SMF

Introduction
Background
In 1952, Schwartz coined the term atrophica idiopathica mucosa oris to describe an oral fibrosing disease he discovered in 5 Indian women from Kenya.1 Joshi subsequently coined the termed oral submucous fibrosis (OSF) for the condition in 1953.2 Oral submucous fibrosis is a chronic debilitating disease of the oral cavity characterized by inflammation and progressive fibrosis of the submucosal tissues (lamina propria and deeper connective tissues). Oral submucous fibrosis results in marked rigidity and an eventual inability to open the mouth. 3,4 The buccal mucosa is the most commonly involved site, but any part of the oral cavity can be involved, even the pharynx. 5 The condition is well recognized for its malignant potential and is particularly associated with areca nut chewing, the main component of betel quid. Betel quid chewing is a habit practiced predominately in Southeast Asia and India that dates back for thousands of years. It is similar to tobacco chewing in westernized societies. The mixture of this quid, or chew, is a combination of the areca nut (fruit of the Areca catechu palm tree, erroneously termed betel nut) and betel leaf (from the Piper betel, a pepper shrub), tobacco, slaked lime (calcium hydroxide), and catechu (extract of the Acacia catechu tree).3 Lime acts to keep the active ingredient in its freebase or alkaline form, enabling it to enter the bloodstream via sublingual absorption. Arecoline, an alkaloid found in the areca nut, promotes salivation, stains saliva red, and is a stimulant. The ingredients and nomenclature of betel quid vary by region as detailed below6,7 :
Pan: This is freshly prepared betel quid (with or without tobacco). Gutka (gutkha, guttkha, or guthka): This is a manufactured version of betel quid with tobacco sold as a single-use sachet. It is primarily used on the Indian subcontinent (ie, India, Pakistan, Bangladesh). Betel quid without tobacco is mostly used in Southeast Asian countries (ie, Taiwan, Myanmar, Thailand, China, Papua New Guinea, Guam). Pan masala: This is a commercially manufactured powdered version of betel quid without tobacco used in the Indian subcontinent. Pan Parag: It is a brand name of pan masala and gutka used in India. Mawa (kharra): This is a crude combination of areca, tobacco, and lime. Mainpuri tobacco: Popular in parts of northern India, Mainpuri tobacco is a mixture of areca nut, tobacco, lime, and various condiments. Depending on local preferences, sweeteners or spices (ie, cardamom, saffron, clove, anise seed, turmeric, mustard) are also added as flavorings.
In most patients with oral submucous fibrosis, areca nut was chewed alone more frequently than it was chewed in combination with pan (ie, betel leaf plus lime plus betel catechu, with or without tobacco)4 or had a higher areca nut content.8
Pathophysiology
The pathogenesis of the disease is not well established, but the cause of oral submucous fibrosis is believed to be multifactorial. A number of factors trigger the disease process by causing a juxtaepithelial inflammatory reaction in the oral mucosa. Factors include areca nut chewing, ingestion of chilies, genetic and immunologic processes, nutritional deficiencies, and other factors. Areca nut (betel nut) chewing
The areca nut component of betel quid plays a major role in the pathogenesis of oral submucous fibrosis.9 In a 2004 study, a clear dose-dependent relationship was observed for both frequency and duration of chewing areca nut (without tobacco) in the development of oral submucous fibrosis.10 Smoking and alcohol consumption alone, habits common to areca nut chewers, have been found to have no effect in the development of oral submucous fibrosis,11 but their addition to areca nut chewing can be a risk for oral submucous fibrosis. 11 Commercially freeze-dried products such as pan masala, guthka, and mawa have higher concentrations of areca nut per chew and appear to cause oral submucous fibrosis more rapidly than self-prepared conventional betel quid, which contains smaller amounts of areca nut.8 Arecoline, an active alkaloid found in betel nuts, stimulates fibroblasts to increase production of collagen by 150%.12 In one study, arecoline was found to elevate the mRNA and protein expression of cystatin C, a nonglycosylated basic protein consistently up-regulated in a variety of fibrotic diseases, in a dose-dependent manner in persons with oral submucous fibrosis.13 In 3 separate but similar studies, keratinocyte growth factor-1, insulinlike growth factor-1, and interleukin 6 expression, which have all been implicated in tissue fibrogenesis, were also significantly up-regulated in persons with oral submucous fibrosis due to areca quid chewing, and arecoline may be responsible for their enhanced expression.14,15,16 Further studies have shown that arecoline is an inhibitor of metalloproteinases (particularly metalloproteinase-2) and a stimulator of tissue inhibitor of metalloproteinases, thus decreasing the overall breakdown of tissue collagen.17 Insertion/deletion 5A polymorphism in the promoter region of the matrix metalloproteinase-3 gene, which results in alteration of transcriptional activities, has also been found in persons with oral submucous fibrosis but not in those with oral squamous cell carcinoma.18 Conversely, insertion/deletion 2G polymorphism in the promoter of the matrix metalloproteinase-1 gene has been implicated in oral squamous cell carcinoma but not oral submucous fibrosis.19 Flavanoid, catechin, and tannin in betel nuts cause collagen fibers to cross-link, making them less susceptible to collagenase degradation.20 This results in increased fibrosis by causing both increased collagen production and decreased collagen breakdown.4 Oral submucous fibrosis remains active even after cessation of the chewing habit, suggesting that components of the areca nut initiate oral submucous fibrosis and then affect gene expression in the fibroblasts, which then produce greater amounts of normal collagen.21 Chewing areca quid may also activate NF-kappaB expression, thereby stimulating collagen fibroblasts and leading to further fibrosis in persons with oral submucous fibrosis.22 Areca nuts have also been shown to have a high copper content, and chewing areca nuts for 5-30 minutes significantly increases soluble copper levels in oral fluids. This increased level of soluble copper supports the hypothesis that copper acts as an initiating factor in persons with oral submucous fibrosis by stimulating fibrogenesis through up-regulation of copper-dependent lysyl oxidase activity.23 Further, a significant gradual increase in serum copper levels from precancer to cancer patients has been documented,24 which may have a role in oral fibrosis to cancer pathogenesis. Ingestion of chilies The role of chili ingestion in the pathogenesis of oral submucous fibrosis is controversial. The incidence of oral submucous fibrosis is lower in Mexico and South America than in India, despite the higher dietary intake of chilies.25 A hypersensitivity reaction to chilies is believed to contribute to oral submucous fibrosis. 4 One study
demonstrated that the capsaicin in chilies stimulates widespread palatal fibrosis in rats,26 while another study failed to duplicate these results.27 Genetic and immunologic processes A genetic component is assumed to be involved in oral submucous fibrosis because of the existence of reported cases in people without a history of betel nut chewing9,28 or chili ingestion.28 Patients with oral submucous fibrosis have been found to have an increased frequency of HLA-A10, HLA-B7, and HLA-DR3.4 An immunologic process is believed to play a role in the pathogenesis of oral submucous fibrosis. 29 The increase in CD4 and cells with HLA-DR in oral submucous fibrosis tissues suggests that most lymphocytes are activated and that the number of Langerhans cells is increased. The presence of these immunocompetent cells and the high ratio of CD4 to CD8 in oral submucous fibrosis tissues suggest an ongoing cellular immune response that results in an imbalance of immunoregulation and an alteration in local tissue architecture. 30 These reactions may be the result either of direct stimulation from exogenous antigens, such as areca alkaloids, or of changes in tissue antigenicity that lead to an autoimmune response.30 Further, the major histocompatibility complex class I chainrelated gene A (MICA) is expressed by keratinocytes and other epithelial cells and interacts with gamma/delta T cells localized in the submucosa. MICA has a triplet repeat (GCT) polymorphism in the transmembrane domain, resulting in 5 distinct allelic patterns. In particular, the phenotype frequency of allele A6 of MICA in subjects with oral submucous fibrosis is significantly higher and suggests a risk for oral submucous fibrosis.31 Some authors have demonstrated increased levels of proinflammatory cytokines and reduced antifibrotic interferon gamma (IFN-gamma) in patients with oral submucous fibrosis, which may be central to the pathogenesis of oral submucous fibrosis.32 Nutritional deficiencies Iron deficiency anemia, vitamin B complex deficiency, and malnutrition are promoting factors that derange the repair of the inflamed oral mucosa, leading to defective healing and resultant scarring.4 The resulting atrophic oral mucosa is more susceptible to the effects of chilies and betel nuts. Other significant factors Some authors have found a high frequency of mutations in the APC gene and low expression of the wild-type TP53 tumor suppressor gene product in patients with oral submucous fibrosis, providing some explanation for the increased risk of oral squamous cell carcinoma development in patients with oral submucous fibrosis.9 Other studies have suggested that altered expression of retinoic acid receptor-beta may be related to the disease pathogenesis.33
Frequency
United States
Oral submucous fibrosis is rare in the United States and is found only in the immigrant members of the South Asian population who chew betel nuts.
International
Worldwide, estimates of oral submucous fibrosis indicate that 2.5 million people are affected, with most cases concentrated on the Indian subcontinent, especially southern India.3 The rate varies from 0.2-2.3% in males and 1.2-4.57% in females in Indian communities.4 Oral submucous fibrosis is widely prevalent in all age groups and across all socioeconomic strata in India. A sharp increase in the incidence of oral submucous fibrosis was noted after pan parag came onto the market, and the incidence continues to increase. Oral submucous fibrosis also occurs in other parts of Asia and the Pacific Islands.3 Migration of endemic betel quid chewers has also made oral submucous fibrosis a public health issue in many parts of the world, including the United Kingdom, South Africa, and many Southeast Asian countries.34
Mortality/Morbidity
Oral submucous fibrosis has a high rate of morbidity because is causes a progressive inability to open the mouth, resulting in difficulty eating and consequent nutritional deficiencies. Oral submucous fibrosis also has a significant mortality rate because of it can transform into oral cancer, particularly squamous cell carcinoma, at a rate of 7.6%.4
Race
Oral submucous fibrosis occurs on the Indian subcontinent, in Indian immigrants to other countries, and among Asians and Pacific Islanders as a result of the traditional use of betel quid endemic to these areas. 3
Sex
The male-to-female ratio of oral submucous fibrosis varies by region, but females tend to predominate. In a study from Durban, South Africa, a distinct female predominance was demonstrated, with a male-to-female ratio of 1:13.35 This was later confirmed by others, with a male-to-female ratio of 1:7.36 In addition, a female predominance in areca nut chewing was also noted in this region. Studies in Pakistan reported a male-tofemale ratio of 1:2.3.4 Conversely, a case-control study of 185 subjects in Chennai, South India revealed a male-to-female ratio 9.9:1.11 In Patna, Bihar (also in India), the male-to-female ratio was 2.7:1.37 With the onset of new commercial betel quid preparations, trends in sex predominance and age of occurrence may shift.
Age
The age range of patients with oral submucous fibrosis is wide and regional; it is even prevalent among teenagers in India. In a study performed in Saipan, 8.8% of teenagers with a mean age of 16.3 years ( 1.5 y) were found to have oral submucous fibrosis.38 Generally, patient age ranges from 11-60 years4,37 ; most patients are aged 45-54 years and chew betel nuts 5 times per day.4
Clinical
History
Symptoms of oral submucous fibrosis include the following3 :
Progressive inability to open the mouth (trismus) due to oral fibrosis and scarring Oral pain and a burning sensation upon consumption of spicy foodstuffs Increased salivation Change of gustatory sensation
Hearing loss due to stenosis of the eustachian tubes Dryness of the mouth Nasal tonality to the voice Dysphagia to solids (if the esophagus is involved) Impaired mouth movements (eg, eating, whistling, blowing, sucking)
Physical
Oral submucous fibrosis is clinically divided into 3 stages,39 and the physical findings vary accordingly, as follows3,4,39 :
Stage 1: Stomatitis includes erythematous mucosa, vesicles, mucosal ulcers, melanotic mucosal pigmentation, and mucosal petechia. Stage 2: Fibrosis occurs in ruptured vesicles and ulcers when they heal, which is the hallmark of this stage. o Early lesions demonstrate blanching of the oral mucosa. o Older lesions include vertical and circular palpable fibrous bands in the buccal mucosa and around the mouth opening or lips, resulting in a mottled, marblelike appearance of the mucosa because of the vertical, thick, fibrous bands running in a blanching mucosa. Specific findings include the following: Reduction of the mouth opening (trismus) Stiff and small tongue Blanched and leathery floor of the mouth Fibrotic and depigmented gingiva Rubbery soft palate with decreased mobility Blanched and atrophic tonsils Shrunken budlike uvula Sinking of the cheeks, not commensurate with age or nutritional status Stage 3: Sequelae of oral submucous fibrosis are as follows: o Leukoplakia is precancerous and is found in more than 25% of individuals with oral submucous fibrosis. o Speech and hearing deficits may occur because of involvement of the tongue and the eustachian tubes.
Causes
The term oral submucosal fibrosis derives from oral (meaning mouth), submucosal (meaning below the mucosa of the mouth), and fibrosis (meaning hardening and scarring).4 Chewable agents, primarily betel nuts (Areca catechu), contain substances that irritate the oral mucosa, making it lose its elasticity. Nutritional deficiencies, ingestion of chilies, and immunologic processes may also have a role in the development of oral submucous fibrosis.3 See Pathophysiology.
Differential Diagnoses
Other Problems to Be Considered
Amyloidosis: Hyalinized stroma can be distinguished from amyloid infiltration by using Congo red and thioflavine T staining under polarized and immunofluorescent light, respectively.
Generalized fibromatosis: Although soft tissue masses are not produced in the usual sense, the fibrosis of oral submucous fibrosis may be confused with generalized fibromatosis. Oral manifestations of scleroderma: Scleroderma can be distinguished by other cutaneous, systemic, and characteristic laboratory findings. A case of localized plaque-type morphea was seen in a patient with longstanding oral submucous fibrosis.40 Oral lichen planus: Wickham striae can mimic atrophy and fibrosis. Anemia: Pale oral mucosa can mimic atrophy and fibrosis.
Workup
Laboratory Studies
No specific laboratory tests are available for oral submucous fibrosis, and abnormalities may be related to secondary nutritional deficiencies. Some oral submucous fibrosis studies have reported the following laboratory findings: o Decreased hemoglobin levels o Decreased iron levels o Decreased protein levels o Increased erythrocyte sedimentation rate o Decreased vitamin B complex levels
Other Tests

