Comparative evaluation of autofluorescence imaging and histopathological investigation for oral potentially malignant disorders in Taiwan

The concept of oral potentially malignant disorders (OPMD) was established by Warnakulasuriya et al., who stated that they are a family of morphological alterations among which some may have an increased potential for malignant transformation. It is also thought to be an indicator of risk for future malignancies elsewhere in the oral mucosa (appearing clinically normal) [1]. In Taiwan, leukoplakia, erythroplakia, and submucous fibrosis are the most frequently observed OPMD in addition to others such as lichen planus [2‐4]. Although the prevalence of OPMD in the general population is debatable, 1–5% has been commonly accepted as the prevalence in the Western countries [5]. Because of cultural differences and the habitual use of carcinogenic products, the prevalence of OPMD can reach 24.4% in certain areas of Taiwan [6].

The current standard for the detection of OPMD, which mainly comprises leukoplakia, erythroplakia, and erythroleukoplakia, is a conventional oral examination (COE) [7]. Although it is insufficient for differentiating categories of OPMD, epithelial dysplasia is often observed in the tissue of patients with OPMDs [8]. Furthermore, compared to more mild histopathological findings of hyperkeratosis or epithelial hyperplasia, epithelial dysplasia is a more conclusive determinant of malignant potential [9, 10], which the malignant transformation is at a rate of 2.2–38.1% [11, 12]. Evidence of dysplasia and even microinvasive carcinoma has been missed when performing a COE [13]. Therefore, epithelial dysplasia after OPMD diagnosis is an important risk factor for poor prognosis and transformation [14], and the precise detection not only in OPMD but also in epithelial dysplasia prior to management is crucial and may prevent advancement in disease progression [15].

In this study, we used a noninvasive, handheld camera device designed to visualize early mucosal changes using the principles of tissue autofluorescence. The autofluorescence camera utilizes blue light in the spectrum of 400–460 nm to detect the difference in luminance between healthy tissue and diseased tissue. Excited fluorophores intrinsic in the oral mucosa result in pale green autofluorescence, indicating normal tissue, whereas abnormal tissue is associated with the loss of autofluorescence, which may be caused by structural changes. For example, the thickening of the epithelium, hyperchromatism, increased cellular or nuclear pleomorphism, and increased microvascularity all lead to the increased absorption or scattering of light; thus, abnormal tissues with such structural changes appear dark in contrast [16, 17]. Because of this loss of autofluorescence in abnormal tissues, autofluorescence imaging is used as an adjunct tool to assess the risk of potentially malignant disorders and select optimal biopsy sites [18‐20].

Patient profiles are presented in Table 1. In 126 patients included in this study, most patients were men (n = 110), and the lesion sites were most frequently located on the buccal mucosa (n = 71).

In Taiwan, oral mucosal disorders are often neglected until the advanced stage and are then usually treated in a final referral medical center. The early diagnosis of OPMD may serve as a preventive measure in the high-risk population [21]. Reviews conducted in the Western countries reveal that approximately 80% of cases of leukoplakia display no evidence of dysplasia, but biopsy indicates the remaining 10–20% are either dysplastic or are already invasive carcinomas [22]. In Taiwan, the occurrence of OPMD correlates highly with the habit of chewing betel nuts [23] and malignant transformation [24]. Attention should specifically be focused on premalignant lesions to prevent disease progression [25]. However, the COE of OPMDs is limited as a diagnostic method for predicting pathological dysplasia [26].

