|Year : 2017 | Volume
| Issue : 4 | Page : 249-253
Comparative Morphological Analysis of Precancerous Lesions and Conditions by Clinical Examination, Chemiluminescence, and Toluidine Blue
MunBhawni Bagga1, Anand C Kumar2, Dipti Bhatnagar1
1 Department of Oral Medicine and Radiology, Maturam Nirmala Devi DAV Dental College and Hospital, Solan, Himachal Pradesh, India
2 Department of Oral Medicine and Radiology, Maaruti College of Dental Sciences, Bengaluru, Karnataka, India
|Date of Submission||21-Aug-2017|
|Date of Acceptance||14-Jan-2018|
|Date of Web Publication||15-Feb-2018|
Department of Oral Medicine and Radiology, MN DAV Dental College and Hospital, Tatul, Solan, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Oral squamous cell carcinoma accounts for up to 50% of malignant tumors in Asian countries and is very common in India, Pakistan, and Taiwan. Currently, the most effective way of combating oral cancer is by early diagnosis followed by adequate treatment. As visualization is the principal strategy used to assess the patient's lesions at risk of malignant transformation, any procedure which highlights such lesions aids the clinician. Aim: The present study was carried out to compare the usefulness and validity of clinical examination, chemiluminescence, and toluidine blue in assessing the precancer. Materials and Methods: The study sample consisted of 100 subjects, 50 each of oral leukoplakia and oral submucous fibrosis. Evaluation of suspicious lesion was carried out by clinical examination, chemiluminescence, toluidine blue with the gold standard of histopathological diagnosis. The chemiluminescent and toluidine blue examination were evaluated by two observers who are unaware of the clinical examination. Results: Chemiluminescence was found to be better than toluidine blue and clinical examination by both the observers (P < 0.05). Chemiluminescence was found to be better in visualization of leukoplakic lesions by both the observers (kappa >0.900). The visualization, exact extent, borders, and surface texture of lesions were revealed accurate by chemiluminescence followed by toluidine blue and clinical evaluation (P < 0.05). The sensitivity and specificity was more reliable for chemiluminescence (75 and 54.7%) than the toluidine blue (57.4 and 44.1%). Our observations suggest that the mild and severe dysplasia of the lesions were well appreciated by chemiluminescence. Conclusion: Chemiluminescence and toluidine blue cannot be compared with histopathology as these are adjunctive aids in early diagnosis of oral precancer and cancer. Hence their adjunctive value is of great importance to be used as a chair-side investigation and for mass screening of oral cancer.
Keywords: Chemiluminescence, oral precancer, toluidine blue
|How to cite this article:|
Bagga M, Kumar AC, Bhatnagar D. Comparative Morphological Analysis of Precancerous Lesions and Conditions by Clinical Examination, Chemiluminescence, and Toluidine Blue. J Indian Acad Oral Med Radiol 2017;29:249-53
|How to cite this URL:|
Bagga M, Kumar AC, Bhatnagar D. Comparative Morphological Analysis of Precancerous Lesions and Conditions by Clinical Examination, Chemiluminescence, and Toluidine Blue. J Indian Acad Oral Med Radiol [serial online] 2017 [cited 2018 Dec 12];29:249-53. Available from: http://www.jiaomr.in/text.asp?2017/29/4/249/225566
| Introduction|| |
Oral squamous cell carcinoma (OSCC) is the eighth most common cancer worldwide and is among three most common types of cancer in South Central Asia. Most malignant tumors of oral cavity are squamous cell carcinoma followed by adenocarcinoma and rarely other types of malignant tumors. In India, the age standardized incidence rate of oral cancer is 12.6 per 100,000 population. Currently, the most effective way of combating oral cancer is by early diagnosis followed by adequate treatment. The critical need for early detection of oral cancer drives to detect potentially malignant and malignant changes in oral cavity. The most commonly occurring oral precancers in Indian population include oral leukoplakia and oral submucous fibrosis (OSMF), out of which 8–10% eventually lead to malignancy. It is therefore important to detect these lesions early to improve the prognosis with the help of better screening by use of various minimally invasive, diagnostic visualization aids such as toluidine blue, ViziLite, Microlux/DL, and VELscope.