Cytologic smears may be performed. A neural networkbased oral precancer stage detection method has been proposed.34 This new technique uses wavelet coefficients from transmission electron micrography images of subepithelial fibrillar collagen in healthy oral submucosa and in oral submucous fibrosis tissues. These wavelet coefficients are used to choose the feature vector, which, in turn, can be used to train an artificial neural network. This trained network is able to classify normal and oral precancer stages (less advanced and advanced) after obtaining the image as an input. This technology is not readily available but could theoretically be used as an adjunct to hematoxylin and eosin histologic evaluations.
Procedures

Currently, oral biopsy for hematoxylin and eosin provides the most definitive diagnosis and is crucial because of the association of oral submucous fibrosis with oral cancer.4 Some authorities have reported benefit with immunohistochemical techniques such as Masson trichrome staining when pathology involved muscle.41 Alteration of cytokeratin expression, as is seen in leukoplakia and oral cancer, has also been noted in oral submucous fibrosis. Increased intensity of staining for pancytokeratin and high molecular weight cytokeratin, aberrant expression of cytokeratin 8, and decreased expression of cytokeratins 5 and 14 suggest their potential as surrogate markers for malignant transformation.42
Histologic Findings
Histologic findings vary according to the stage of the disease. Very early stage Fine fibrillar collagen, marked edema, large fibroblasts, dilated and congested blood vessels, and inflammatory infiltrates (primarily polymorphonuclear leukocytes and eosinophils) are found. Early stage Early hyalinization is characterized by thickened collagen bundles, moderate numbers of fibroblasts, and inflammatory cells (primarily lymphocytes, eosinophils, and plasma cells). Moderately advanced and advanced stages Dense bundles and sheets of collagen, thick bands of subepithelial hyalinization extending into the submucosal tissues (replacing fat or fibrovascular tissue), decreased vascularity, no edema, and inflammatory cells (lymphocytes and plasma cells) are found. Oral submucous fibrosis is generally characterized by diffuse hyalinization of the subepithelial stroma with pigment incontinence from the overlying epithelial melanin.43 Other histologic findings include an atrophic epithelium and intercellular edema, with or without hyperkeratosis, parakeratosis, or orthokeratosis; epithelial dysplasia (25% of patients who underwent biopsy); squamous cell carcinoma histologically identical to typical squamous cell carcinomas; chronic inflammation and fibrosis in the minor salivary glands in the area of quid placement; and atrophy of the underlying muscle.29 Ultrastructural changes in oral submucous fibrosis include an increase in collagen type I; however, fibrils retain the normal structure.44
Staging
In addition to the above clinical staging, in 1995 Khanna and Andrade40 developed a group classification system for the surgical management of trismus.
Group I: This is the earliest stage and is not associated with mouth opening limitations. It refers to patients with an interincisal distance of greater than 35 mm. Group II: This refers to patients with an interincisal distance of 26-35 mm. Group III: These are moderately advanced cases. This stage refers to patients with an interincisal distance of 15-26 mm. Fibrotic bands are visible at the soft palate, and pterygomandibular raphe and anterior pillars of fauces are present. Group IVA: Trismus is severe, with an interincisal distance of less than 15 mm and extensive fibrosis of all the oral mucosa. Group IVB: Disease is most advanced, with premalignant and malignant changes throughout the mucosa.
Treatment
Medical Care
The treatment of patients with oral submucous fibrosis depends on the degree of clinical involvement. If the disease is detected at a very early stage, cessation of the habit is sufficient. Most patients with oral submucous fibrosis present with moderate-to-severe disease. Moderate-to-severe oral submucous fibrosis is irreversible. Medical treatment is symptomatic and predominantly aimed at improving mouth movements. Treatment strategies include the following4 :
Steroids: In patients with moderate oral submucous fibrosis, weekly submucosal intralesional injections or topical application of steroids may help prevent further damage. Placental extracts: The rationale for using placental extract in patients with oral submucous fibrosis derives from its proposed anti-inflammatory effect,45 hence, preventing or inhibiting mucosal damage. Cessation of areca nut chewing and submucosal administration of aqueous extract of healthy human placental extract (Placentrex) has shown marked improvement of the condition.46 Hyaluronidase: The use of topical hyaluronidase has been shown to improve symptoms more quickly than steroids alone. Hyaluronidase can also be added to intralesional steroid preparations. The combination of steroids and topical hyaluronidase shows better long-term results than either agent used alone.47 IFN-gamma: This plays a role in the treatment of patients with oral submucous fibrosis because of its immunoregulatory effect. IFN-gamma is a known antifibrotic cytokine. IFN-gamma, through its effect of altering collagen synthesis, appears to be a key factor to the treatment of patients with oral submucous fibrosis, and intralesional injections of the cytokine may have a significant therapeutic effect on oral submucous fibrosis.48 Lycopene: Newer studies highlight the benefit of this oral nutritional supplement at a daily dose of 16 mg. Mouth opening in 2 treatment arms (40 patients total) was statistically improved in patients with oral submucous fibrosis. This effect was slightly enhanced with the injection of intralesional betamethasone (two 1-mL ampules of 4 mg each) twice weekly, but the onset of effect was slightly delayed.49 Pentoxifylline: In a pilot study, 14 test subjects with advanced oral submucous fibrosis given pentoxifylline at 400 mg 3 times daily were compared to 15 age- and sex-matched diseased control subjects. Statistical improvement was noted in all measures of objective (mouth opening, tongue protrusion, and relief from fibrotic bands) and subjective (intolerance to spices, burning sensation of mouth, tinnitus, difficulty in swallowing, and difficulty in speech) symptoms over a 7-month period.50 Further studies are needed, but this could be used in conjunction with other therapies.
The role of these treatments is still evolving. The US Food and Drug Administration has not yet approved these drugs for the treatment of oral submucous fibrosis.
Surgical Care
Surgical treatment is indicated in patients with severe trismus and/or biopsy results revealing dysplastic or neoplastic changes. Surgical modalities that have been used include the following:
Simple excision of the fibrous bands: Excision can result in contracture of the tissue and exacerbation of the condition. Split-thickness skin grafting following bilateral temporalis myotomy or coronoidectomy: Trismus associated with oral submucous fibrosis may be due to changes in the temporalis tendon secondary to oral submucous fibrosis; therefore, skin grafts may relieve symptoms.29
Nasolabial flaps and lingual pedicle flaps: Surgery to create flaps is performed only in patients with oral submucous fibrosis in whom the tongue is not involved.51 Use of a KTP-532 laser release procedure was found to increase mouth opening range in 9 patients over a 12-month follow-up period in one study.52
Consultations

