Noninvasive diagnostic adjuncts for the evaluation of potentially premalignant oral epithelial lesions: current limitations and future directions

Potentially premalignant oral epithelial lesions (PPOELs) are a group of clinically suspicious conditions, of which a small percentage will undergo malignant transformation. PPOELs are suboptimally diagnosed and managed under the current standard of care. Dysplasia is the most well-established marker to distinguish high-risk PPOELs from low-risk PPOELs, and performing a biopsy to establish dysplasia is the diagnostic gold standard. However, a biopsy is limited by morbidity, resource requirements, and the potential for underdiagnosis. Diagnostic adjuncts may help clinicians better evaluate PPOELs before definitive biopsy, but existing adjuncts, such as toluidine blue, acetowhitening, and autofluorescence imaging, have poor accuracy and are not generally recommended. Recently, in vivo microscopy technologies, such as high-resolution microendoscopy, optical coherence tomography, reflectance confocal microscopy, and multiphoton imaging, have shown promise for improving PPOEL patient care. These technologies allow clinicians to visualize many of the same microscopic features used for histopathologic assessment at the point of care. Potentially premalignant oral epithelial lesions (PPOELs) are a group of clinically suspicious conditions, of which a small percentage will undergo malignant transformation. PPOELs are suboptimally diagnosed and managed under the current standard of care. Dysplasia is the most well-established marker to distinguish high-risk PPOELs from low-risk PPOELs, and performing a biopsy to establish dysplasia is the diagnostic gold standard. However, a biopsy is limited by morbidity, resource requirements, and the potential for underdiagnosis. Diagnostic adjuncts may help clinicians better evaluate PPOELs before definitive biopsy, but existing adjuncts, such as toluidine blue, acetowhitening, and autofluorescence imaging, have poor accuracy and are not generally recommended. Recently, in vivo microscopy technologies, such as high-resolution microendoscopy, optical coherence tomography, reflectance confocal microscopy, and multiphoton imaging, have shown promise for improving PPOEL patient care. These technologies allow clinicians to visualize many of the same microscopic features used for histopathologic assessment at the point of care. Statement of Clinical RelevanceExisting methods to evaluate potentially premalignant oral epithelia lesions often lead to suboptimal diagnosis and management, increasing the global oral cancer burden. Novel in vivo microscopy technologies allow clinicians to visualize microscopic tissue features at the point of care to facilitate evidence-based patient care. Existing methods to evaluate potentially premalignant oral epithelia lesions often lead to suboptimal diagnosis and management, increasing the global oral cancer burden. Novel in vivo microscopy technologies allow clinicians to visualize microscopic tissue features at the point of care to facilitate evidence-based patient care. Over 300,000 new cases of oral cancer are diagnosed annually worldwide, with particularly high incidence rates in South and Southeast Asia, Europe, Latin America, and the Caribbean and Pacific nations. Risk factors for oral cancer include tobacco use, excessive consumption of alcohol, and betel quid chewing. The 5-year survival rate for oral cancers is only about 50%, largely because oral cancers are most commonly diagnosed in advanced stages of disease.1Ferlay J. Soerjomataram I. Dikshit R. et al.Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.Int J Cancer. 2015; 136: E359-E386Crossref PubMed Scopus (21251) Google Scholar, 2Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer.Oral Oncol. 