The clinical application of toluidine blue has been shown to be selective for staining of premalignant and malignant lesions. In the oral cavity, toluidine blue has been shown to be related to genetic changes, i.e., allelic loss or loss of heterozygosity that are associated with progression of oral premalignancy to cancer. We attempt to identify the risk of dysplasia because it is nowhere well documented. From previous literature, we found that no staining indicated no dysplasia, partial staining, i.e., <25% staining of lesion, revealed mild dysplasia, equivocal staining (50%) revealed moderate dysplasia, and complete staining revealed severe dysplasia.
The ViziLite capsule or chemiluminescent light stick comprises an outer flexible plastic capsule containing aspirin or acetyl salicylic acid and an inner fragile glass vial containing hydrogen peroxide. Activation of the capsule is achieved by flexing it, wherein the inner fragile glass vial ruptures releasing the hydrogen peroxide. The chemicals react to produce light of the blue-white color with a wavelength ranging from 430 to 580 nm. The light lasts for approximately 10 min.
The mechanism of action of chemiluminescence is that the blue-white light is absorbed by the cells of the normal mucosa and is reflected by cells with abnormal nuclei, including dysplastic and neoplastic cells. The normal mucosa appears blue, whereas abnormal mucosal areas reflect the light (due to higher nuclear/cytoplasmic ratio of epithelial cells) and appear more acetowhite with brighter, sharper, and more distinct margins. In chemiluminescence lightish blue light was reflected from the mucosal lesions in no dysplasia, regular borders with blue defined white light in mild dysplasia, diffuse blue-white light with regular/irregular borders in moderate dysplasia, and in severe dysplasia it appears to be acetowhite with irregular borders.,
Although histologic examination of tissue from a biopsy is the gold standard for diagnosing oral cancer, but with all cancers early detection is the key to successful treatment and reduction in morbidity. So, our aim of the present study was to compare the usefulness and validity of chemiluminescence and toluidine blue in assessing the precancerous lesions diagnosed by clinical and histopathological examination. This study is the first report of the use of chemiluminescent light source application in OSMF.
| Materials and Methods|| |
The study sample consisted of 100 subjects, 50 each of oral leukoplakia (Group A) and OSMF (Group B) reporting to Outpatient Department of Oral Medicine and Radiology. Among the subjects, minimum age was 15 years and maximum was 72 years, with the mean age of 34.92 years (S.D. ± 13.11). The study was conducted according to Human Ethics Guidelines approved by institution, following which the written consent was obtained from each patient, and on clinical grounds the oral leukoplakia and OSMF groups were formed.
Suspicious lesions were first identified with a conventional visual examination under incandescent projected light. Lesions were measured and data regarding the conventional visual examination was recorded. The site was determined according to the extent and severity of involvement. This was followed by an oral rinse with a 1% acetic acid solution for 30–60 s before expectorating. Room lights were dimmed, the oral cavity was examined, and each visually identified lesion was re-evaluated using a chemiluminescent light (Zila Pharmaceuticals, Phoenix, Arizona, USA). Lesions were measured and data were recorded.
All visualized lesions were then swabbed with a 1% acetic acid solution followed by a local application of pharmaceutical grade toluidine blue (Himeda Pharmaceuticals, Mumbai, India) using a presoaked swab. A local swab application with 1% acetic acid was then used to remove any excess toluidine blue. Visual examination was repeated under standard incandescent light to identify toluidine blue stain retention for each previously identified lesion and findings were recorded. All visualized lesions were then biopsied using punch technique and histopathological grades of dysplasia were evaluated.
All the lesions were recorded for location, extent, visibility, borders, and surface texture.
Visibility criteria was recorded on four-point Likert scale as: Not visible, Poorly visible, Visible, and Clearly visible. Borders were designated either diffuse or sharp  and surface texture characteristics were recorded as granular, smooth, rough, and crusting. Photography of each lesion was done at each examination using Sony Cybershot DSC-T7 digital camera. The chemiluminescent and toluidine blue examination were evaluated by two observers who are unaware of the clinical examination.
Chi-squared analysis was used for comparison and for the purposes of determining the sensitivity and specificity information for toluidine blue; any stain retention (complete or incomplete) was considered positive staining. All analyses were executed using Statistical Package for the Social Science, version 15.0 (SPSS Inc., Chicago, Illinois, USA).
| Results|| |
A total of 100 (50 in each group) patients were enrolled in the study, out of which 100 lesions were examined. The study population consisted of 93% males and 7% females in which location of oral precancer was widely distributed as involvement of buccal mucosa (67%), oral commissures (10%), and multiple sites (13%). All the subjects in the study group were involved in some form of deleterious habits. In Group A, 28 (56%) patients were betel nut quid mixed with lime chewers, but in Group B, areca nut chewers were 30 (60%). Correlating the grades of dysplasia with deleterious habits, we found betel nut quid mixed with lime is associated with more risk of dysplasia [Table 1].