Consult an ear, nose, and throat specialist for evaluation of dysplasia and close follow-up monitoring for the development of oral cancer. Consult a plastic surgeon for patients with severe trismus, in whom reconstructive surgery may be possible.
Diet
Dietary focus should be on reducing exposure to the risk factors, especially the use of betel quid, and correcting any nutritional deficiencies, such as iron and vitamin B complex deficiencies.3
Activity
Physical therapy using muscle-stretching exercises for the mouth may be helpful in preventing further limitation of mouth movements. This is often combined with medical and surgical therapy.
Medication
The goals of pharmacotherapy are to reduce morbidity and to prevent complications. In addition to the medications listed below, placental extract has been used experimentally at a dose of 50 mcg/m2 SC 3 times per week if the patient's body surface area (BSA) is greater than 0.52 m2 or 1.5 mcg/kg/dose SC 3 times per week if the BSA is less than or equal to 0.5 m2.
Corticosteroids
Can be used in pharmacologic doses for their anti-inflammatory and immunosuppressant properties and their effects on blood and lymphatic systems in the palliative treatment of various diseases.
Dexamethasone (Decadron)
For various inflammatory diseases. Decreases inflammation by suppressing migration of polymorphonuclear leukocytes and reducing capillary permeability.
Dosing Interactions Contraindications Precautions
Adult
4 mg IV/IM (suggested in studies)

Pediatric
Base dose on severity of disease and response rather than age, body weight, or BSA
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautions
Triamcinolone acetonide (Aristocort, Kenaject)
Suppresses immune system by reducing activity and volume of lymphatic system. Treats inflammatory mucosal lesions that are responsive to steroids. Decreases inflammation by suppressing the migration of polymorphonuclear leukocytes and by reversing capillary permeability.
Adult
Dental paste (for oral inflammatory or ulcerative lesions): Apply thin film bid/tid pc and hs IM: 40-80 mg (studies have used 10 mg/mL diluted in 1 mL of lidocaine 2% to avoid tissue irritation and facilitate proper distribution of drug)
Pediatric
Not established
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautions
Betamethasone valerate (Diprosone)
For inflammatory reactions responsive to steroids. Decreases inflammation by suppressing migration of polymorphonuclear leukocytes and by reversing capillary permeability. Affects production of lymphokines and has inhibitory effect on Langerhans cells.
Adult
Suggested dose: 0.05% topically q6h for 3 wk

Pediatric
Not established
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautionsExtravasation
antidotes
Can enhance diffusion of locally irritating or toxic drugs in the management of intravenous extravasation.
Hyaluronidase (Wydase Injection)
Stimulates hydrolysis of hyaluronic acid, one of the chief ingredients of tissue cement, which offers resistance to diffusion of liquids through tissues. Used to aid in absorption and dispersion of injected drugs.
Adult
150 U added to vehicle solution and administered SC/ID

Pediatric
Administer as in adults
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautionsInterferons
Naturally produced proteins with antiviral, antitumor, and immunomodulatory actions. Alpha-, beta-, and gamma-interferons may be given topically, systemically, or intralesionally.
Interferon gamma (Actimmune)
Believed to act via ability to counteract cell surface expression of proinflammatory or proadhesion molecules on immune cells, among other effects. More studies needed to fully understand mechanisms of action.
Adult
BSA >0.5 m2: 50 mcg/m2 SC 3 times/wk BSA <0.5 m2: 1.5 mcg/kg/dose SC 3 times/wk
Pediatric
Not established
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautionsAntioxidant
Has been found to possess antioxidant and antiproliferative properties in animal and laboratory studies, although activity in humans remains controversial. Adult dose below suggested from 1 study. 49
Lycopene (LycoRed)
Considered an antioxidant and has antiproliferative properties in animal and laboratory studies, although activity in humans remains controversial.
Adult
16 mg/d PO (suggested in study)

Pediatric
Not established
DosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicationsPrecautionsDosingInteractionsContraindicati onsPrecautionsVasodilator
Methylxanthine derivative that has vasodilating properties and may increase mucosal vascularity. Adult dosage suggested by Rajendran et al.50
Pentoxifylline (Trental)
Methylxanthine derivative that has vasodilating properties and may increase mucosal vascularity.
Adult
400 mg tid as coated, sustained-release tab

Pediatric
No known indicated pediatric dosage in relation to oral submucous fibrosis
Follow-up
Further Inpatient Care
Ensure nutritional intake is not compromised postoperatively.
Further Outpatient Care
Regular physical examinations, biopsy specimen analysis, and cytologic smear testing should be scheduled to detect oral dysplasia or carcinoma, especially in patients with severe oral submucous fibrosis. Patients with surface leukoplakias require close follow-up monitoring and repeat biopsies. Patients with dysplasias and carcinomas should receive routine treatment for these entities.53
Complications
Oral dysplasias and squamous cell carcinomas are complications of oral submucous fibrosis. In patients with oral submucous fibrosis, the risk of developing oral carcinoma is 7.6% over a 10-year period.3 If the palatal and paratubal muscles are involved in patients with oral submucous fibrosis, conductive hearing loss may occur because of functional stenosis of the eustachian tube. 54 Patients with oral submucous fibrosis who require anesthesia for trismus correction, resection, and reconstructive (oncoplastic) surgery may have difficulty during laryngoscopy and intubation of the trachea.55
Prognosis
No treatment is effective in patients with oral submucous fibrosis, and the condition is irreversible.56 Reports claim improvement of the condition if the habit is discontinued following diagnosis at an early stage.46 Patients with oral submucous fibrosis have an increased risk of developing oral cancer. The malignant potential and the origin of cancer are attributed to the generalized epithelial atrophy associated with oral submucous fibrosis.56 Tobacco is the component of the quid believed to be most associated with cancer development. However, the carcinogenic property of the areca nut was discovered after noticing that cancer occurred in patients who chewed the nut without tobacco.21 In vitro, betel nut extracts increase the rate of cell division, reduce cell cycle time, induce DNA strand breaks, and induce unscheduled DNA synthesis.57 Whether the use of tobacco in addition to areca nuts is responsible for the increased risk of oral cancer is controversial because evidence is conflicting. 58
Patient Education

Instruct patients regarding the importance of discontinuing the habit of chewing betel quid. Inform patients that eliminating tobacco from the quid product may reduce the risk of oral cancer. Instruct patients to avoid spicy foodstuffs. Instruct patients to eat a complete and healthy diet to avoid malnutrition. Instruct patients regarding maintaining proper oral hygiene and scheduling regular oral examinations. Intervention studies and public health campaigns against oral habits linked to oral submucous fibrosis may be the best way of controlling the disease at the community level. Educate the community regarding the local adverse effects of chewable agents, which although not inhaled, are still not harmless. For excellent patient education resources, visit eMedicine's Cancer and Tumors Center. In addition, see eMedicine's patient education article Cancer of the Mouth and Throat.
Miscellaneous
Medicolegal Pitfalls
Lack of appropriate follow up for screening and periodic biopsy of suspicious regions of the oral mucosa can lead to missed early premalignant lesions and possible progression to cancer.
Special Concerns
Watch for signs that indicate malignant change, which include the following: o An unhealing ulcer in the lesion o Lesion undergoing red changes (erythroplakia) o A burning sensation in the mouth o An exophytic mass o A lump in the neck o Difficulty in chewing, swallowing, or speaking
Discovery of Novel Biomarkers in Oral Submucous Fibrosis by Microarray Analysis

