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ORIGINAL ARTICLE Table of Contents  
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A comparison of proliferative capacity of reticular and erosive variants of oral lichen planus by argyrophilic nucleolar organizer regions method


1 Department of Oral Pathology and Microbiology, D Y Patil University School of Dentistry, Navi Mumbai, Maharashtra, India
2 Department of Oral Pathology and Microbiology, Peoples Dental Academy, Bhopal, Madhya Pradesh, India
3 Department of Oral Medicine and Radiology, D Y Patil University School of Dentistry, Navi Mumbai, Maharashtra, India
4 Department of Oral Pathology and Microbiology, Bharati Vidyapeeth Dental College and Hospital, Navi Mumbai, Maharashtra, India
5 Department of Prosthetic Dentistry, D Y Patil University School of Dentistry, Navi Mumbai, Maharashtra, India
6 Department of Orthodontics, D Y Patil University School of Dentistry, Navi Mumbai, Maharashtra, India

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Date of Submission09-Oct-2020
Date of Acceptance11-Nov-2020
Date of Web Publication05-Jan-2023
 

  Abstract 


Background: Oral lichen planus (OLP), an immune mediated disorder, has been recognized since 1869 and is presented as any one of the six variants. Reticular and erosive are the most frequently encountered. Its proliferative capacity can give some information regarding its progression. We adopted the argyrophilic nucleolar organizer regions (AgNORs) method because of its simplicity to use and dependable results. We evaluated AgNORs in basal, suprabasal, and squamous cell layers. We also compared these three layers within two variants, reticular, and erosive. Materials and Methods: Thirty clinically diagnosed patients of OLP were included in the study. Reticular and erosive variants were included in our study. This was followed by hematoxylin and eosin staining and later by the AgNOR method. The mean number of AgNORs per nucleus was calculated. Results: Thirteen males and 17 females were the gender distribution. Twenty-three (76.67%) had reticular pattern and seven (23.33%) had erosive pattern. The basal cell layer had the highest mean AgNOR compared to suprabasal and squamous layers. Even among, erosive and reticular variants, the former had higher mean AgNOR counts. Discussion: Our results suggest that the inflammatory infiltrate close to the epithelial cells can alter the proliferation index for the pattern of protein synthesis of these cells. Moreover, the high proliferative index in OLP can be related to a specific immunologic response. Conclusion: We conclude that AgNOR can be used as a proliferative marker in earlier lesions to detect the severity.

Keywords: Argyrophilic nucleolar organizer regions, erosive, lichen planus, proliferation, reticular


How to cite this URL:
Gotmare SS, Gupta AA, Waghmare M, Kavle P, Rathod A, Sonawne S, Pereira T. A comparison of proliferative capacity of reticular and erosive variants of oral lichen planus by argyrophilic nucleolar organizer regions method. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Feb 8]. Available from: https://www.jmau.org/preprintarticle.asp?id=362482