2009; 45: 309-316Abstract Full Text Full Text PDF PubMed Scopus (2119) Google Scholar Oral cancers are typically preceded by potentially premalignant oral epithelial lesions (PPOELs), a group of clinically suspicious conditions, including leukoplakia, erythroplakia, submucous fibrosis, and lichen planus.3Warnakulasuriya S. Johnson N.W. Van Der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa.J Oral Pathol Med. 2007; 36: 575-580Crossref PubMed Scopus (977) Google Scholar Although the majority of PPOELs do not progress to cancer, distinguishing high-risk PPOELs from low-risk PPOELs is difficult. As a result, under the current standard of care, PPOELs are suboptimally diagnosed and managed. In this article, we will discuss the limitations of the existing methods of PPOEL risk assessment, including biopsy and noninvasive diagnostic adjuncts. Then, we will introduce in vivo microscopy (IVM), a novel group of technologies that could help clinicians overcome these limitations by allowing them to visualize microscopic, histology-like features of PPOELs at the point of care. The most widely accepted marker to assess the risk of a PPOEL eventually undergoing malignant transformation is the presence and grade of dysplasia in the lesion. Dysplasia is defined as the presence of specific epithelial architectural and cytologic changes and is graded as mild, moderate, or severe based on the depth and severity of the changes. It is frequently assumed that oral carcinogenesis involves PPOELs that undergo a gradual progression beginning with hyperplasia and evolving through stages of mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ (CIS), and finally carcinoma after cellular invasion through the basement membrane. In reality, it is likely that in some cases, the course of oral cancer does not occur in such an orderly manner. PPOELs with dysplasia are considered non-obligate precursors of oral squamous cell carcinoma (OSCC), indicating that not all dysplastic PPOELs will progress to invasive cancer. PPOELs containing dysplasia are more likely to undergo malignant transformation, and the risk increases as the grade of dysplasia increases. One recent meta-analysis estimated the malignant transformation rate of all leukoplakia, regardless of dysplasia, at 3.4%, with results of individual studies ranging from 0.13% to 34.0%.4Warnakulasuriya S. Ariyawardana A. Malignant transformation of oral leukoplakia: a systematic review of observational studies.J Oral Pathol Med. 2016; 45: 155-166Crossref PubMed Scopus (221) Google Scholar Lesions containing dysplasia have a higher transformation rate. Bouquot et al.5Bouquot J.E. Speight P.M. Farthing P.M. Epithelial dysplasia of the oral mucosa—diagnostic problems and prognostic features.Curr Diagn Pathol. 2006; 12: 11-21Abstract Full Text Full Text PDF Scopus (86) Google Scholar estimated that less than 5% of mild dysplasia cases undergo eventual malignant transformation compared with 3% to 15% for moderate dysplasia and 16% (range 7%-50%) for severe dysplasia or CIS. A 2009 meta-analysis estimated the transformation rate as 12.1% (confidence interval [CI] 8.1%-17.9%) for dysplastic lesions with a 10.3% rate (CI 6.1%-16.8%) for mild to moderate dysplasia and 24.1% (CI 13.3%-39.5%) for severe dysplasia and CIS.6Mehanna H.M. Rattay T. Smith J. McConkey C.C. Treatment and follow-up of oral dysplasia—a systematic review and meta-analysis.Head Neck. 2009; 31: 1600-1609Crossref PubMed Scopus (268) Google Scholar Differences in patient population and interobserver variation in diagnosis, treatment, and follow-up likely account for the inconsistent estimates. Despite ubiquitous use, dysplasia is an imperfect risk marker because at its core, carcinogenesis is driven by the accumulation of somatic mutations and epigenetic changes. The relationship between these key drivers of carcinogenesis and the dysplasia phenotype is unclear.7Barnes L. Eveson J.W. Reichart P. Sidransky D. Tumours of the oral cavity and oropharynx.in: World Health Organization Classification of Tumours. 9th ed. IARC Press, Lyon, France2005: 178Google Scholar Reports of OSCC arising from nondysplastic mucosal areas, the presence of genetic alterations in histologically normal epithelium adjacent to carcinoma, and the high recurrence rates after retinoic acid-induced regression of dysplasia provide evidence for the phenotype–genotype disparity.8Reibel J. Prognosis of oral pre-malignant lesions: significance of clinical, histopathological, and molecular biological characteristics.Crit Rev Oral Biol Med. 2003; 14: 47-62Crossref PubMed Scopus (444) Google Scholar, 9Brennan J.A. Mao L. Hruban R.H. et al.Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck.N Engl J Med. 1995; 332: 429-435Crossref PubMed Scopus (668) Google Scholar, 10Mao L. Papadimitrakopoulou V. Shin D.M. et al.Phenotype and genotype of advanced premalignant head and neck lesions after chemopreventive therapy.JNCI J Natl Cancer Inst. 1998; 90: 1545-1551Crossref PubMed Scopus (125) Google Scholar Thus, much effort has been devoted to the discovery of molecular biomarkers capable of distinguishing progressive PPOELs from nonprogressive PPOELs (reviewed elsewhere in this focus issue). Although genetic loss of heterozygosity is considered a better marker for predicting the malignant progression risk of a PPOEL, it has not been integrated into day-to-day clinical practice.11William W.N. Papadimitrakopoulou V. Lee J.J. et al.Erlotinib and the risk of oral cancer.JAMA Oncol. 2016; 2: 209Crossref PubMed Scopus (92) Google Scholar A number of studies have attempted to identify biomarkers to predict which patients are likely to develop OSCC after the diagnosis of PPOEL with dysplasia, but so far, no such biomarkers have been validated and prospectively shown to predict malignant transformation risk. Therefore, the degree of dysplasia will remain the key determinant for assessing the malignancy risk of PPOELs until emerging biomarkers are validated and integrated into clinical use. The current clinical gold standard for predicting the cancer progression risk of a PPOEL requires biopsy and microscopic evaluation of the resulting hematoxylin-and-eosin (H&E)–stained tissue section by a trained oral and maxillofacial or head and neck pathologist to determine the presence and grade of dysplasia or carcinoma. Conventional oral examination (COE) alone is insufficient for risk stratification. COE is generally effective for lesion identification, but not for the ensuing clinical workup for treatment planning. Once an oral lesion has been discovered, it must be classified as a PPOEL or a nonsuspicious lesion, a distinction that many general dental practitioners (GDPs) are not sufficiently trained to make.12Patel K.J. De Silva H.L. Tong D.C. Love R.M. Concordance between clinical and histopathologic diagnoses of oral mucosal lesions.J Oral Maxillofac Surg. 2011; 69: 125-133Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Seoane et al.13Seoane J. Warnakulasuriya S. Varela-Centelles P. Esparza G. Dios P. Oral cancer: experiences and diagnostic abilities elicited by dentists in North-western Spain.Oral Dis. 2006; 12: 487-492Crossref PubMed Scopus (43) Google Scholar assessed 32 GDPs in Northwestern Spain by showing them photographs of 50 oral mucosal lesions, including 31 benign lesions, 12 PPOELs, and 7 cases of OSCC. The GDPs distinguished OSCC and PPOELs from benign lesions with only 57.8% sensitivity and 53% specificity, a result only marginally better than random guessing. Specialists are likely more capable of distinguishing PPOELs from nonsuspicious lesions.12Patel K.J. De Silva H.L. Tong D.C. Love R.M. Concordance between clinical and histopathologic diagnoses of oral mucosal lesions.J Oral Maxillofac Surg. 2011; 69: 125-133Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Once a lesion has been classified as a PPOEL, classifying it as dysplastic or nondysplastic based on COE is extremely difficult regardless of training level. A 2012 meta-analysis estimated that COE had 93% sensitivity but only 31% specificity for the identification of dysplasia or carcinoma.14Epstein J.B. Güneri P. Boyacioglu H. Abt E. The limitations of the clinical oral examination in detecting dysplastic oral lesions and oral squamous cell carcinoma.J Am Dent Assoc. 2012; 143: 1332-1342Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Most of the studies had been performed in specialty clinics, and inclusion criteria ranged from including only PPOELs and OSCC to including any oral mucosal lesion. More recently, a retrospective analysis of 1003 oral lesions at a tertiary medical center found that oral and maxillofacial surgeons distinguished dysplastic or cancerous lesions from benign lesions with a sensitivity of 48.6% and specificity of 98.1%.15Forman M.S. Chuang S.