Mast cell expression in oral submucous fibrosis: A systematic review and metanalysis

Background: Oral submucous fibrosis (OSMF) is a chronic disorder associated with reduced mouth opening, burning sensation and listed as potentially malignant disorder. The role of mast cells in initiation and progression of this condition has been debated in last few years. It is imperative to understand the definitive role of mast cells and subsequently identify a possible cost-efficient treatment modality for OSMF. This review aimed to study the role of mast cells in OSMF by framing a research question that assessed the mast cell count (MCC), their degranulation and immunohistochemical analyses. We performed a comprehensive search of PubMed, EBSCOhost and general Google search that conceded 26 studies from which 15 articles were finalized for the review. The individual study syntheses revealed increased MCC in OSMF as compared to controls. Also, there was decreased MCC with the progression of OSMF. However, the metanalysis showed high level of heterogeneity as three studies out of eight studies found reduced MCC in disease when compared with controls. There is definite increase in mast cell in OSMF although the cell count falls with the advancement of OSMF. This increases the scope for further research to identify exact mechanism by which mast cells contribute to fibrosis and conduct the drug trials that can inhibit the mechanism. Key words:Oral submucous fibrosis, mast cell count, degranulated mast cells, stages of OSMF.Oral submucous fibrosis, mast cell count, degranulated mast cells, stages of OSMF.


Introduction
Oral submucous fibrosis (OSMF) is a potentially malignant disorder (PMD) affecting oral mucosa and oropharynx and is frequently seen in Southeast Asia, especially the Indian subcontinent. This condition has an obscure etiology and cannot be cured completely, only symptomatic medical management can be rendered. Nonetheless, Areca nut is implicated as the main etiological factor, areca alkaloids strongly involve in the pathogenesis of OSMF (1,2). Over the last three decades, numerous studies had been performed to identify the molecular etiopathogenesis of OSMF in-e572 cluding mast cell infiltration and mast-related biomolecules (3)(4)(5). Sir Paul Ehrlich (1877) discovered a granular cell of loose connective tissue called mast cells; their functional behavior was almost completely linked to allergic conditions. Mast cells are bone-marrow-derived cells, and their mechanism is found to be complex, well-orchestrated, and multifunctional playing a significant role in the immune system (6,7). Mast cell progenitors exit the blood circulation to mature and inhabit the connective tissue, at the interface between the host and the outside environment, principally in subepithelial regions and in the connective tissue surrounding nerves, blood vessels, lymphatics, and mucus glands (7). Presently, it is well known that these cells play numerous roles in physiologic and pathological conditions including cancer angiogenesis (8).
Mast cells are strong contributors in the formation of classical proangiogenic factors like fibroblast growth factor 2, vascular epithelial growth factor, and non-classical factors like tryptase and chymase (9,10). The current information on mast cells, their count, and the various tumor-related angiogenic factors is quite largely available (8,11). Also, their count and role in certain PMDs like oral lichen planus have been studied extensively (12). However, the information about mast cells and their role in the pathogenesis of OSMF is sparse. With this viewpoint, the current systematic review was undertaken to delineate mast cell count (MCC) and their possible clinicopathologic implications in OSMF.

-Objectives
The primary objective of the present review was to assess the relationship between mast cell density/count and OSMF. Secondary objectives included if additional parameters like micro-vessel density, stage-wise MCC analysis in OSMF, and comparison with any other PMD or oral squamous cell carcinoma (OSCC) are estimated.

-PICOS
The keywords for the systematic review were defined as PICO format: i) Population: "OSMF patients" ii) Intervention: "mast cell density or count immunohistochemistry/serum analysis " iii) Comparison: "healthy controls", "normal healthy individuals" iv) Outcomes: Assessment of MCC We constructed a primary research question using mentioned keywords: Is there any increase in MCC/density in OSMF? The secondary research questions included: a) "is there any degranulation of mast cells?' and "is there any variation in the count with the progression of the disease".