  Introduction Top


The surveillance of the immune apparatus has an important function of destroying aberrant host cells. The immune system on certain situations may act as a “self-destroyer” when it loses its ability to distinguish between self and foreign, causing immune-mediated disorders. Oral lichen planus (OLP) is said to fall under this category.[1] OLP, mucosal counterpart of cutaneous lichen planus (LP), occurs bilaterally and is characterized as white striations or plaques in varied forms.[2] Erasmus Wilson, in 1869, was the first to describe and coin the name “Lichen Planus” followed by Wickham describing the characteristic pattern of grayish-white lines and dots representing the arrangement of papules.[3] Reticular, papular, plaque-like, erosive, atrophic, and bullous are the recognized variants of OLP.[4] The results of a meta-analysis by Aghbari et al. projected the rate of malignant transformation 1.1% in pooled proportion from a total of 57 studies constituting 20,095 patients.[5] The epithelium of OLP shows pathognomic sawtooth rete ridges with basal cell degeneration and Civatte bodies in the basal cell layer. The malignant potential of OLP is still controversial, and studies have indicated the presence of dysplasia in its initial presentation.[6] Some debate revolves whether they are actual OLPs with dysplasia or, in fact, they may be dysplastic leukoplakias with the lichenoid appearance or lichenoid dysplasias.[7] The clinical and histopathological presentation of OLPs may show alterations, which may be subtle and subjective demanding a more accurate and dependable results. The proliferative capacity of a lesion can be indicative of its “irreversible progression” and can be intuitive about its prognosis. This can be accomplished by Ki-67, proliferating cell nuclear antigen (PCNA), and argyrophilic nucleolar organizer regions (AgNORs). Evaluation with PCNA involves some difficulties because it is fixation dependent and its positivity is reduced in 24 h and is almost eliminated after 72 h. AgNORs have this subtle advantage over PCNA.[8] AgNORs are nothing but proteins attached to nucleolar organizer regions (NORs) and were first recognized by Heintz and McClintock (1934). The number of NORs is directly proportional to the intensity of cellular proliferation, making quantitative assessment of NORs a potent marker to differentiate benign from malignant cells.[9] We performed a study to compare the proliferative capacity of OLP by counting and comparing AgNORs in basal, suprabasal, and spinous layers. These counts were also compared with that of normal nucosa (NM). The intention of this study was to assimilate the results and find out if there is any correlation between the proliferative capacity of OLP and its aggressive behavior.


  Materials and Methods Top


The current prospective study was carried out under the efficient concordance of two departments, namely Department of Oral and Maxillofacial Pathology (Government Dental College and Hospital, Mumbai) and Department of Dermatology (Lokmanya Tilak Municipal Medical College and Hospital, Sion, Mumbai). About 63 patients were screened for OLP. Based on the discrete clinical features of visibly evident papules in either a linear or reticular pattern, patients were selected. Areas of ulceration were also considered for patients with a history of papules in the same region. Patients with a medical history and who are undergoing treatment were excluded.

Thirty clinically diagnosed patients of OLP (Group I) were included in the study. The study was restricted to two forms of OLP reticular (Group [Ia] most common type) and erosive (Group [Ib] the only symptomatic variant). Patients were informed regarding the surgical biopsy procedure and were explained explicitly about the diagnostic procedure. Written consents were taken from each patient and their attendants. Five healthy mucosal samples were taken as control (Group II). This study had clearance from the Institutional Ethical Committee as well as the Research Advisory Committee (IEC- GDC/IEC/1088/2018 Dated - 06 March 2018).

The staining protocol for AgNOR mentioned by Bukhari et al. was adopted.[10] Four microns thick sections were cut and were deparafinized, after which they were passed to descending grades of alcohol. This was followed by rehydration by ultrapure distilled water, incubation with acid alcohol for 6 min, and distilled water rinsing several times. The sections were then treated with silver nitrate solution (consisting of two parts of a 50% solution of silver nitrate and one part 2% gelatin in 1% formic acid solution) in a dark humidified chamber for 38 min at room temperature. The sections were then thoroughly washed by ultrapure distilled water, dehydrated by increasing grades of alcohol and the slides were mounted using coverslips. The corresponding tissues were also stained for hematoxylin and eosin (H and E).

Interpretation

AgNOR staining – Black to brown dots

Background stain – Yellow brown

H and E staining:

Nucleus – Blue

Cytoplasm – Pink

One hundred cells in the epithelium (basal, parabasal, and spinous cells) were counted and assessed for AgNOR dots in the magnification of ×1000. Intra-nucleolar, as well as extra-nucleolar dots, were counted. In the case of clusters, there were considered as one dot. The mean number of AgNORs per nucleus was calculated. The mean of OLP and NM was compared. Among OLPs, we also compared the mean between basal cell layer, suprabasal, and spinous cell layer. Student's “t”-test was applied for statistical analysis.