1. Ning Li1, 2. Xinchun Jian1, 3. Yanjia Hu1, 4. Chunjiao Xu2, 5. Zhigang Yao3 and 6. Xiaohuan Zhong4 + Author Affiliations
1. Departments of 1Oral and Maxillofacial Surgery, 2Oral Medicine, and 3Oral Pathology, Xiangya Hospital, and 4Department of Stomatology, The Second Affiliated Hospital of Xiangya Medical College, Central South University, Changsha, China
1. Requests for reprints: Xinchun Jian, Xiangya Hospital, Central South University, Xiangya Road, Changsha 410008, China. Phone: 86-731-4327475; Fax: 86-731-4805086. E-mail: jianxinchun@hotmail.com
Next Section
Abstract
Oral submucous fibrosis (OSF) is a high-risk precancerous condition of the oral cavity. Areca nut chewing is its key etiologic factor, but the full pathogenesis is still obscure. In this study, microarray analysis was used to characterize the mRNA changes of 14,500 genes in four OSF and four normal buccal mucosa samples to identify novel biomarkers of OSF. Five candidate genes with the most differential changes were chosen for validation. The correlation between clinicopathologic variables of 66 OSF patients and the expression of each gene was assessed by immunohistochemistry. The microarray analysis showed that 661 genes were up-regulated (fold value >2) and 129 genes were down-regulated (fold value <0.5) in OSF (q < 0.01). The top three up-regulated genes [Loricrin,
Cartilage oligomeric matrix protein (COMP), Cys-X-Cys ligand 9 (CXCL9)] with the largest fold changes and the top two down-regulated genes [keratin 19 (KRT19), cytochrome P450 3A5 (CYP 3A5)] with the most significantly differential changes in OSF were chosen as candidate biomarkers. In
immunohistochemical results, the expression of Loricrin and COMP showed statistically significant association with histologic grade of OSF ( P = 0.03 and 0.006, respectively). COMP was found to be
overexpressed frequently in patients with the habit of areca nut chewing for more than 4 years ( P = 0.002). CYP 3A5 was revealed an inverse correlation with histologic grade (P = 0.04). This pilot study showed that five novel genes might play important roles in the pathogenesis of OSF and may be clinically useful for early detection of OSF. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2249 59)
Oral submucous fibrosis
Novel biomarker Microarray analysis
Areca nut
Previous SectionNext Section
Introduction
Oral submucous fibrosis (OSF) is a chronic, insidious, and progressive oral mucosal disease that primarily affects any part of the oral cavity (1). It is characterized by a juxta-epithelial inflammatory reaction followed by progressive fibrosis of the lamina propria and the underlying submucosal layer, with associated epithelial atrophy. This always leads to the stiffness of the oral mucosa and the restriction of mouth opening, eventually impairing the ability to eat, the ability to speak, and dental care (2). Although the etiology of OSF is obscure, evidence has shown that it is a precancerous disorder related to the habit of chewing areca nut, either alone or as a component of betel quid (3). There are 600 million people worldwide amounting to 10% to 20% of the world's popu lation who chew raw areca nut or in any processed form (4). Several case-control studies provide overwhelming evidences that areca nut is the main risk factor for OSF in Hunan of China (5, 6). Thus, the disease is now a public health issue in many parts of the world. OSF carries a high risk of transition to oral cancer. In an epidemiologic study in India, the malignant transformation rate was 7.6% over a period of 17 years (7). Moreover, more than 2,400 new cases of oral cancer arising from OSF are diagnosed every year in Taiwan due to the prevalent use of betel quid (8). In another study by Jian, three cases of oral cancer were found in 147 cases of OSF in Hunan (9). Therefore, early diagnosis of this potentially malignant oral lesion is very important and effective. Much efforts have been devoted into researching the underlying molecular mechanisms of OSF. However, understanding the differences in gene expression between OSF and normal tissue is important for the research. High-throughput oligonucleotide microarrays can perform analysis of such differences at a transcriptional level to know expression profiles for thousands of genes simultaneously and to characterize the biological behaviors of cell in a single experiment. To explore OSF biology and search for genes with differential expression to represent diagnostic as well as therapeutic biomarkers for OSF, we used microarray analysis to screen genes deregulated in OSF and
shed some light on the molecular mechanism for its etiology and pathogenesis. Five novel genes identified in our study may be clinically useful for detection of OSF. Their functional implication of pathogenesis could provide a basis for better understanding of the molecular mechanisms underlying the development of OSF. Previous SectionNext Section
Materials and Methods

Patients and Tissue Samples
Under an ethical guideline of the Central South University Ethics Committee, 29 patients with clinically defined OSF lesions were recruited from the Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Changsha, China. Among them, eight patients who had previous local treatments for oral mucosa or underwent systemic diseases (hepatitis B, diabetes) were excluded, and two patients refused to accept the biopsy. Eventually, from the remaining 19 untreated primary patients, we obtained 19 biopsy mucosa samples of the buccal lesion area, which is the mainly affected site of OSF. All the patients had the habit of areca nut chewing. Because there is no way to collect the matched sample in the same patient because of the special characteristics of OSF, 14 unmatched normal buccal mucosa (NBM) tissues were procured from 14 healthy volunteers without the habit of areca nut chewing and systemic diseases, who underwent surgery for third molar impactions or road traffic accidents. Informed consent was obtained from all donors. Immediately after surgical removal and wash, each sample was divided into two parts: one part was fixed in 10% neutral-buffered formalin for 24 to 48 h and embedded in paraffin for pathologic review by an experienced investigator according to the concept of Pindborg and Sirsat (2); the residual sample was snap frozen with liquid nitrogen and stored in a 80C refrigerator after we removed partial submucous tissue to ensure the same thickness of all biopsies (2 mm). In this procedure, the removal of part connective tissue could lead to losing some information of genes in deeper connective tissues but still remain the key parts (epithelium, lamina propria, and adjacent connective tissue) of OSF mucosa for our study. After a final pathologic review, 14 of 19 biopsies were pathologically confirmed as OSF lesion, two samples were oral lichen planus, two were inflammatory tissues, and the last was scored for dysplasia. All control samples were assessed as NBM tissues. In addition, 66 paraffin-embedded buccal mucosa specimens of OSF patients with the habit of areca nut chewing were randomly drawn and reconfirmed from the files of the Department of Oral Pathology between March 2007 and June 2007. Patient age, gender, duration of areca nut chewing, duration of OSF disease, and histopathologic grade were used as clinicopathologic variables (Supplementary Table S1).
Isolation of RNA from Tissue Samples

Total RNA was extracted from OSF and normal biopsies using Trizol reagent (Invitrogen). RNA quality and purity were assessed by 1% agarose gel electrophoresis. Total RNA with A260/A280 >1.8 was used for microarray experiments.
Microarray Experiments and Data Mining

