Coexisting Histopathological Features in Oral Leukoplakia, Quid Induced Lichenoid Lesions and Oral Submucous Fibrosis - An Analysis

Introduction

Oral potentially malignant disorders (OPMD) are a group of mucosal disorders which are at increased risk of developing into oral squamous cell carcinoma. The most common OPMDs are leukoplakia, oral lichen planus, oral submucous fibrosis (OSF) and erythroplakia.¹

OSF is a chronic potentially malignant disease affecting any part of the oral cavity involving pharynx sometimes and has malignant transformation rate of 7-10% into oral squamous cell carcinoma. OSF is most prevalent in areas where betel nut chewing is a widespread habit.² Clinically OSF presents as burning sensation of the mouth, blanching of the oral mucosa with reduced mouth opening. Histopathologically, OSF is characterized by atrophy of the epithelium with dense collagen fibers in the lamina propria and submucosa together with inflammatory cell infiltration.³

Leukoplakia is a white precancerous lesion which is clinically seen as homogenous or non-homogenous types. Histopathological features of leukoplakia vary from hyperkeratinization with no dysplasia to dysplasia and carcinoma.⁴

Oral mucosal lesions and conditions have been reported in association with quid chewing habit. Preparation of quid contains tobacco and areca nut, other substances like betel leaf, slaked lime and condiments are also added. Quid is kept in contact with oral mucosa, either actively sucked or chewed.⁵Contact oral lichenoid lesions have been recognized in patients with quid chewing habit and these have been given the term quid induced or associated lichenoid lesions.⁶ Clinically quid induced lichenoid lesion (QILL) presents as fine or thick, white, parallel, wavy lines which do not crisscross and may be seen radiating from the central erythematous area.⁷ Sub epithelial band of inflammatory cells and basal cell degeneration are the histopathological changes.^8,9

Leukoplakia, lichenoid lesion and OSF are chronic inflammatory conditions and immunologically mediated. All the three lesions show association with one or the other form of quid. Issac et al., for the first time histologically identified lichenoid lesion features in OSF cases without any clinical evidence of lichenoid features.¹⁰ Based on this study, Yanduri S et al (2015) found the concomitant features in OSF and quid induced lichenoid lesion cases at histological level. They were able to identify fibrosis in QILL cases and lichenoid features in OSF cases.³

Based on these findings, present study was done including leukoplakia along with OSF and QILL to study the concurrent features histopathologically and to identify if there is any presence of combination of these lesions clinically. Leukoplakia being the most prevalent and clinically encountered potentially malignant lesion was added to the present study.

Materials and Methods

Source of data for the study consisted of retrospective study samples. Retrospective study samples comprised of hematoxylin and eosin stained slides of histopathologically proven cases of OSF (n=60), leukoplakia (n=60) and QILL (n=20). Cases of OSF and QILL with evidence of dysplasia or squamous cell carcinoma were excluded from the study. Samples were retrieved from the archives of the Department of Oral and Maxillofacial Pathology from the year 2019 to 2021.

Clinically and histopathologically diagnosed cases of oral submucous fibrosis, quid induced lichenoid lesion and oral leukoplakia were included in the study. The study was conducted for 2 months. The slides were evaluated for the following criteria:

• Presence or absence of lichenoid features in OSF:

Lichenoid features like presence of band of inflammatory cell infiltration immediately below the epithelium, liquefactive degeneration of cells of the basal layer. Other features like saw tooth rete ridges, colloid bodies, reduplication of basement membrane and melanin incontinence were also noted down,8,9 and grading of OSF was done.¹¹

• Presence or absence of OSF features in QILL cases:

Features of OSF like fibrosis of the connective tissue along with the features of lichenoid reaction were noted down.

• In leukoplakia cases:

Search for presence or absence of OSF features like fibrosis in the connective tissue, hunt for lichenoid features like band of inflammatory infiltration immediately below the epithelium, liquefactive degeneration of cells of the basal layer, saw tooth rete ridges, colloid bodies, reduplication of basement membrane and melanin incontinence was done in leukoplakia cases along with grading of leukoplakia.