-K. August M. The accuracy of clinical diagnosis of oral lesions and patient-specific risk factors that affect diagnosis.J Oral Maxillofac Surg. 2015; 73: 1932-1937Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar These studies demonstrated the ineffectiveness of COE for PPOEL risk stratification, although the specific balance of sensitivity and specificity may vary, in part, as a result of differences in the definition of a positive COE. Once the decision has been made to perform biopsy on a PPOEL, the clinician must select a biopsy site, which should represent the area of the lesion most likely to contain dysplasia or carcinoma. The presence and grade of dysplasia and invasive carcinoma frequently vary throughout a lesion, and dysplasia may even be present in clinically normal mucosal areas outside its visible boundaries. Excisional biopsy can be performed for smaller lesions and could prevent sampling bias, but the risk of incomplete excision of malignant lesions exists and the procedure is excessively aggressive in the case of benign lesions. For these reasons, incisional biopsy is typically preferred, but it does not assess an entire lesion. This sampling bias can lead to underdiagnosis or misdiagnosis, particularly in cases of multifocal, large, or nonhomogeneous PPOELs. Goodson et al.16Goodson M. Kumar A. Thomson P. P171. Oral precancer excision is required for definitive diagnosis: incisional vs excisional biopsies in oral leukoplakia management.Oral Oncol. 2011; 47: S128-S129Abstract Full Text Full Text PDF Google Scholar analyzed 152 patients with a single leukoplakic lesion treated with laser excision, a mean of 4.43 months after a preoperative incisional biopsy, and found that 50% of the incisional biopsy diagnoses were upgraded on the basis of the excised specimen. Chen et al.17Chen S. Forman M. Sadow P.M. August M. The diagnostic accuracy of incisional biopsy in the oral cavity.J Oral Maxillofac Surg. 2016; 74: 959-964Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar assessed 80 oral mucosal lesions and found that incisional biopsy missed 6 OSCC cases. Underdiagnosis of dysplasia was not specifically discussed. In the largest sampling bias study, Lee et al.18Lee J.-J. Hung H.-C. Cheng S.-J. et al.Factors associated with underdiagnosis from incisional biopsy of oral leukoplakic lesions.Oral Sur Oral Med Oral Pathol Oral Radiol Endod. 2007; 104: 217-225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar retrospectively found that incisional biopsy underdiagnosed 29.5% of leukoplakic lesions in 200 patients who underwent surgical resection within 30 days of the initial incisional biopsy. In 11 of the 24 patients with a diagnosis of carcinoma after surgery, the initial diagnosis based on incisional biopsy was no dysplasia or mild dysplasia. The 42 patients who underwent multiple incisional biopsy procedures had a lower rate of underdiagnosis (11.9%). Additionally, the authors found higher rates of underdiagnosis in nonhomogeneous lesions than in homogeneous lesions. The authors defined homogeneous leukoplakia as a white lesion with a uniform flat thin appearance or a white lesion with shallow cracks within a smooth, wrinkled, or corrugated surface of constant texture. Nonhomogeneous leukoplakia was defined as an irregularly flat, nodular, or exophytic white or white and red lesion. There are several other limitations associated with biopsy (Table I). Multiple steps are required to prepare the biopsy sample before it is suitable for microscopic diagnosis. A skilled specialist is required to perform the biopsy procedure properly, and a trained pathologist is required to interpret the histopathologic findings. These requirements make it necessary for care providers to follow up with patients for several days after their initial visit, limiting the use of biopsy in resource-poor clinical sites. Grading dysplasia is subjective and involves high levels of interobserver and intraobserver variance, even among oral and maxillofacial or head and neck pathology specialists.19Speight P.M. Update on oral epithelial dysplasia and progression to cancer.Head Neck Pathol. 