Most adjuncts for assisting clinicians in daily OPMD- and cancer-related work are considered most suitable for use in secondary-care facilities, such as the current study site; however, the more effective purpose of these adjuncts is to assist the specialists in selecting biopsy sites for cancer and OPMD surveillance [19]. Autofluorescence imaging as an adjunctive tool has gained popularity as a modality because its physical effect is exerted without the need of other assisting agents [27]. When the oral mucosa is illuminated with blue excitation light with a wavelength of 400–460 nm, the targeted normal oral mucosa containing abundant endogenous autofluorescent substances, such as collagen and flavin adenine dinucleotide, emits green fluorescence with a wavelength of 515 nm [28]. Dysplasia is associated with alterations in the stromal architecture, which cause the loss of autofluorescence [29]. Abnormal tissue with the loss of autofluorescence appears dark in contrast; this may be because of lower levels of endogenous autofluorescent substances in the abnormal tissue than in the surrounding tissue [30]. This effect may be explained by a decrease in the main source of cellular fluorescence, namely flavin adenine dinucleotide, in tissues with dysplasia [31]. As the collagen cross-links and basal lamina are destroyed, glucose may be consumed in malignant tissue even in an aerobic environment; this is called the Warburg effect [32‐34].

The main location of OPMD was (according to incidence) the buccal mucosa, followed by the gingiva, tongue, and floor of the mouth [35‐37]. In our study, the location of the lesion was similar to conventional locations, which were mostly the buccal mucosa (67.6%), followed by the gingiva (13.2%) (Table 1). These data are comparable to the findings of a recent large population-based study in southern Taiwan [38]. We noted the highest occurring lesion was leukoplakia, and this is also similar to a recent large population study done in Taiwan [39]. Leukoplakia occurred in 25.5% of patients, which is a quarter of the study population, and erythroplakia and erythroleukoplakia occurred in 19.9% of patients. The group of “others” (Table 2), which were considered by the specialist in the COE to be benign, occurred in 38.1% of patients. Hyperkeratosis, hyperplasia, and inflammation were most reported by the pathologist.

Our comparative results showed a disappointing lack of specificity in the autofluorescence examination performed before excluding the non-OPMD features from the COE groups of the oral mucosal disorder checklist (Table 3). The specificity was 35.42% as the result because FVL was observed in the majority of the clinically diagnosed cases of non-OPMD. Low specificity was also observed in the study conducted by Awan et al.; they noted that the FVL findings were positive in the majority of the benign cases that may be mistakenly diagnosed as OPMD by the nonspecialist [40]. This lack of specificity creates drawbacks and remains a constant problem to other studies[41‐43]. In a recent meta-analysis, Luo et al. demonstrated an overall superiority in accuracy in detection of OPMD compared with other aerodigestive lesions using autofluorescence examinations. Additionally, approaching the diagnosis with algorithms could ensure the specificity in general practice [44].

Several studies utilizing autofluorescence examination for mucosal screening have found increased rates of detection of epithelial dysplasia in the high-risk group but with a substantial number of false positives [45‐47]. Pigmented, vascular, and inflammatory lesions are especially likely to present with LAF [16]. In our study, the clinical diagnoses of “others” in OPMD (Table 2) show that 48 cases in 38.1% of total identifications were benign lesions, including benign fractional keratosis, aphthous ulcer, and candidiasis thrush. After the exclusion of these non-OPMD cases using the current standard oral mucosal disorder checklist in Taiwan along with autofluorescence imaging, the specificity for dysplasia increased to 72.73% (Table 4). Moreover, the overall accuracy for dysplasia in OPMD cases improved after using the current standard oral mucosal disorder checklist in Taiwan in combination with autofluorescence imaging. However, with the understanding of oral mucosal disorders, COE protocol can aid in differentiating benign lesions from OPMDs, and proficient autofluorescence examination is required for proper FVL assessment before biopsy to identify dysplasia.

Digital autofluorescence imaging can be used as an adjunct tool of clinical value, assisting clinical specialists in OPMD surveillance and biopsy. The results suggest that the autofluorescence imaging could be considered as an aid when targeting mucosal dysplasia in high-risk patients, such as those with OPMD. Proper examination protocol along with autofluorescence imaging should be considered for decision-making regarding the biopsy site or in repeat OPMD follow-ups. However, the device alone may not identify lesions without the standard COE and histopathological examination. Future large population–based trials to assess the benefits of autofluorescence technology are warranted.