The slides were analyzed using a conventional light microscope and diagnosis was confirmed followed by grading. The cases of oral leukoplakia were graded into mild, moderate and severe epithelial dysplasia based on the criteria given by WHO (2005).¹² OSF grading was based on Pindborg JJ and Sirsat SM (1966).¹¹ Diagnosis and grading was confirmed after taking consensus from two Oral pathologists. Clinical details like age, gender, type of habit, duration and frequency of habit along with clinical diagnosis were put into a table after reviewing the slides.

Statistical analysis

Data obtained were statistically analyzed by Chi-square test, Spearman and Pearson correlation test.

Results

OSF Cases

Out of 60 cases, 84.6% of the cases were males and 15.4% were females. Average age of the study group was 47.7 years and the most common habit was gutka chewing (91.7%). Most common site of OSF was buccal mucosa. More than 50% of the cases showed moderately advanced stage on histopathological grading. Gutka chewing was the most common habit in the moderately advanced stage of OSF cases. We could not find any positive relation between duration and frequency of the habit.

OSF Cases Showing Lichenoid Features

Around 25% of the OSF cases, that is 15 out of 60 cases showed lichenoid features histopathologically. Average age was 48 years. Gutka chewing was seen in 14 cases and only one case showed pan chewing habit. The mean duration of habit and frequency were 12 years and six times daily, respectively. Thirteen cases showed moderately advanced OSF and two cases showed early stage OSF on histopathological grading. Gender distribution included 13 male and two female patients. Six cases out of 15 cases showed lichenoid reactions, found to have an associated white lesion which was diagnosed clinically as leukoplakia. Other nine cases did not reveal any associated secondary lesion clinically. Features of lichenoid reactions like basal cell degeneration, sub epithelial band of inflammatory cells, reduplication of basement membrane, colloid bodies, saw tooth rete ridges were evident in all the cases. Reduplication of basement membrane showed border line significant results (p<0.05). The inflammatory band was found to be dense in some cases and diffuse and perivascular in others. The main type of inflammatory cells present were lymphocytes. Other inflammatory cells like mast cells and neutrophils were also seen (Table 1 and 2) (Figure 1). We could not find any statistically significant result in this category.

QILL Cases

Twenty cases of QILL were considered, out of which more than 50% of the patients were above the age of 40 years, with male predominance. Buccal mucosa was the most common affected site. Chief habit was found to be gutka chewing. Duration of the habit was between 1 to 10 years.

QILL Cases Showing Fibrosis

Eight out of 20 cases showed features of fibrosis in the connective tissue. Mean age of patients was 36.5 years. All the eight cases were males. Gutka habit was seen in seven patients and one case had the habit of areca nut chewing. Mean duration of habit was seven years with frequency being three times per day. Clinically none of the cases showed evidence of fibrosis. Inflammatory cells were seen in diffuse form in most of the cases and lymphocytes being the most. Histopathologically, five cases showed subepithelial fibrosis and three cases showed fibrosis in the deeper parts of the connective tissue. No significant results were seen between the presence of fibrosis and frequency of habit. Other features of QILL cases like basal cell degeneration, melanin incontinence, colloid bodies and saw tooth rete ridges were also seen. (Table 3 and 4) (Figure 2)

Leukoplakia Cases

Sixty leukoplakia cases were studied. The average age was 44.8 years. 60% of the cases were male patients with main habit being gutka chewing. The second common habit was areca nut and paan. Duration of habit ranged from 1 to 10 years. Frequency of habit varied from 3 to 10 times per day. Buccal mucosa was the most common site (63%). On histopathological grading, more than 50% of the cases showed moderate grade of epithelial dysplasia which were compatible with clinical diagnosis and gutka chewing was the most common habit in these cases.

Leukoplakia Cases Showing Fibrosis

Out of 60 cases, two cases (3.3%) showed features of fibrosis in the connective tissue. Both the cases were female patients; one had the habit of gutka chewing and the other paan chewing. Clinically there was no evidence of any other lesion. Histopathologically, bundles of collagen fibers were seen indicating fibrosis subepithelially in both the cases. Both the cases were diagnosed as hyperkeratotic lesion with mild epithelial dysplasia which were compatible with clinical diagnosis. (Table 5 and 6) (Figure 3)