Material and Methods
A systematic review of data published on mast cell count and the OSMF was performed following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines.
-Literature searches and Eligibility criteria The target population was OSMF patients who participated directly or indirectly (retrospective tissue analysis) for mast cell counting. PubMed, EBSCOhost, and Google search engines were searched using Boolean terms "mast cell", and "Oral Submucous fibrosis" or "OSMF". These terms were specified to "all fields" in the Advanced Search builder for PubMed and EBSCO. The search presented 14 articles from PubMed, 7 from EBSCOhost, and 9 from Google search until December 2021. While four articles were common in PubMed and EBSCOhost searches, those retrieved from Google search were not indexed in major indexing and abstracting systems.
-Data collection process The decision to include the articles was made by reading the article title, abstract, and text containing the search terms and retrieved 26 articles for the review. The inclusion criteria were the presence of search terms, MCC performed between the study group and controls, and any additional data showing the relationship between mast cells and OSMF. The general review articles, prevalence studies, and other molecular studies on OSMF found during the search were excluded from the study. -Data extraction Both the authors independently extracted the data from each study and included items as the first author, year of publication, staining technique, key features, and inference of the study. Further, sample sizes, means, and standard deviations values of MCCs from each selected study were tabulated. In addition, data for the expression of mast cells in various stages of OSMF, other PMDs and OSCC were recorded. Any disagreement or data extraction errors were settled by reviewing the subtleties of that specific article by following the research question, inclusion and exclusion criteria, and study results. -Quality assessment Both the authors critically reviewed the selected articles for clarity of focused question, selection of study populations (no. of cases and controls), and study design. Further, validation of exclusion criteria, comparison of data to establish statistical differences, and applicability of result were assessed among these studies. The relevant information collected was based on the SIGN methodology checklist version 2.0 for case-control studies (Scottish Intercollegiate Guidelines Network) (13).
-Synthesis methods The statistical analyses were performed using the Review Manager computer program (RevMan) version 5.4 for academic use recommended by Cochrane Collaboration, 2020 (14). The standard deviation for one study was calculated from the mean and p-value following the sixth chapter of Cochrane Handbook Guidelines (15).

Results
-Study selection Studies were selected for two data sets: key salient features exhibiting search terms and fulfilling the parameters of the primary research question. Data were also extracted if the studies further addressed the secondary research questions.
-Study characteristics Study characteristics included the presence of a comparison of MCC between OSMF and normal tissue. Additional features like comparisons of MCCs with PMDs and OSCCs were also taken into consideration. -Risk of bias in studies Methodological assessment of studies selected as evidence was based on the SIGN methodology checklist (13). The checklist focused on specific aspects of the study design that research has shown to have a significant effect on the risk of bias in the results and conclusions. Eight studies were of high quality whereas seven studies were acceptable after the methodology checklist for case-control studies. Two papers published only data for the OSMF group and failed to address the primary research question. Thus, 15 studies were included in the review and eight high-quality studies were selected for metanalysis.
-Results of syntheses A meticulous search yielded a total of 26 articles that were scrutinized according to inclusion and exclusion criteria. We excluded six articles from PubMed (4 general reviews), all three from EBSCOhost, and 2 from Google search as they did not match with the PICO format. Thus 15 articles were selected for final analysis as given in the flowchart (Fig. 1) as per PRISMA guidelines.

Author Staining Inference
Sirsat (16) Toluidine Blue In the early stages, due to strong reaction of the tissue to the irritant, the mast cell counts are highest. As the tissue becomes less reactive and hyaline, the mast cells decrease and are often fewer than the normal mucosa.
Ankle ( Khatri (29) Toluidine Blue and IHC The number of mast cells increased and remained constant with progression of OSMF. IHC was more specific and reliable technique for mast cell expression.

Chavan (19)
Toluidine Blue MCC was higher in early stages and reduces with progression of disease. Histamine release from degranulated of the mast cells in the early stages lead to fall in count of mast cells in the more advanced stage.

Paul (20)
Toluidine blue Increased degranulated mast cells play a significant role in early oral submucous fibrosis, their role in modulation of angiogenesis in later stages is insignificant.