  Results Top


Thirty cases of OLP were evaluated clinically, histopathologically and histochemically by AgNORs. Five cases from NM were taken as control from patients undergoing extractions. The age of the patients ranged between 30 and 60 years, with the highest frequency noted between 30 and 40 years (43.33%). The gender distribution was 13 males and 17 females. Only 3 out of 30 patients had concurrent cutaneous lesions. Twenty-three (76.67%) had reticular pattern and seven (23.33%) had erosive pattern [Table 1] and [Figure 1]a and [Figure 1]b. The H and E stained sections confirmed the diagnosis of OLP [Figure 2]a and [Figure 2]b. The mean number of AgNORs in OLP [Figure 3]a, [Figure 3]b and [Figure 4]a, [Figure 4]b and NM were counted in the 3 cell layers (basal, suprabasal, spinous) and tabulated [Table 2]. The results were highly significant. Among OLP, reticluar and erosive forms were evaluated at the same 3 cellular levels and the results were tabulated [Table 3]. The means of AgNOR count in only the basal cell layer were statistically significant when reticular and erosive variants were considered.
Figure 1: (a) Intraoral picture of reticular variant of oral lichen planus, and (b) shows erosive variant

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Figure 2: (a) Photomicrograph of haematoxylin and eosin (×10) stained section of erosive oral lichen planus showing ulcerated epithelium and a band of juxta-epithelial presence of chronic inflammatory cells. (b) Photomicrograph of hematoxylin and eosin section (×10) of reticular variant showing hyperparakeratosis, saw tooth reter edges and band of juxta-epithelial presence of chronic inflammatory cells

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Figure 3: Photomicroghraph showing argyrophilic nucleolar organizer regions stained sections showing reticular oral lichen planus (a) ×10, and (b) ×40

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Figure 4: Photomicroghraph showing argyrophilic nucleolar organizer regions -stained sections showing erosive oral lichen planus (a) ×10, and (b) ×40

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Table 1: Gender and clinical variants distribution

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Table 2: Comparison of mean argyrophilic nucleolar organizer regions counts between different layers of oral lichen planus

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Table 3: Comparison of mean argyrophilic nucleolar organizer regions counts between different layers of oral lichen planus in the two clinical variants