Leukoplakia Cases with Lichenoid Reaction

Around 16.6% (10 cases) of the total 60 leukoplakia cases showed lichenoid reaction. Average age of patients was 48.9 years. More than 50% were male patients with gutka chewing being the main habit. Lichenoid features like basal cell degeneration and melanin incontinence were seen in all the cases. Colloid bodies, reduplication of basement membrane and saw tooth rete ridges were also recognized. Lymphocytes were the main inflammatory cells, other cells being neutrophils and mast cells (Table 7 and 8) (Figure 4). Out of these 10 cases, five cases were of mild epithelial dysplasia, three were of moderate epithelial dysplasia and two were of severe epithelial dysplasia, all showing hyperkeratinization and all the 10 cases were compatible with the clinical diagnosis of leukoplakia.  

Discussion

Quid is a substance or a mixture of substances containing two basic ingredients, tobacco or areca nut or mixture of both in raw or processed forms. It is usually placed in contact with the mucosa of the oral cavity, either actively sucked or chewed.¹³ About 10-20% of the world population are betel quid chewers, mostly concentrated in South and South Eastern Asia. Different types of quid preparations are available around the world. In India, preparations available include gutka, pan (betel quid), pan masala, mawa, tobacco lime preparation, and snuff. Gutka is the most common form of eating quid in India, which is a mixture of dry ingredients like tobacco, areca nut and spices without betel leaf packed in the form of sachets and sold commercially.¹⁴

According to a survey, areca nut chewing in India is estimated to be 30% in men and 7% in women and 36.5% of men and 8.4% of women in the age group of 15 to 49 years chew some form of tobacco. Even though quid chewing is the most prevalent habit in India, studies related to oral lesions associated with the habit are inadequate.¹⁵ Daftary (1980) in the epidemiological study of oral cancer and precancer in the Indian population in Kerala identified and reported cases of lichen planus like lesions in the betel nut chewers.¹⁶ A study by Ismail SB et al., (2007) on oral lichen planus and lichenoid reaction reported that betel and tobacco chewing plays a major role in the development of lichenoid reaction and stated that they are potentially malignant.⁹ A study by Tejaswi et al., (2019) confirmed the strong association of betel nut and tobacco chewing with various lesions such as OSMF, leukoplakia, chewer’s mucosa, lichenoid reaction, and chemical burn.⁷

A fresh clinical term called as ‘betel-quid lichenoid lesion’ was given by Zain et al., (1996) to describe an oral lichen planus like lesion associated with the betel quid habit in a workshop on oral mucosal lesions related to betel quid, areca nut and tobacco chewing habits.⁶ In 2008, for the first time, Issac et al., histopathologically reported findings of lichenoid features in 35 cases of oral submucosal fibrosis, without any presence of lichenoid lesion clinically.¹⁰ Yanduri S et al., (2015) in the study showed that the evidence of lichenoid features like sub epithelial inflammatory band, basal cell degeneration and saw tooth rete ridges along with the characteristic features of OSF seen in OSF cases, and fibrosis either subepithelially or in the deeper connective tissue was seen in cases with lichenoid reaction, though there were no clinical features of OSF in these patients.³

With the available reports on concomitant lesions, a retrospective study was done in search and analysis of concurrent histopathological features in oral leukoplakia, quid induced lichenoid lesions and oral submucous fibrosis.

Around 25% of the OSF cases, that is 15 out of 60 cases showed lichenoid features histopathologically. The histopathological features seen were sub epithelial inflammatory band, basal cell degeneration and saw tooth rete ridges along with the characteristic features of OSF. Clinical information showed that six cases out of 15 demonstrated evidence of leukoplakia along with OSF, which was similar to study conducted by Sarita et al., where three out of seven cases showed clinical evidence of leukoplakia in OSF cases. The remaining nine cases did not reveal any clinical evidence of secondary lesion like in Issac’s study. However, all the cases showed lichenoid changes at histopathological level.

The next part of the study was to look for features of fibrosis in cases which were diagnosed as lichenoid lesions, both clinically and histopathologically. Eight cases of QILL out of 20 showed fibrosis which was 40% of the total cases. Fibrosis in these QILL cases were seen in either subepithelial or deeper parts of the connective tissue. But we could not reveal any clinical presence of OSF in these QILL cases.

We took a step forward and included leukoplakia in the study. We examined for the features of fibrosis and lichenoid reaction in clinically and histopathologically confirmed leukoplakia cases.