A global expression analysis of buccal mucosa samples from 4 OSF patients (4 males; age range 2236 years old; median age 29.5 years old; the histopathologic stage early stage in 1 case, moderately
advanced stage in 2 cases, advanced stage in 1 case), and 4 healthy volunteers (4 males; age range 16-30 years old; median age 20.7 years old) was done in microarray analysis. Briefly, total RNA from buccal mucosa of either the 4 volunteers or the 4 OSF patients was prepared to cRNA, biotin labeled, and hybridized to commercially available high-density oligonucleotide microarrays (human genome U133A 2.0 Gene Chips, Affymetrix, Inc.), containing 18,400 transcripts and 14,500 genes. Then, the arrays were scanned using GeneChip Scanner 3000 (Affymetrix). The hybridization data were analyzed using GeneChip Operating software (GCOS 1.4). The raw signals of individual probes for the eight arrays were normalized against the chip with median raw signal intensity using the dChip software (dChip2006). To identify genes with the most differentially altered expression in OSF, we focused on the results of supervised analysis with the Significance Analysis of Microarrays (SAM) software. Normalized expression values from dChip analysis were used for a two class paired SAM analysis. The SAM software estimated the false discovery rate and generated a q value for each gene, which can indicate a more significantly differential expression than the P value. In the present study, the SAM false discovery rate accepted in the analysis was <0.01, and those showing at least a 2-fold induction or 0.5-fold repression (q < 0.01) in their expression were selected as deregulated genes in our study. Three up-regulated genes with the most significantly differential changes and top two down-regulated genes in OSF versus NBM were selected for next validation. Besides, clustering analysis was done using Gene Cluster 3.0 software and TreeView 1.6 with average linkage method and correlation.
Validation Studies
For semiquantitative reverse transcription-PCR (RT-PCR), 1 g of total RNA each from 10 normal and 10 OSF samples was used as template for reverse transcription using PrimeScrip 1st Strand cDNA synthesis Kit (Takara Bio, Inc.). We then used gene-specific primers for five genes: Loricrin, forward 5-CAGGTCACTCAGACCTCGT-3, reverse 5-CCTCCAGAGGAACCACCT-3, product sizes 200 bp;
COMP,
product
forward sizes
5-ACGTGGTCTTGGACACAAC-3, 121 bp;
reverse 201
5-TCATAGTCCTCTGGGATGGT-3, reverse forward 55bp;
CXCL9,
forward
5-CTGTGGCCAGAATTTAAACC-3, sizes
ATGCAAGGTAAGTGGGTCAC-3,
product
KRT19,
AGGGTCTTGAGATTGAGCTG-3, reverse 5-CTCACTATCAGCTCGCACAT-3, product sizes 161 bp;
CYP 3A5, forward 5-ACACCCTTTGGAACTGGAC-3, reverse 5-GAAGAAGTCCTTGCGTGTCT-3, product sizes 154 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification from the
same cDNA samples served as internal controls, whose specific primer was forward 5 ACCACAGTCCATGCCATC-3, reverse 5-TCCACCACCCTGTTGCTG-3, product sizes 452 bp. The PCR products were visualized by UV illumination on a 1.5% agarose gel. Signals were captured with the Multi Genus Bio Imaging System and signal intensity was analyzed by the GeneTools software (Synoptics, Ltd.). For Western blotting, proteins (30 g) of OSF samples and normal controls we re separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. After being blocked, filters were incubated with the following primary antibody: rabbit polyclonal anti-Loricrin (Abcam; 1:1,000 dilution), rat monoclonal anti-COMP (Genetex; 1:1,000 dilution), mouse monoclonal anti-CXCL9 (R&D; 2 g/mL dilution), mouse monoclonal anti-KRT19 (Abcam; 1:1,000 dilution), and rabbit
polyclonal anti-CYP3A5 (Biomol; 1:2,000 dilution). Then, the horseradish peroxidase conjugated secondary antibody (Santa Cruz Biotechnology) was applied onto the filter at 1:2,000 dilution. As an internal control, samples were probed with mouse monoclonal anti-actin antibody (BD Biosciences). Bands were visualized using the ECL system (Amersham) and signal intensity was analyzed by the Bandscan software (Glyko).
Immunohistochemistry
Immunohistochemical studies were done using the avidin-biotin-peroxidase method. Briefly, serial 3-m-thick sections were mounted on silanized slides. After being deparaffinized and rehydrated, the sections were subjected to 2 min heat-induced antigen retrieval or 15 min pepsin antigen retrieval (only for KRT19). After being blocked by 3% hydrogen peroxide, the sections were incubated with primary antibodies: polyclonal anti-Loricrin (Abcam; 1:500 dilution), monoclonal anti-COMP (Genetex; 1:10 dilution), monoclonal anti-CXCL9 (R&D; 10 g/mL dilution), monoclonal anti-KRT19 (Abcam; 1:1,000 dilution), and polyclonal anti-CYP3A5 (Biomol; 1:2,000 dilution). Then, the slides were incubated for 30 min with the biotinylated IgG (Santa Cruz Biotechnology). Antigen-antibody complexes were visualized with 3,3-diaminobenzidine. Subsequently, the slides were counterstained with Mayer's hematoxylin, differentiated, dehydrated, and mounted. The known Loricrin-positive hyperkeratotic skin, COMP-positive synovium of articular genu, CXCL9-positive oral mucosa of lichen planus, KRT19-positive breast carcinoma, and CYP 3A5-positive lung cancer served as positive controls. A routinely processed OSF section without the primary antibody served as a negative control in each staining series. The slides were examined and scored independently by two investigators blinded to the clinicopathologic data. We defined such criteria for each antigen: Given that Loricrin is normally undetectable by immunohistochemistry in nonkeratinized mucosa (10), any expression was considered positive, regardless of the number of positive cells and staining intensity; tissues showing immunostaining of CXCL9, KRT19, and CYP 3A5 were considered positive irrespective of staining intensity if more than 10% of cells displayed staining; tissues showing immunostaining of COMP was considered positive irrespective of staining intensity if more than 10% of lamina propria had continuous and unfragmented staining because COMP staining was present in areas with very few cells.
Statistical Analysis
Statistical analysis of RT-PCR and Western blotting intensity data was done using Student's t test. The associations between expression of any two proteins as well as between a immunohistochemical result and a clinicopathologic variable were tested by 2 test. Pearson contingency coefficient (r) shows the strength of correlation. A P value of <0.05 was considered significant. The statistical analysis was carried out by using the SPSS for Windows 13.0 program (SPSS, Inc.). Previous SectionNext Section
Results
Identification of Differentially Expressed Genes in OSF
Using a q value of <0.01, the expression pattern of genes either with 2-fold induction or with 0.5fold repression in OSF specimens relative to normal controls was considered as differential regulation. Based on these selection criteria, 661 up-regulated and 129 down-regulated probe sets in OSF with statistically different expression levels were selected by SAM analysis (Supplementary Table S2). The top 30 genes with increased expression and the top 20 ones with decreased expression in OSF relative to NBM were listed in Tables 1 and 2 . Loricrin (46.990-fold, q = 0.006),
COMP (45.764-fold, q = 0.000), and CXCL9 (29.330-fold, q = 0.005) showed the most significantly up-regulated changes, and the top two differentially down-regulated genes were KRT19 (0.063-fold, q = 0.004) and CYP 3A5 (0.129-fold, q = 0.009) as revealed by microarray analysis.
View this table:
In this window In a new window

Table 1. List of the top 30 genes with increased expression in OSF versus NBM
View this table:

Table 2. List of the top 20 genes with decreased expression in OSF versus NBM
Cluster Analysis
The eight samples were grouped using hierarchical clustering with the 790 probe sets identified the differential expression genes in OSF. The resulting expression map was visualized with Treeview 1.6. As expected, four OSF samples clustered separately from the normal samples, meaning that the differential expression genes could distinguish OSF from normal tissues (Fig. 1 ).
View larger version:

Figure 1.
In this page In a new window
Download as PowerPoint Slide
Cluster analysis of gene expression in OSF. Hierarchical clustering of 4 OSF samples and 4 normal tissues using the 790 differential gene sets. The results are expressed as a heat diagram: red, overexpression; green, underexpression. The vertical axis represents genes, and those with the most similar patterns of expression were grouped adjacent to one another. The horizontal axis denotes biopsy samples, and those with the most similar patterns of overall gene expression are placed adjacent to one another.
mRNA Expression of the Five Genes by RT-PCR

To determine the validity of the microarray analysis, we did RT-PCR to confirm the mRNA level of five genes. In concordance with microarray results, OSF tissues expressed significantly more transcript for Loricrin (14.606-fold, P = 0.001), COMP (4.755-fold, P = 0.002), and CXCL9 (3.054fold, P = 0.001) and less for KRT19 (0.184-fold, P = 0.001) and CYP 3A5 (0.321-fold, P = 0.002) compared with the normal controls (Fig. 2A and B ). Because the tissue samples we used in RT-PCR were different from those in the array, heterogeneity of tissues led to the some differences between array data and RT-PCR results, mainly in the fold values.

Figure 2.
Analysis of mRNA and protein levels of Loricrin, COMP, CXCL9, KRT19, and CYP 3A5 genes in OSFs compared with normal controls. A. RT-PCR analysis of five genes of representative five couples of samples [5 OSF (O) and 5 NBM samples (N)]. M, DNA marker. B. mRNA ratios of Loricrin, COMP,
CXCL9, KRT19, and CYP 3A5 in OSF versus normal controls. The gene expression level was normalized by GAPDH as internal control. C. Western blotting analysis of five proteins of representative three couples of samples (3 OSF and 3 NBM). D. Protein ratios of COMP, CXCL9, KRT19, and CYP 3A5 in OSF versus normal controls. The protein expression level was normalized by using -actin as internal control.
Evaluation of Each Protein Expression by Western Blotting

To verify whether the alterations of genes at the level of transcription ultimately result in the alterations at the level of translation, we conducted Western blotting for the five proteins. In agreement with differential mRNA expression, staining for COMP (P = 0.032) and CXCL9 (P = 0.001) revealed a stronger protein level in OSF than in normal control, whereas KRT19 ( P = 0.022) and CYP 3A5 (P = 0.019) protein levels were significantly weaker in OSF than in normal mucosa. However, we did not get a statistically significant result of Loricrin staining ( P > 0.05), possibly because of its unique insolubility and limited expression characteristics in established cell culture systems and any epithelial tissue (10, 11) or limited sample sizes in the study. A representative Western blotting result was presented in Fig. 2C and D.
Expression of Loricrin
No detectable expression of Loricrin was shown (Fig. 3A ) in all normal samples. However, 42 (63.6%) of 66 OSF cases exhibited intensively brown staining for Loricrin almost limited to the upper spinous epithelial layer and sometimes in the keratinocyte layer. The staining was predominantly in the cytoplasm, whereas occasionally both cytoplasmic and nuclear immunoreactivity were observed (Fig. 3B). A significant increase in Loricrin expression was observed in OSF lesions in comparison with NBM specimens (2 = 18.756, P = 0.149 104; Supplementary Table S3).