2007; 1: 61-66Crossref PubMed Scopus (160) Google Scholar, 20Speight P.M. Abram T.J. Floriano P.N. et al.Interobserver agreement in dysplasia grading: toward an enhanced gold standard for clinical pathology trials.Oral Surg Oral Med Oral Pathol Oral Radiol. 2015; 120 (e2): 474-482Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The 2017 World Health Organization criteria for dysplasia requires assessment of numerous subjective factors with no objective method to weigh them.21El-Naggar A. Chan J. Grandis J. Takata T. Slootweg P. WHO Classification of Head and Neck Tumours.4th ed. IARC Press, Lyon, France2017Google Scholar Discrepancies are particularly common with regard to cases of PPOELs diagnosed with mild dysplasia because of the difficulty in distinguishing mild dysplasia from reactive and reparative atypia associated with inflammation and ulcerations, respectively. Finally, invasive biopsy is associated with morbidity and cost.Table ILimitations of biopsySampling bias as a result of site selectionTrained clinician required to perform biopsy correctlyTrained pathologist and processing facilities required for diagnosisLengthy time (days) to diagnosisInterobserver and intraobserver variancePatient morbidity and discomfort Open table in a new tab The limitations discussed above have motivated ongoing attempts to develop diagnostic adjuncts to assist with PPOEL evaluation. In particular, there is a clinical need for diagnostic adjuncts that can augment COE to (1) help clinicians decide which lesions need biopsy by distinguishing high-risk PPOELs that harbor dysplasia or cancer from low-risk PPOELs and other mucosal lesions; (2) identify the highest risk sites within a PPOEL for biopsy guidance; and (3) longitudinally monitor PPOELs to decide if repeat biopsy procedures are necessary. At present, diagnostic adjuncts should not replace COE—biopsy of PPOELs with sufficient clinical suspicion is recommended regardless of results obtained from adjuncts, nor should they replace biopsy for definitive diagnosis. The ideal diagnostic adjunct would provide accurate correlation with dysplasia and cancer, provide results immediately at the point of care, and evaluate a large area for biopsy guidance. Additionally, the diagnostic adjunct should be minimally-invasive, involve low cost, require few consumables, require minimal training to use, and allow objective interpretation. Common diagnostic adjuncts include toluidine blue, brush biopsy with cytology, acetowhitening with chemiluminescence, and autofluorescence imaging. Numerous authors have reviewed the evidence on these adjuncts,22Lingen M.W. Abt E. Agrawal N. et al.Evidence-based clinical practice guideline for the evaluation of potentially malignant disorders in the oral cavity.J Am Dent Assoc. 2017; 148: 712-727Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 23Lingen M.W. Kalmar J.R. Karrison T. Speight P.M. Critical evaluation of diagnostic aids for the detection of oral cancer.Oral Oncol. 2008; 44: 10-22Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 24Macey R. Walsh T. Brocklehurst P. et al.Diagnostic tests for oral cancer and potentially malignant disorders in patients presenting with clinically evident lesions.Cochrane Database Syst Rev. 2015; (CD010276)Google Scholar so we only briefly discuss them in the context of above criteria (Table II).Table IIExisting diagnostic adjunctsTechniqueSensitivitySpecificityTime to resultSize of area assessedCostTraining requiredInvasivenessObjective interpretationBiopsyGold standardGold standardDaysSmallHighHighHighNoCytology/brush biopsyHighHighDaysSmallMediumMediumModeratePartiallyToluidine blueOSCC: HighLowImmediateLargeLowMediumMinimalNoDysplasia: LowAcetowhitening/chemiluminescenceLowLowImmediateLargeMediumMediumMinimalNoAutofluorescence imagingHighLowImmediateLargeMediumMediumNoneNo, but potentially yesOSCC, oral squamous cell carcinoma. Open table in a new tab OSCC, oral squamous cell carcinoma. Use of the vital dye toluidine blue consists of an initial rinse of the oral cavity with acetic acid followed by toluidine blue. It is thought that the dye has an affinity for DNA, so increased DNA levels seen in dysplasia and carcinoma lead to greater staining.25Sridharan G. Shankar A.A. Toluidine blue: a review of its chemistry and clinical utility.J Oral Maxillofac Pathol. 