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  Discussion Top


OLP is a distinct entity with characteristic clinical and histopathological manifestations. Although this disorder was introduced in 1869 by Erasmus Wilson, its etiology has been debated very passionately. The etiology includes genetic (human leukocyte antigen-associated genetic factors), infective and autoimmune mechanisms.[11] Other associated factors in the etiology of OLPs are dental materials, drugs, infectious agents (hepatitis C virus, Helicobacter pylori), bowel disease, food allergies, stress, habits, trauma, diabetes, hypertension.[12] The recent reports on the pathogenesis of OLP assume that this disorder is the result of damage to basal keratinocytes by cluster of differentiation-8 (CD-8). This further leads to apoptosis. The antigen responsible to activate cytotoxic T-cells is the other associated factors previously described. The increased quantities of Langerhans cells due to overproduction of cytokines leads to higher amounts of interferon-alpha. This, in turn, activates cytotoxic cells.[13] Different molecular markers have different roles. p53, CK-19 positivity is considered as a precancerous potential. Lymphocytic markers (CD5, CD 19, CD20, CD27, and CD38), Heat shock proteins, Metalloproteinases are being investigated.[14],[15] The proliferative cell markers have gained importance since years. Of these, we preferred AgNORs over PCNA and Ki-67 because of its simplicity to perform and affordability. Ki-67, PCNA are the most widely used proliferative markers as they provide the percentage of proliferating cells entering the cell cycle. On the other hand AgNOR count reflects the rapidity of cell proliferation by providing information regarding the duration of the cell cycle.[16] We attempted this study to find the proliferative capacity of the epithelial cells with the AgNOR method. Through this study, we also summarized age and gender predilection. We found that females (56.67%) were commonly affected than males which were in accordance with various studies.[2],[3],[17] Eighteen patients (60%) reported to have stress and 6 patients (20%) had associated systemic illness (diabetes mellitus + hypertension). A study carried out by Varghese et al. in 2016 observed that 57% of patients with erosive LP gave a history of stress and 6.55% had associated systemic illness (diabetes + hypertension).[18] Our study showed similar results. According to Suresh et al., nervous system markers (catecholamines; adrenaline, noradrenaline, and dopamine), Endocrine system markers (cortical and aldosterone), and Immune system (T-cells, B-cells and natural killer cells, immunoglobulin's) are altered in situations of stress, anxiety, depression. This plays a key role in the development of OLP and may also worsen the clinical and pathological picture.[19],[20] Our study showed more number of reticular variant of OLP (23 cases) as compared to erythematous variant (7 cases), among a total of 30 cases. We also noticed greater discomfort in the later variant. Such results were observed in various other studies and our study was in concordance.[18],[21],[22] The investigators concluded that the reticular form is the commonest form and is seen as interlacing white keratotic lines (Wickham's striae) with an erythematous border. The striae are typically present bilaterally on the buccal mucosa, mucobuccal fold and gingiva commonly. The erythematous variety is the second-most common type of OLP, which shows a mixed erythematous and ulcerated lesion, surrounded by finely radiating keratotic striae. The histopathological findings in our study showed basal cell layer liquefaction degeneration and juxtaepithelial infiltrate as constant findings. The atrophic epithelium was seen in erosive variety. The other findings were hyperparakeratosis/hyperorthokeratosis, saw tooth rete ridges. Our results showed similarity with previous studies.[3],[11],[23] Techniques to detect proteins are routinely assessed to observe the proliferation of normal and altered cells and also to understand biologic behavior and prognosis of oral lesions. A number of advanced methods are available to detect early changes at the cellular level. AgNOR method is one such relatively inexpensive and easy procedure which can be performed in a single step. NORs are loops of deoxyribonucleic acid that contain ribosomal gene, which are important in the synthesis of protein. The AgNORs are NOR-associated acidic nonhistone proteins which are stained by the silver staining method.[24] AgNOR staining was performed, and we observed mean AgNOR count was higher in all three-layer basal (3.04), suprabasal (1.969), and spinous (1.592) when compared to control group basal (1.52), suprabasal (1.094), and spinous (0.938). These results were highly significant. We also noted decrease in the number of AgNORs from basal to suprabasal to spinous cell layers in both normal and OLP. These results were highly significant and were in accordance with other studies.[1],[25],[26] However, Carli et al. showed different results which may be because authors performed AgNOR counts in cytology, which may have influenced their results.[27] Our study proves that there is increased proliferative activity in OLP compared to normal. According to Taniguchi et al., increased cellular proliferation is likely to be a secondary phenomenon due to the damage inflicted on keratinocytes by infiltrating mononuclear cells in the submucosa.[28],[29] However, Coleman A et al. analyzed AgNOR counts in odontogenic cysts and ameloblastomas and came to the conclusion that the AgNOR count may not be a good indicator of cell proliferation. It may represent variations in metabolic or transcriptional activity.[30] Khan et al. found that increased synthesis of keratinocytes leads to the release of many cytokines.[30],[31] On the basis of these observations it may be assumed that increase in mean AgNOR count could be an indicator of variations in metabolic and transcriptional activity. We also observed increased AgNOR count in basal cell layer in the erosive variant (3.26) when compared with reticular (2.96). To summarize, our results suggest that the inflammatory infiltrate close to the epithelial cells can alter the proliferation index for the pattern of protein synthesis of these cells. Moreover, the high proliferative index in OLP can be related to a specific immunologic response.


  Conclusion Top


Mean AgNOR count was higher in OLP compared to normal. The mean AgNOR in the basal cell layer exceeded the other cell layers, and the erosive variant showed higher proliferation as depicted by the mean AgNOR count. Hence, we conclude that AgNOR can be used as a proliferative marker in earlier lesions to detect the severity. This can be used as a cheap, easy, and one-step procedure.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Swati Shrikant Gotmare,
Department of Oral Pathology and Microbiology, D Y Patil University School of Dentistry, Nerul, Navi Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_104_20



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