About 3.3% (two cases) of the total leukoplakia cases showed fibrosis in the connective tissue. One case showed fibrosis in the sub epithelial region and other in the deeper part of the connective tissue. But clinical data revealed no evidence of fibrosis. Both the cases were previously diagnosed as hyperkeratotic lesion with mild epithelial dysplasia compatible with clinical findings.

Around 16.6% (10 cases) of the total leukoplakia cases showed lichenoid features histologically. Features like basal cell degeneration, melanin incontinence and reduplication of basement membrane were seen in all the cases. Subepithelial band of inflammatory cells were seen as diffused, localized and at perivascular location. Lymphocytes were the main inflammatory cells present, mast cells and neutrophils were also seen. There was no clinical evidence of any secondary lesion.

All the three lesions were chronic inflammatory diseases. Disease process and progression of these lesions were inadequately stated. Even though the study had limited number of cases, based on the results we can hypothesize that, despite the presence of only one lesion clinically, likelihood of emergence of lichenoid features or fibrosis may occur at histological level. These results provide insight into the fact that these can be the preliminary modifications in the disease process. It would be significant to check for the development of lichenoid lesion in individuals with OSF and leukoplakia cases clinically at follow up stages where only lichenoid features were seen at histological level. In the same way, it would be interesting to verify for the development of fibrosis clinically in the lichenoid lesion and leukoplakia cases at a later stage, where only fibrosis was seen at histological level. Though it depends on the continuation of habit by the patients, more studies are required to confirm these findings.

Oral cancer is the sixth most common cancer occurring in the world and it is the most aggressive, diverse and multifaceted entity. Oral carcinogenesis is a multistep process and the underlying mechanism needs further evaluation.^17,18,19 Li et al., (2003) found that oral lichenoid lesion in OSF is an intense area of immunological reaction which might cause malignant changes. They also confirmed that there was an increase in the inflammatory cytokine (CXCL9) when OSF and quid induced lichenoid lesions were present together which shows that the presence of concomitant lesions share common pathologic process. Increase in the expression of inflammatory cytokines in tumour epithelial cells has been confirmed and highlighted in the initiation and progression of oral premalignant and malignant lesions in several studies.²⁰ Ekanayaka RP et al., (2016) demonstrated that in patients with OSF occurring along with leukoplakia, there is a higher chance of progression to early malignant change.²¹

A study by Chen PH (2017) showed that betel quid usage has a multidimensional health effect with increased rate of risk for oral premalignant disorders, cancers of oral cavity, pharynx, larynx and esophagus.²² Areca nut is the major constituent of betel quid which chemically consists of mainly alkaloids and polyphenols. Alkaloids like arecoline and arecaidine are confirmed to show carcinogenicity and mutagenicity. Lime, one of the content of the quid, interacts with the polyphenols of areca nut to release reactive oxygen species which plays a major role in the process of carcinogenesis. Nitrosomine substances released during betel quid chewing like N-nitrosoguvacine (NGC), N-nitrosoguvacoline (NGL), 3-methylnitrosaminopropionaldehyde (MNPA), and 3-methylnitrosaminopropionitrile (MNPN) are known to be carcinogenic. MNPN is known to cause abnormal cell proliferation and carcinogenesis.^14,22 Hence, arecanut or betel quid associated lesions should be thoroughly evaluated, diagnosed and should be uncompromisingly treated in order to reduce the risk of malignant transformation.

All the three lesions have common etiological agents, and are time and dose dependent with potential for malignant transformation and field cancerization. Even the normal appearing mucosa around the precancerous lesion can be a spot for genetic mutation. Histological presence of concomitant lesions without the clinical evidence of secondary lesion may indicate initial changes in secondary disease process, thereby increasing the malignant potentiality. However, stopping the habit can cause reversal of the disease process, reducing the malignant transformation.

Conclusion

This study highlights the possibility of concurrent histopathological features in three lesions, despite the clinical presence of only one lesion. Once histopathological presence of concomitant features is confirmed, proper diagnosis and management of the patient is needed in order to prevent malignant transformation. However, more inter institutional studies and use of molecular markers are required for further evaluation.

Sources and funding

Nil

Conflict of interest

None