Figure 3.
Representative immunohistochemical staining for five proteins. A. Negative Loricrin staining in NBM (original magnification, 100). B. Strong cytoplasmic and nuclear staining of Loricrin in the upper spinous epithelial layer and keratinocyte layer of OSF (original magnification, 200). C. Negative
COMP staining in NBM (original magnification, 100). D. Strong staining of COMP in the juxtaepithelial lamina propria and deeper connective tissue of OSF (original magnification, 100). E. Weak to negative CXCL9 staining in NBM (original magnification, 100). F. Strong staining of CXCL9 in the cytoplasm of inflammatory cells and endothelial cells of OSF (original magnification, 400). G. Strong KRT19 staining in the cytoplasm of basal cells of NBM (original magnification, 100). H. Weak to little KRT19 staining in basal cells of OSF (original magnification, 400). I. Strong CYP 3A5 staining in the membrane and cytoplasm of spinous epithelial cells as well as the cytoplasm of endothelial cells of NBM (original magnification, 100). J. Weak to little staining of CYP 3A5 in endothelial cells and spinous epithelial cells of OSF (original magnification, 100).
Expression of COMP
Little to no staining of COMP was found in 14 normal biopsies (Fig. 3C); however, intense signal for COMP was observed in 36 (54.5%) of the 66 OSF cases, which was present in the juxta-epithelial lamina propria with or without deeper connective tissue. However, COMP staining was shown in areas with very few cells, most likely cytoplasm of fibroblasts as judged by the shape and location of the cells (Fig. 3D). A significant increase in COMP immunopositivity was observed in OSF lesions compared with NBM specimens (2 = 13.884, P = 0.194 103; Supplementary Table S3).
Expression of CXCL9 Protein

The staining of CXCL9 protein was observed mainly in the cytoplasm of inflammatory cells and endothelial cells throughout the superficial layer of connective tissue, especially in lamina propria. The connective tissue in 1 (7.1%) of 14 NBM showed very faint CXCL9 expression (Fig. 3E). Fortythree (65.2%) of 66 OSF samples exhibited intense staining of CXCL9 protein (Fig. 3F). The expression of CXCL9 protein was significantly stronger in OSF lesions than in NBM specimens ( 2 = 15.703, P = 0.741 104; Supplementary Table S3).
Expression of KRT19 Protein

All NBM stained continuously and strongly for KRT19 in virtually the cytoplasm of basal cells (Fig. 3G). Only 7 (10.6%) of 66 OSF samples showed faint and fragmented staining in the cytoplasm of basal cells or no staining (Fig. 3H). A significant decrease in KRT19 staining was observed in OSF lesions versus NBM specimens (2 = 47.677, P = 0.503 1011; Supplementary Table S3).
Expression of CYP 3A5 Protein

CYP 3A5 staining was detected in the membrane and/or cytoplasm of spinous epithelial cells and cytoplasm of endothelial cells. Twelve (85.7%) of 14 NBM specimens showed very intense CYP 3A5 staining in spinous epithelial cells, and all normal controls showed intense staining in endothelial cells (Fig. 3I). In OSF cases, only 5 of 66 OSF samples (7.6%) showed faint staining in the cytoplasm of spinous epithelial cells; 33 of 66 OSF samples (50%) showed very faint staining of endothelial cells
(Fig. 3J). The expression of CYP 3A5 protein was weaker in OSF lesions than in NBM specimens ( 2 = 5.986, P = 0.014; Supplementary Table S3).
Correlation between the Expression and Clinical Pathologic Variables in OSF

As shown in Table 3 , there is a significantly positive correlation in OSF between Loricrin-positive expression and histologic grade (early stage and moderately advanced stage, 2 = 0.230, P = 0.030,
r = 0.295) between positive expression of COMP and duration of areca nut chewing ( 2 = 10.000, P = 0.002, r = 0.379) as well as histologic grade (moderately advanced stage and advanced stage, 2 = 7.580, P = 0.006, r = 0.397). A significantly inverse correlation was found between CYP 3A5 expression and histologic grade (early stage and moderately advanced stage, 2 = 4.180, P = 0.040, r = 0.281; moderately advanced stage and advanced stage, 2 = 4.370, P = 0.040, r = 0.302).
However, no significant association of the expression of positive CXCL9 or KRT19 with clinicopathologic variables was shown by the 2 test.
View this table:

Table 3. Correlation between the expression of each protein and clinicopathologic variables in OSF ( n = 66)
Correlation between the Expression of Loricrin, COMP, CXCL9, KRT19, and CYP 3A5
A significant inverse correlation was found only between COMP and CYP 3A5 expression in OSF (2 = 3.911, P = 0.048, r = 0.243; Supplementary Table S4). No clear correlation between the expression of any other two proteins was found in OSF (data not shown), possibly because of the limited sample size in our immunohistochemical study. Previous SectionNext Section
Discussion
To the best of our knowledge, there are limited reports that focus on OSF by means of microarray technology. Kao et al. (12) revealed 18 up-regulated genes and 10 down-regulated genes in OSF compared with normal controls by using microarrays with 1,316 genes, but the limited genes detected could lead to ignoring many significant genes of OSF. Meanwhile, any relationship between individual gene and clinicopathologic factor was never clarified in their experiment. Toward this end, in our study, a microarray with 14,500 genes was first used to identify a list of 790 genes that best represented the gene expression differences between OSF and NBM. Among these deregulated genes, some were previously reported by other groups (13-15); some were not yet observed in OSF but within oral squamous cell carcinoma (16-18); and a number of genes had not been described in any reports on both OSF and oral squamous cell carcinoma. However, there are still some different results between our data and other researchers' reports (12). We consider that microarray analysis for human tissue is very complex, which has extremely numerous biological informations hard to be
fully discovered by a single experiment. A systemic collection and analysis for the sorted complementary data from various research groups will benefit us in making global profiles of OSF. Moreover, the difference of races and region distributions, the different processed methods of betel quid, as well as the different procedure of tissue collection and management may contribute to the distinction among various laboratories. Notable genes in our present study were those with the largest expression changes in OSF compared with NBM, which were thought to be represented diagnostic and therapeutic biomarkers for OSF. The top three differentially up-regulated genes (Loricrin, COMP, and CXCL9) and the top two significantly down-regulated genes (KRT19 and CYP 3A5) were selected as the candidate genes. However, the long lists of data of microarray analysis help little in the understanding of clinical characteristics of OSF. Analysis of gene expression in correlation with clinical or phenotypic variations by our immunohistochemical experiment may indicate biologically meaningful changes of the five novel genes in OSF patients. Inferences based on our findings and evidence from others studies are discussed below. Loricrin, as a protective barrier to protect from environmental hazards, is one of the differentiation markers and the major protein of the cornified envelope of terminally differentiated keratinocyte (19). The abundance of Loricrin in keratinizing epithelia subject to considerable mechanical stress, such as human foreskin epidermis (20), has led to the assumption that expression of Loricrin is essential for the function of these tissues. Obviously, the coarse fibers of areca nut are the key mechanical stress to the epithelium of oral mucosa of OSF patients; thus, it is plausible to hypothesize to find corresponding increase of Loricrin expression in OSF samples. Although Loricrin is normally incorporated into the cornified envelope within 2 hours of synthesis in the differentiating keratinocyte (21), the enhanced expression of extractable Loricrin (not cornified envelope incorporated) in protein extracts from OSF, in the present study, indicated that it was a protective effect of oral mucosa against the persistently mechanical irritation of areca nuts. Moreover, Loricrin in OSF was identified to be localized in the upper spinous epithelial layer and keratinocyte layer corresponding to its location in keratinizing epidermis (22), which could also provide support for our above-mentioned hypothesis. A significant difference of Loricrin staining between early stage and moderately advanced stage of OSF was found but not between moderately advanced stage and advanced stage. The reason for this result might be, on the one hand, the limited number (n = 13) of advanced-stage OSF samples we used in the present study; on the other hand, it is possible that Loricrin would be a kind of protein with limited capacity against the persistently mechanical stress of areca nut in the advanced stage of OSF lesion; however, additional experiments will be necessary to get a definitive answer. Cartilage oligomeric matrix protein (COMP) is an abundant, noncollagenous, extracellular matrix protein as a member of the thrombospondin gene family that modulates the cellular phenotype during tissue genesis and remodeling (23), which was previously found likely to bind to several extracellular matrix proteins, including type I, type II, type IX collagen, and fibronectin (24, 25). COMP was shown to be localized mainly in the extracellular matrix of chondrocytes, synovium, tendons, and ligaments (26). However, other authors also assigned COMP to dermal fibroblasts in
vitro (27), in skin wound tissue but not normal skin (28), and in scleroderma dermal fibroblasts (29). In the present study, COMP was verified to be up-regulated significantly in the submucous tissue of
OSF, whereas immunohistochemical analysis also showed that COMP in OSF was stained in the area with the kinds of collagens deposited consistent with the staining region of COMP in scleroderma (29), possibly pointing to the similar mechanisms of collagen deposition between the two diseases. Thus, it might be excessive COMP secreted by fibroblasts that subsequently facilitate the deposition of collagen in OSF and promote the development of OSF by binding to matrix components. Moreover, the increased expression of fibrogenic cytokines, namely TGF-, was found in OSF tissues (30), whereas a previous study also indicated that COMP could be induced to overexpress by TGF- treatment (29), suggesting that increased TGF- might also provoke the synthesis of COMP in OSF. In addition, autoimmunity has been examined as an etiologic factor for OSF (31), whereas COMP has been cited as potential autoantigens in patients with rheumatoid arthritis (32). It may thus raise the possibility that there is an autoimmune response to COMP within the subepithelial connective tissue of some patients with OSF. Nonetheless, additional studies will be necessary to elucidate the question of whether COMP is synthesized by fibroblasts in the connective tissue of OSF rather than other types of cell. Chemokines are constitutively expressed or stimulated by inflammatory processes (33), which play important roles as regulators of leukocyte migration, adhesion, and activation during inflammatory diseases, angiogenesis, tumor rejection, rejection of organ transplants, and HIV infection (34, 35). Among a variety of chemokines, CXCL9 (monokine induced by IFN-, MIG) functions as a potent chemoattractant for tumor-infiltrating lymphocytes (36), activated natural killer cells, and TH1 lymphocytes of peripheral blood lymphocytes (37), and has been shown to inhibit neovascularization (38) and contribute to a variety of inflammatory disorders. Strong expression of CXCL9 was shown in both basal keratinocytes and dermal mononuclear cells of lichen planus lesions (39), as well as exclusively restricted to macrophages and vessel-associated cells in the papillae tips of psoriasis lesion (40). One of the hallmarks of OSF is a juxta-epithelial and diffuse mononuclear cell infiltration in the lamina propria. Accordingly, in present study, the enhanced expression of CXCL9 was found in the inflammatory cells of lamina propria of OSF, indicating that CXCL9 might contribute to an ascending chemotactic gradient in the subepithelial connective tissue and the pronounced recruitment of inflammatory cells in OSF. Furthermore, our data showed that CXCL9 protein was stained in endothelial cells of OSF microvessels, hypothesizing that the expression of CXCL9 in endothelial cells, on the one hand, contributes to the infiltration of inflammatory cells to adhere to endothelial cells and, on the other hand, might have a detrimental effect in the repair process of the vasculature and result in the eventual atresia of OSF microvessels. Interestingly, another study showed that IFN-, which is considered as an exclusive regulator to induce CXCL9, was shown to have little or no expression in biopsies from OSF tissues compared with normal controls (41). This raises the question whether there might be other extracellular stimuli in OSF to modulate the expression of CXCL9. However, the hypothesis has been supported by recent studies (42, 43). Indeed, this is a topic that deserves investigation on the concretely regulating mechanisms of CXCL9 gene in OSF with reduced IFN- expression. KRT19, which is expressed exclusively by epithelial cells and cancers derived from them, is the smallest member of the cytoplasmic intermediate filament protein family and has a wide tissue distribution (44). Previous studies have suggested that KRT19 expression might be linked to the retention of proliferative (stem cell) potential or undifferentiated character in oral nonkeratinized
mucosa, hair follicle, and skin (45-47). In normal nonkeratinized mucosa, KRT19 was detectable in the basal cell layer, whereas there was no detectable KRT19 in normal keratinized mucosa (45). In the present study, the expression of KRT19, whether in the mRNA or protein level, was shown to be significantly weaker in the OSF basal cell layer, which is the proliferative invasive layer, than in normal buccal nonkeratinized mucosa, indicating that the self-renewing capacity of the basal cell layer of OSF through stem cells was obviously inhibited, likely by the chemical irritation of areca nut that subsequently depressed the constant regeneration of OSF mucosa and promoted the atrophy of oral epithelium, which is known as one of characteristic pathologic features of OSF. Eventually, the atrophic epithelium of OSF with the decreased repair rate would potentially be more vulnerable and subject to facilitating the more hazardous chemical substances of areca nut to permeate through this narrow epithelial strip into the connective tissue and causing further deterioration of OSF lesion. In the present immunochemical data, no significant correlation was found between the KRT19 expression and the histopathologic grade of OSF, which might suggest that the proliferative potential of oral epithelium would be obviously depressed by some unknown mechanism from the early stage of the lesion. These results lend strong support to the idea that enhancing the renewing ability of mucosa from the early stage would be a new field for the therapy of OSF. The cytochrome P450 enzymes are a very large gene family of constitutive and inducible monooxygenase enzymes that metabolize many lipophilic, biologically active endogenous and xenobiotic substrates, including a large number of therapeutic drugs and toxic environmental chemicals (48, 49). CYP 3A5 enzyme is one of the main P450 families involved in xenobiotic metabolism, including the metabolism of various carcinogens and several current anticancer drugs, which has been identified in several normal tissues including colon, lung, and anterior pituitary gland. Among numerous chemical constituents of areca nut, alkaloid is the most important agent to undergo nitrosation and give rise to N-nitrosamines, which might have cytotoxic and mutagenic effects on cells of the oral mucosa, whereas CYP 3A5 enzyme activities can just metabolize the activation of these procarcinogens (50). However, in the present study, it seems to be irrational that the expression of CYP 3A5 in OSF was found significantly depressed versus NBM, whereas the reason would be the possibility that the central capacity of CYP 3A5 to metabolize these procarcinogens in OSF mucosa was depressed likely by the persistently and excessively chemical stress whose strength has exceeded the defending capacity of NBM from areca nut constituents. Thus, the depressed ability of OSF mucosa to defend the stress of active endogenous and xenobiotic substrates would open more doors for more toxicities of areca nut to affect more cells in the OSF mucosa and further deteriorate the disease. Moreover, in the present study, the presence of CYP 3A5 protein in the spinous epithelial cells showed an apparent depression from the initial stage; the endothelial cells revealed a gradual reduction correlated with the histopathologic grade, which is possible because the epithelium of mucosa is the first place to respond to the xenobiotic toxicity of areca nut, whereas the endothelial cells are not so sensitive to the toxicity as the epithelium because of their deeper location. In conclusion, the techniques of microarray analysis provide a dramatic tool to screen novel pathopoiesis-associated genes in OSF research. In the present microarray analysis, Loricrin, COMP,
CXCL9, KRT19, and CYP 3A5 were the genes with the most significantly differential deregulation and
their functional implications could provide more novel and valuable explanations on the distinctive
pathologic changes and pathopoiesis of OSF. Moreover, based on the above-mentioned notions, we presume that some important changes of the mucosa epithelium in OSF might play a vital role in the development of OSF and subsequently result in the typical pathologic changes of the subepithelial connective tissue of OSF. However, we may need more clinical studies involving larger numbers of clinical specimens and more quantitative analysis to explore the potential application of these markers in clinical management of OSF patients. Otherwise, in the present study, we were unable to completely eliminate the contribution of alcohol and smoking to the alteration of gene expression because a great majority of areca nut chewers here were also heavy drinkers and/or smokers. Thus, in a further study, OSF patients without alcohol and smoking habits will be collected to address the roles of the deregulated genes only caused by areca nut chewing. Previous SectionNext Section
Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Previous SectionNext Section
Acknowledgments
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Prof. Weixin Hu and Dr. Haitao Zeng (Research Center of Molecular Biology of Central South University) for their technical assistance. Previous SectionNext Section
Footnotes