2012; 16: 251-255Crossref PubMed Scopus (247) Google Scholar Toluidine blue is low cost, provides immediate results, and can be used to assess the entire oral cavity but is limited by false positive results for inflammatory lesions or ulcers, low sensitivity for dysplasia, and subjective interpretation. An oral biopsy brush can be used to remove transepithelial cells with minimal invasion, and the cells are transferred to a slide and evaluated cytologically. Cytologic smears can then be evaluated for cellular atypia. When performed properly, brush biopsy is potentially the most accurate adjunct. A meta-analysis of cytology reported sensitivity and specificity of 91%.24Macey R. Walsh T. Brocklehurst P. et al.Diagnostic tests for oral cancer and potentially malignant disorders in patients presenting with clinically evident lesions.Cochrane Database Syst Rev. 2015; (CD010276)Google Scholar However, brush biopsies can only assess a small region of the oral mucosa, do not provide results for days, and are not reliable for evaluating PPOELs with thick keratin layers. Acetowhitening entails rinsing the oral cavity with acetic acid and then using a chemiluminescent light to look for mucosal areas with a white appearance indicating a PPOEL. This approach can assess large regions at the point of care, but studies have demonstrated poor sensitivity and specificity.26Mehrotra R. Singh M. Thomas S. et al.A cross-sectional study evaluating chemiluminescence and autofluorescence in the detection of clinically innocuous precancerous and cancerous oral lesions.J Am Dent Assoc. 2010; 141: 151-156Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 27Farah C.S. McCullough M.J. A pilot case control study on the efficacy of acetic acid wash and chemiluminescent illumination (ViziLite) in the visualisation of oral mucosal white lesions.Oral Oncol. 2007; 43: 820-824Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar ViziLite (Zila Pharmaceuticals Inc., Phoenix, AZ) is an example of an acetowhitening and chemiluminescence product. Autofluorescence imaging (AFI) is based on the concept that dysplasia and cancer cause measurable changes in tissue autofluorescence, defined as fluorescence intrinsic to tissue. Epithelial fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and stromal fluorophores, such as collagen and elastin, are the primary contributors to autofluorescence in the normal oral mucosa. Dysplasia and cancer are typically accompanied by a large loss of green autofluorescence, along with a small increase in red autofluorescence. Certain changes, such as increased metabolism, increased nuclear area and pleomorphism, increased epithelial thickness, increased vascularization, breakdown of collagen cross-links, and production of fluorophores by bacteria, contribute to this effect. Several AFI devices have become commercially available in the past decade, including the VELscope (LED Dental, Atlanta, GA), Identafi (StarDental-DentalEZ, Englewood, CO), and OralID (Forward Science, Stafford, TX). Clinical use typically involves illumination of the tissue with blue or violet light in a darkened room, allowing the clinician to visualize tissue autofluorescence. Normal mucosal areas appear bright, whereas suspicious areas exhibit loss of fluorescence and appear dark. Interestingly, loss of fluorescence frequently extends beyond the visible borders of a lesion, and these extensions often harbor dysplasia and loss of heterozygosity.28Poh C.F. Zhang L. Anderson D.W. et al.Fluorescence visualization detection of field alterations in tumor margins of oral cancer patients.Clin Cancer Res. 2006; 12: 6716-6722Crossref PubMed Scopus (235) Google Scholar A randomized controlled trial is underway to investigate whether AFI can be used to delineate margins during surgical resection of OSCC to reduce recurrence rates.29Poh C.F. Anderson D.W. Durham J.S. et al.Fluorescence visualization–guided surgery for early-stage oral cancer.JAMA Otolaryngol Neck Surg. 2016; 142: 209Crossref PubMed Scopus (42) Google Scholar The advantages of AFI include high sensitivity for dysplasia and cancer, capability to assess large areas of the oral mucosa