones, antimicrobial elements, and cytokines.40 The saliva collection is easy, safe, noninvasive, and inexpensive. In recent years, interest has grown in miRNAs (found in various biological fluids, including saliva) being the latter considered as potential markers for diagnosing, prognosis, and evaluating the effectiveness of treating multiple diseases.6 It is likely that miRNA expression profiling not only allows the identification of neoplastic tissue and its histological origin but also discriminates between different subtypes of malignant lesions.40 Regarding inflammation, understanding the role of miRNAs in its regulation could be important in helping understand the pathogenesis of a large group of diseases. Among the diagnostic methods based on vital staining, RB staining and Lugol’s iodine staining must be cited. RB staining was used to delineate the extent of corneal and conjunctival neoplasms. Therefore, such characteristics of RB have enlightened us to perform surveying research on premalignant and malignant lesions of the oral cavity. Lesions more stained by RB had a higher likelihood of being OSCC or epithelial dysplasia than those less stained.45 Thus, RB staining might have the potency to be used as a diagnostic aid to detect oral premalignant or malignant lesions for clinicians. With regard to the methods based on diagnostics with optical systems, particular interest is given to methods based on fluorescence/autofluorescence and NBI.64 In soft tissues, potentially malignant lesions and tumor lesions can be detected. Thus, the optical fluorescence system allows for simple, noninvasive, real-time diagnosis and identification of structures and alterations in the oral cavity, revealing lesions that are not easily detectable with lighting.73 NBI is a new optical technology already widely applied in diagnosing gastrointestinal lesions. Unlike the epidermal tissue, the mucous membrane of the oral cavity has few keratinized layers and lacks appendages deriving from the outlet of the minor salivary glands.64 Hence the capillaries in the papillae of the connective tissue under the epithelium are hardly observed from the external mucosal surface. While in the healthy oral mucosa, the pegs of the epithelial network and the connective tissue papillae are regularly connected, in cancer, this connection becomes irregular.76 Therefore, visually, the capillaries will assume an irregular and dense distribution. By observing a tumor lesion under magnification, the proliferation of capillaries can be recognized as a characteristic spotting of the tissue. Another method used for the early diagnosis of oral premalignant lesions is chemiluminescence.77 Many systems use this method; the 2 most used are the ones based on luminol and based on peroxyoxalate. Regardless of the system, blue-white light is absorbed by healthy cells and reflected by cells with abnormal nuclei, including dysplastic and neoplastic cells. The acetic acid rinse putatively removes debris and disrupts the glycoprotein barrier on the epithelium’s surface, allowing light penetration.34 Variable dye uptake was observed between exophytic and ulcerated SCC. The dye showed excellent retention and staining in ulcerates compared to exophytic lesions due to the increased intercellular spaces that allow for better dye 18 Ear, Nose & Throat Journal 00(0) penetration.85,86 In conclusion, chemiluminescent light is useful as an additional diagnostic tool for oral cancer care and follow-up Potentially Malignant Epithelial Lesions (PMELs) of subjects treated for the same. A significant limitation in diagnosing oral potentially malignant lesions, such as oral leukoplakia, is the need for more awareness and knowledge among dental and medical professionals. Despite the availability of various techniques for oral examination, the challenge persists due to a limited understanding of oral leukoplakia and its diagnostic process.87-92 Bridging these gaps by enhancing awareness and knowledge is crucial to facilitate early detection and prevent the progression of OSCC and other potentially malignant lesions in the oral cavity. Subjectivity in diagnosis due to visual interpretation and the variability in lesion appearance further complicate accurate identification.93,96 In addition, sampling bias during biopsy procedures, the absence of reliable predictive biomarkers, limited accessibility to specialized care, and patient compliance with follow-up appointments all contribute to the challenges in achieving early and precise diagnoses. Overcoming these limitations requires standardized diagnostic criteria, diagnostic techniques, biomarker research advancements, improved accessibility to specialized care, and enhanced patient education and engagement. Conclusion The early diagnosis of oral premalignant conditions is crucial for minimizing invasive surgical intervention, providing a better prognosis, and improving the quality of life for patients. Currently, several diagnostic tools for screening are available, enhancing the characterization of suspicious lesions. Today, surgical biopsy and histology remain the primary therapeutic choices, but the advent of salivary biomarkers presents promising new techniques. Scientific progress is continually modernizing diagnostic procedures to facilitate early detection of oral cancer and reduce diagnostic delay. Although any light-based diagnostic device could aid in diagnosing oral mucosal lesions, chemiluminescence examination can delineate oral lesions more effectively, as the edges of the lesions exhibit improved brightness and clarity. Other emerging techniques include OCT and molecular imaging, which offer high-resolution imaging capabilities. Moreover, when combined with quantitative autofluorescence analysis, the autofluorescence-based system could differentiate between tumors and benign oral dysplasia. Acknowledgments None. Data Availability No new data were created. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. Ethical Approval The study was conducted in accordance with the Declaration of Helsinki. Grant Number No grant was associated. Informed Consent Informed consent was waived for the review study. ORCID iDs Jerome Rene Lechien https://orcid.org/0000-0002-0845-0845 Carlos Chiesa-Estomba https://orcid.org/0000-0001-9454-9464 Antonino Maniaci https://orcid.org/0000-0002-1251-0185 Thomas Radulesco https://orcid.org/0000-0002-5939-5372 Trial Registration The trial registration was waived for the review study. References 1. Mortazavi H, Safi Y, Baharvand M, Jafari S, Anbari F, Rahmani S. Oral white lesions: an updated clinical diagnostic decision tree. Dent J (Basel). 2019;7(1):15. doi:10.3390/ dj7010015 2. 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