In comparison of chemiluminescence and toluidine blue, there was increased illumination by chemiluminescence (P ≤ 0.05) [Table 2]. There was increased visibility in oral leukoplakia with chemiluminescence and toluidine blue. Chemiluminescence lesional borders did not always coincide with their clinical outlines viewed under dental light, in the sense that they often extended beyond the clinically identified outline. Both chemiluminescence and toluidine blue provide delineation of sharp borders [Table 2].
|Table 2: Comparison between diagnostic tools among the observers based on parameters|
Click here to view
The interobserver agreement on visibility, surface texture, and border parameter were 91.4 and 100%; 89.4 and 92.5%; and 86.7 and 94.9% among chemiluminescence and toluidine blue, respectively [Table 3]. On comparison of chemiluminescence and toluidine blue with histopathology, no significant correlation was observed [Table 4]. The sensitivity, specificity, and accuracy for chemiluminescence was 75, 54.7, and 68%, whereas for toluidine blue it was 57.4, 44.1, and 44.0%, respectively [Graph 1].
|Table 3: Inter observer agreement and reliability on chemiluminescence and toluidine blue among the observers|
Click here to view
|Table 4: Comparison of chemiluminescence and toluidine blue with histopathology|
Click here to view
| Discussion|| |
As the incidence and death rate because of cancer have shown a sharp acceleration since the last two decades, more intense efforts are required to fight against this life-threatening disease. We assessed the adjunctive utility of a chemiluminescent examination and application of a toluidine blue stain for detecting serious pathology associated with dysplasia when compared with the traditional clinical examination of the oral cavity. The representative cases in [Figure 1] and [Figure 2] depict oral precancer screening by clinical [Figure 1]a and [Figure 2]a, chemiluminescence [Figure 1]b and [Figure 2]b, and toluidine blue examination [Figure 1]c and [Figure 2]c followed by histopathological outcome [Figure 1]d and [Figure 2]d.
|Figure 1: Verrucous leukoplakia- (a) Clinical examination, (b) Chemiluminescence examination, (c) Toluidine blue examination, and (d) Histopathology|
Click here to view
|Figure 2: OSMF- (a) Clinical examination, (b) Chemiluminescence examination, (c) Toluidine blue examination, and (d) Histopathology revealing mild dysplasia|
Click here to view
In Indian population, male predominance was present due to prevalence of habits in males, and buccal mucosa (67%) was the commonest site for oral precancer, due to quid placement in the same region as suggested by previous studies.,, We observed high risk of dysplasia was present in older age group because longer exposure to etiologic agent were more prone for malignant transformation. Our findings suggest that betel nut quid mixed with lime is associated with more risk of dysplasia.
All the four parameters, visibility, size, borders, and surface texture, were first assessed by clinical examination, then by the chemiluminescence and toluidine blue. As OSMF was a generalized condition, we selected the area of interest according to severity of the condition. The visualization of lesions is improved by chemiluminescence followed by toluidine blue in comparison to clinical examination because in chemiluminescence acetowhite blue light was reflected from keratotic epithelium with increased nuclear cytoplasmic ratio;, however, in toluidine blue absolute contrast was present adjacent to normal oral mucosa. Also, we found dark royal blue staining is associated with more risk  of dysplasia, then pale blue stain. The size parameter was crucial, as it shows the actual extent of the lesion in oral cavity which aids in biopsy site selection and helps in treatment planning of the patients. Our observation revealed that the lesion in most of the cases often extended beyond the clinically identified outline when viewed under chemiluminescence in accordance with Ram and Siar (2005).
Chemiluminescence was capable of delineating sharp borders in all the groups, which is in agreement with Epstein et al. Oral leukoplakia was identified well by chemiluminescence as it is defined patch, and the application of a cytoplasmic dehydration agent such as acetic acid aids in visualization due to changes in refractile properties. Whereas the borders with toluidine blue, however, are not as sharp, but they provide an excellent contrast that aids in biopsy. It is a well-known fact that surface texture is a key in diagnosis of early asymptomatic oral carcinoma. Both chemiluminescence and toluidine blue showed improved surface characteristics of precancer as both the aids give clear appearance of the surface characteristics. The interobserver reliability and agreement was found to be good for both the methods.