Grant support: National Natural Sciences Foundation of China grant 30572044. Note: Supplementary data for this article are available at Cancer Epidemiology Biomakers and Prevention Online (http://cebp.aacrjournals.org/).
o o o
Accepted June 10, 2008. Received December 14, 2007. Revision received May 20, 2008.
Previous Section
References
1.
Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking, and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24:4503.
CrossRefMedline 2.
Pindborg JJ, Sirsat SM. Oral submucous fibrosis. Oral Surg Oral Med Oral Pathol 1966;22:76479.
CrossRefMedline 3.
Pindorg JJ. Oral submucous fibrosis: a review. Ann Acad Med Singapore 1989;18:603 7.
Medline 4. Reichart PA, Philipsen HP. Betel and Miang. Vanishing Thai habits. 2nd ed. Bangkok: White Lotus Ltd. Co; 2005. 5.
Jian XC, Shen ZH, Liu SF. Oral submucous fibrosiscase reports. J Clin Stomatol 1985;1:123.
6.
Jian XC, Liu SF, Shen ZH, et al. A clinical study of oral submucous fibrosis. Chin J Stomatol 1989;24:29902.
7.
Murti PR, Bhonsie RB, Pindborg JJ, Daftary DK, Gupta PC, Mehta FS. Malignant transformation rate in oral submucous fibrosis over a 17-year period. Community Dent Oral Epidemiol 1985;13:3401.
CrossRefMedline 8.
Jin YT, Tsai ST, Wong TY, Chen FF, Chen RMY. Studies on promoting activity of Taiwan betel quid ingredients in hamster buccal pouch carcinogenesis. Eur J Cancer B Oral Oncol 1996;32B:3436.
9.
Jian XC, Peng JY, Tang ZG. Three cases of oral cancer associated with oral submucous fibrosis. West China J Stomatol 2000;18:1301.
10.
Katou F, Shirai N, Kamakura S, Tagami H, Nagura H, Motegi K. Differential expression of cornified cell envelope precursors in normal skin, intraorally transplanted skin and normal oral mucosa. Br J Dermatol 2003;148:898905.
Medline 11.
Hohl D, Lichti U, Breitkreutz D, Steinert PM, Roop DR. Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. J Invest Dermatol 1991;96:4148.
CrossRefMedline 12.
Kao WB, Shieh YD, Hsia YJ, Shieh TY. The micro-array analysis of genetic change and proteins identification of oral submucous fibrosis. Int J Oral Maxillofac Surg 2005;34:139.
13.
Tsai CH, Yang SF, Chen YJ, Chou MY, Chang YC. The upregulation of insulin-like growth factor-1 in oral submucous fibrosis. Oral Oncol 2005;41:9406.
Medline 14. Yang SF, Hsieh YS, Tsai CH, Chou MY, Chang YC. The upregulation of type I
plasminogen activator inhibitor in oral submucous fibrosis. Oral Oncol 2003;39:367 72.
Medline 15.
Utsunomiya H, Tilakaratne WM, Oshiro K, et al. Extracellular matrix remodeling in oral submucous fibrosis: its stage-specific modes revealed by immunohistochemistry and in situ hybridization. J Oral Pathol Med 2005;34:49807.
Medline 16.
Liu SY, Lin MH, Yang SC, et al. Areca quid chewing enhances the expression of salivary matrix metalloproteinase-9. J Formos Med Assoc 2005;104:1139.
Medline 17. Liu CJ, Lui MT, Chen HL, Lin SC, Chang KW. MICA and MICB overexpression in oral
squamous cell carcinoma. J Oral Pathol Med 2007;36:437.