at the point of care, nonrequirement of consumables, and noninvasiveness. Commercially available systems rely on subjective interpretation of autofluorescence, but AFI offers the potential for more objective interpretation. Unfortunately, AFI is limited by false positive results. Lesions of various etiologies have different autofluorescent properties (Table III); most commonly, inflammatory benign lesions also often exhibit a loss of fluorescence. Keratin is autofluorescent,30Pavlova I. Williams M. El-Naggar A. Richards-Kortum R. Gillenwater A. Understanding the biological basis of autofluorescence imaging for oral cancer detection: high-resolution fluorescence microscopy in viable tissue.Clin Cancer Res. 2008; 14: 2396-2404Crossref PubMed Scopus (199) Google Scholar and in the authors' experience, hyperkeratinized high-risk PPOELs such as proliferative verrucous leukoplakia may not show loss of fluorescence even in the presence of dysplasia or cancer. The VELscope is approved for use by the U.S. Food and Drug Administration as an adjunct to enhance the visualization of oral mucosal abnormalities but not as a tool for risk stratification. AFI may have clinical utility for risk assessment during longitudinal monitoring of patients with known high-risk PPOELs or previous history of cancer.Table IIIEffects of tissue changes on autofluorescenceAdapted with permission from Vigneswaran and El-Naggar. Early detection and diagnosis of oral premalignant squamous mucosal lesions. In: Wong BJ, Ilgner J, eds. Biomedical Optics in Otorhinolaryngology. New York: Springer; 2016:601-618.Histologic assessmentAutofluorescence featureEpithelial hyperplasiaNo changeDysplasiaComplete or partial lossInvasive carcinomaComplete lossVerruciform hyperkeratosisNo change or increaseInfectiousComplete or partial lossVascular lesionsComplete or partial lossSubmucous fibrosisEnhancedAmalgam pigmentation (tattoo)Complete lossFocal melanosisComplete lossHairy leukoplakia / candidiasisRed to orange spectrum Open table in a new tab It is clear from the above discussion that no existing diagnostic adjunct meets all of the ideal criteria. The adjuncts that provide immediate results from large regions (toluidine blue, acetowhitening and chemiluminescence, and AFI) suffer from limited accuracy because they do not directly assess the microscopic features used to diagnose dysplasia and cancer. Brush biopsy allows for direct assessment of cellular atypia but provides delayed results from small regions. Accordingly, the most recent American Dental Association guidelines included a conditional recommendation against the use of cytologic adjuncts; autofluorescence imaging; tissue reflectance adjuncts, such as chemiluminescence; and vital staining adjuncts, such as toluidine blue, for the assessment of clinically evident lesions.22Lingen M.W. Abt E. Agrawal N. et al.Evidence-based clinical practice guideline for the evaluation of potentially malignant disorders in the oral cavity.J Am Dent Assoc. 2017; 148: 712-727Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar In recent years, a class of optical imaging technologies known as IVM has emerged as a promising new class of diagnostic adjunct. IVM combines the strengths of existing adjuncts by enabling high-resolution, microscopic imaging of intact tissue for disease detection and diagnosis at the point of care. IVM produces images of microscopic tissue features by measuring tissue optical properties, such as reflectance, scattering, absorption, and fluorescence emission, which are frequently altered in disease states. IVM has been proposed for a range of clinical applications, including disease diagnosis, disease risk stratification, longitudinal monitoring of patients, and surgical margin delineation. IVM instrumentation has been integrated into many form factors, such as endoscopes, catheters, needles, and benchtop devices, allowing for their use in a variety of anatomic locations. Currently, an IVM technique called optical coherence tomography (OCT) is the standard of care in ophthalmology for retinal imaging.31Adhi M. Duker J.S. Optical coherence tomography—current and future applications.Curr Opin Ophthalmol. 2013; 24: 213-221Crossref