Husain (21) Toluidine Blue
With the advancement of the grades mast cell number decreased in OSMF.
Telagi (22) Toluidine Blue Increased mast cells play a key role in mediating the cross links between external angiogenic agents and local immunological factors. Granular mast cells decreased with progression.

Paul (23)
Toluidine blue The increased degranulated MCC has a profound effect on the burning sensation in early stages of OSMF.

Dutta (24)
Toluidine blue Increased mast cells found in OSMF than in controls.
Kumar (  Seven studies provided the sample size, mean and standard deviation for both groups, for one article standard deviation was calculated from the p-value (Table  2). Thus, a Meta-analysis based on data from 8 studies was performed using the RevMan for academics. The synthesis calculated the pooled average effect size, the standard deviation, and 95% confidence intervals. The forest plot revealed a significant variation in the MCC between OSMF and control groups (effect size = 7.35, p-value < 0.00001, z= 95% confidence interval: -0.37 to 15.07) (Fig. 2). However, the high I2 value of 100% indicates a high level of heterogeneity between the studies included.

Discussion
OSMF is a chronic progressive disorder of the oral cavity, oropharynx, and esophagus which is induced pri-marily by betel nut through its alkaloids. The disease exhibits fibrosis that continues to cause difficulty in mouth opening, a burning sensation, and possesses high chance of malignant transformation. There are studies that have described etiopathogenesis through molecular pathways, trace elements, and mast cells (25,28,30   Mast cells were found more in OSMF as compared to leukoplakia and OSCC immunologic factors (4). Telagi et al. attributed the increase in mast cells to vesicle formations and subsequent itching sensation due to histamine release (22). Further, Pujari et al. supported increased MCC to the association between mast cells and fibroblasts in the pathogenesis of OSMF (18).

Author/ Other features
A high level of heterogeneity in metanalysis was due to three studies (19,20,26) that showed negative inver- Two studies observed an increase in MCC with the progression of the disease and predicted the possible role of mast cells in the initiation and progression of the disease however, no definite mechanism is proposed (18,25 (26).
Six studies included other PMD and/or oral squamous cell carcinomas for comparative evaluation of mast cells in OSMF (4,17,22,24,27,28). The PMD included leukoplakia and lichen planus. Except for Dutta et al. (24), all the studies reported increased count in PMD and OSCC compared to OSMF. One article performed MCC and lipid profiles in OSMF patients but there was no correlation mentioned in the study (21). The present review disclosed that the MCC had a definite role in the pathogenesis of OSMF. However, the exact mechanism of these cells in initiation and progression was nonconclusive. Although certain studies tried to correlate the count with various stages and there was a significant fall in MCC with the severity of OSMF, the possible roles of cross-talks or entry of other biomarkers are obscure. OSMF possesses a high malignant transformation rate (30), therefore there is a need to identify proangiogenic biochemicals in the tissue microenvironment that stimulate mast cell proliferation in OSCC. The systematic review puts forward various research e577 opportunities for further biomolecular research in mast cell-related serine proteins (Chymase and tryptase), histamine, and diamine oxidase. The plasma levels of these molecules can give an insight to the systematic aspect of mast cells in OSMF. Mast cells in subepithelial tissues are considered gatekeepers, the chronic inflammatory response may change them to destructors. As Pujari et al. debated the therapeutic implications that can target the mast cell-endothelial cell axis to influence mast cell secretions to control inflammation and fibrotic changes. Drugs that can stabilize mast cells need to be considered for future treatment modalities (18).

Conclusions
From this systematic review, it is evident that MCC is increased in OSMF that decreases with the progression of the disease. But the exact mechanism of action and biomolecular pathogenesis is obscure. There is a lack of data about the variation in MCC in pretreatment and post-treatment setting. Authors believe that there is a need for pre-and post-treatment studies targeting mast cells and correlating the outcomes with the MCC. Further, there is a need to understand the exact mechanism of action by mast cells in initiating malignant transformation in OSMF so that a target drug therapy can be instituted through clinical trials.