Sensitivity, specificity, and percentage accuracy was higher for chemiluminescence than toluidine blue in all the groups and are in agreement with previous literature. As the sensitivity was more than specificity, we suggest these aids as useful diagnostic adjunct but not a substitute for histopathology. An attempt has been made to correlate the chemiluminescence and toluidine blue findings with histopathological findings in the study populations. Our observation suggests that chemiluminescence is better in detection of mild and severe dysplasia as suggested previously by Huber et al. and Epstein et al.
| Conclusion|| |
Chemiluminescence and toluidine blue are useful noninvasive methods for early detection of oral cancer but both cannot be compared with histopathology. Their adjunctive value is of great importance and should always be used as a chair-side investigation and for mass screening of oral cancer. Chemiluminescence is better than toluidine blue but the manufacturers have to cut down the cost of the product for its use in mass screening. We recommend future studies with larger sample size on all types of precancerous population with two or more expert clinicians to evaluate the full potential of chemiluminescence and toluidine blue for target screening of high-risk group individuals.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Petersen PE. Strengthening the prevention of oral cancer: The WHO perspective. Community Dent Oral Epidemiol 2005;33:397-9.
Bettendorf O, Piffkò J, Bànkfalvi A. Prognostic and predictive factors in oral squamous cell cancer: Important tools for planning individual therapy. Oral Oncol 2004;40:110-9.
Silverman S. Early diagnosis of oral cancer. Cancer 1988;62:1796-9.
Farah CS, McCullough MJ. 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;43:820-4.
McIntosh L, McCullough MJ, Farah CS. The assessment of diffused light illumination and acetic acid rinse (Microlux/DLTM) in the visualisation of oral mucosal lesions. Oral Oncol 2009;45:227-31.
Epstein JB, Silverman S, Epstein JD, Lonky SA, Bride MA. Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and toluidine blue. Oral Oncol 2008;44:538-44.
Zhang L, Williams M, Poh CF, Laronde D, Epstein JB, Durham S, et al
. Toluidine blue staining identifies high-risk primary oral premalignant lesions with poor outcome. Cancer Res 2005;65:8017-21.
Ram S, Siar CH. Chemiluminescence as a diagnostic aid in the detection of oral cancer and potentially malignant epithelial lesions. Int J Oral Maxillofac Surg 2005;34:521-7.
Huber MA, Bsoul SA, Terezhalmy GT. Acetic acid wash and chemiluminescent illumination as an adjunct to conventional oral soft tissue examination for detection of dysplasia: A pilot study. Quintessence Int 2004;35:378-84.
Massad LS, Lonky NM, Mutch DG, Mann WJ, Blanco JS, Vasilev SA, et al.
Use of speculoscopy in the evaluation of women with atypical Papanicolaou smears. Improved cost effectiveness by selective colposcopy. J Reprod Med 1993;38(3):163-9.
Mashberg A, Samit AM. Early detection, diagnosis, and management of oral and oropharyngeal cancer. CA Cancer J Clin 1989;39:67-88.
Neville BW, Damm DD. Epithelial pathology. Damm DD, Bouquot JE, Neville BW, Allen C. Oral and Maxillofacial Pathology. 2nd
edition. New Delhi, India: Elsevier; 2002. p. 337-45.
Pindborg JJ, Chawla TN, Srivastava AN, Gupta D, Mehrotra ML. Clinical aspects of oral submucous fibrosis. Acta Odontol Scand 1964;22:679-91.
Shiau YY, Kwan HW. Submucous fibrosis in Taiwan. Oral Surg Oral Med Oral Pathol 1979;47:453-7.
Teruo A, Masahi Y, Hitoshi I. Oral leukoplakia related to malignant transformation. Oral Sci Int 2006;3:45-55.
Jeng JH, Kuo ML, Hahn LJ, Kuo MY. Genotoxic and non-genotoxic effects of betel quid ingredients on oral mucosal fibroblasts in vitro
. J Dent Res 1994;73:1043-9.
Gandolfo S, Pentenero M, Broccoletti R, Pagano M, Carrozzo M, Scully C. Toluidine blue uptake in potentially malignant oral lesions in vivo
: Clinical and histological assessment. Oral Oncol 2006;42:89-95.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]