Medline 18.
Tsai WC, Tsai ST, Ko JY, et al. The mRNA profile of genes in betel quid chewing oral cancer patients. Oral Oncol 2004;40:41826.
CrossRefMedline 19.
Kozo Y, Daniel H, Wesley M, et al. The human loricrin gene. J Biol Chem 1992;267:180606.
Abstract/FREE Full Text 20.
Candi E, Melino G, Mei G, et al. Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein. J Biol Chem 1995;270:2638290.
Steinert PM, Marekov LN. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J Biol Chem 1995;270:1770211.
Ishida YA, Takahashi H, Iizuka H. Loricrin and human skin diseases: molecular basis of loricrin keratodermas. Histol Histopathol 1998;13:81926.
Medline 23.
Oldberg A, Antonsson P, Lindblom K, Heinegrd D. COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins. J Biol Chem 1992;267:22346 50.
Di Cesare PE, Chen FS, Moergelin M, et al. Matrix-matrix interaction of cartilage oligomeric matrix protein and fibronectin. Matrix Biol 2002;21:46170.
CrossRefMedline 25.
Holden P, Meadows RS, Chapman KL, Grant ME, Kadler KE, Briggs MD. Cartilage oligomeric matrix protein interacts with type IX collagen, and disruptions to these interactions identify a pathogenetic mechanism in a bone dysplasia family. J Biol Chem 2001;276:604655.
Hedbom E, Antonsson P, Hjerpe A, et al. Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage. J Biol Chem 1992;267:6132 6.
Hummel KM, Neidhart M, Vilim V, et al. Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids. Br J Rheumatol 1998;37:721 8.
Smith RK, Zunino L, Webbon PM, Heinegrd D. The distribution of cartilage oligomeric matrix protein (COMP) in tendon and its variation with tendon site, age and load. Matrix Biol 1997;16:25571.
CrossRefMedline 29.
Farina G, Lemaire R, Korn JH, Widom RL. Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts. Matrix Biol 2006;25:21322.
CrossRefMedline 30.
Haque MF, Harris M, Meghji S, Barrett AW. Immunolocalization of cytokines and growth factors in oral submucous fibrosis. Cytokine 1998;10:7139.
CrossRefMedline 31.
Chiang CP, Hsieh RP, Chen TH, et al. High incidence of autoantibodies in Taiwanese patients with oral submucous fibrosis. J Oral Pathol Med 2002;31:4029.
CrossRefMedline 32.
Krenn V, Souto-Carneiro MM, Kim HJ, et al. Histopathology and molecular pathology of synovial B-lymphocytes in rheumatoid arthritis. Histol Histopathol 2000;15:7918.
Medline 33.
Proudfoot AE. Chemokine receptors: multifaceted therapeutic targets. Nat Rev Immunol 2002;2:10615.
CrossRefMedline 34.
Sallusto F, Mackay CR, Lanzavecchia A. The role of chemokine receptors in primary, effector, and memory immune responses. Annu Rev Immunol 2000;18:59320.
CrossRefMedline 35.
Liao F, Rabin RL, Yannelli JR, Koniaris LG, Vanguri P, Farber JM. Human Mig chemokine: biochemical and functional characterization. J Exp Med 1995;182:1301 4.
Rollins BJ. Chemokines. Blood 1997;90:90928.

FREE Full Text 37.
Gasperini S, Marchi M, Calzetti F, et al. Gene expression and production of the monokine induced by IFN- (MIG), IFN-inducible T cell chemoattractant (I-TAC), and IFN--inducible protein-10 (IP-10) chemokines by human neutrophils. J Immunol 1999;162:492837.
Angiolillo AL, Sgadari C, Taub DD, et al. Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. J Exp Med 1995;182:1552.
Spandau U, Toksoy A, Goebeler M, Brcker EB, Gillitzer R. MIG is a dominant lymphocyte-attractant chemokine in lichen planus lesions. J Invest Dermatol 1998;202:10039.
40.
Goebeler M, Toksoy A, Spandau U, Engelhardt E, Brcker EB, Gillitzer R. The CXC chemokine Mig is highly expressed in the papillae of psoriatic lesions. J Pathol 1998;183:8995.
41.
Haque MF, Meghji S, Khitab U, Harris M. Oral submucous fibrosis patients have altered levels of cytokine production. J Oral Pathol Med 2000;29:1238.
CrossRefMedline 42.
Ohmori Y, Hamilton TA. A macrophage LPS-inducible early gene encodes the murine homologue of IP-10. Biochem Biophys Res Commun 1990;168:12617.
CrossRefMedline
43.
Mikihiroi, Yoshihiro O. Constitutive nuclear factor B activity is required to elicit interferon--induced expression of chemokine CXC ligand 9 (CXCL9) and CXCL10 in human tumour cell lines. J Biochem 2003;376:393402.
44.
Haque MF, Meghji S, Khitab U, Harris M. The human keratin genes and their differential expression. Curr Top Dev Biol 1987;22:534.
Medline 45.
Lindberg K, Rheinwald JG. Suprabasal 40 kd keratin (K19) expression as an immunohistological marker of premalignancy in oral epithelium. Am J Pathol 1989;134:8998.
Abstract 46. Lindberg K, Rheinwald JG. Three distinct keratinocyte subtypes identified in human oral
epithelium by their patterns of keratin expression in culture and in xenografts. Differentiation 1991;45:23041.
47.
Michel M, Trk N, Godbout MJ, et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 1996;109:101728.
Abstract 48.
Nebert DW, Russell DW. Clinical importance of the cytochromes P450. Lancet 2002;360:115562.
CrossRefMedline 49.
Danielson PB. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab 2002;3:56197.
CrossRefMedline 50.
Parke DV. The cytochromes P450 and mechanisms of chemical carcinogenesis. Environ Health Perspect 1994;102:8523.
Medline
In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study C Ponranjini Vedeswari1, S Jayachandran1, S Ganesan2 1 Department of Oral Medicine and Radiology, Government Dental College and Hospital, Chennai, Tamil Nadu, India 2 Department of Medical Physics, Anna University, Chennai, Tamil Nadu, India Click here for correspondence address and email
Date of Submission Date of Decision Date of Acceptance Date of Web Publication
17-Jun-2008 02-Feb-2009 03-Mar2009 30-Oct-2009
Abstract Aims and Objectives: To compare the autofluorescence spectra of oral submucous fibrosis (OSF) with normal mucosa, the autofluorescence spectra of OSF before and after treatment with intralesional dexamethasone and hyaluronidase, the clinical improvement following treatment with the changes in autofluorescence spectra and to prove that autofluorescence spectroscopy is a good method for diagnosis and assessment of treatment effectiveness in OSF. Materials and Methods: The study was conducted at the Department of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, Chennai and Division of Medical Physics and Lasers, Department of Physics, Anna University, Chennai in 20 patients seeking medical management for symptomatic OSF and 20 patients who had dental caries only without any oral mucosal diseases and oral habits were used as normal controls. Their ages ranged from 20 to 40 years, including both male and female. In vivo fluorescence emission spectra were obtained using a handheld optical fiber probe attached to a Fluoromax-2 spectrofluorometer. Results: The fluorescence spectrum of OSF had an intense fluorescence emission at 385 nm with a secondary emission peak at 440 nm compared with that of the normal oral mucosa. The average fluorescence spectrum of the post treated OSF mucosa had a lesser intensity around 385 nm and a higher intensity around 440 nm than that of the pre treated OSF mucosa, thereby mimicking the normal oral mucosa. All the three clinical parameters (maximal mouth opening, tongue protrusion and the severity of burning sensation) showed a high statistical significance, with P < 0.001, as in the case of classification of pre treated OSF mucosa from the post treated OSF mucosa using the autofluorescence technique. Conclusion: The change in the fluorescence emission spectrum for both normal and OSF mucosa before and after treatment can be explained by analyzing the changes in the fluorescence intensity of the endogenous fluorophores. Keywords: Autofluorescence spectroscopy, dexamethasone, hyaluronidase, OSF How to cite this article: Vedeswari CP, Jayachandran S, Ganesan S. In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study. Indian J Dent Res 2009;20:261-7
How to cite this URL: Vedeswari CP, Jayachandran S, Ganesan S. In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study. Indian J Dent Res [serial online] 2009 [cited 2010 Feb 4];20:261-7. Available from: http://www.ijdr.in/text.asp?2009/20/3/261/57354 When biologic tissue interacts with light, it becomes excited and reemits light of varying colors (fluorescence) [1],[2] , which can be detected by sensitive spectrometers. Compared with normal tissues, different diseased tissues contain different morphohistologic characteristics and intrinsic fluorophores that give rise to different fluorescence emission spectra when the tissues are excited at a suitable wavelength. [1],[3] The physical and chemical properties of a tissue can be evaluated by analyzing the intensity and the character of light emitted in the form of fluorescence. [9],[10] Oral submucous fibrosis (OSF) is characterized by increased collagen deposition in the subepithelial connective tissue layer and markedly atrophic oral epithelium. It has been demonstrated that, under the range of a 300-360 nm excitation wavelength, the emission band at 380-400 nm is mainly attributed to the presence of collagen, whereas that at 440-460 nm is mainly due to the presence of nicotinamide adenine dinucleotide (NADH). [6],[7] Conventional submucosal injections of dexamethasone plus hyaluronidase results in symptomatic improvement in 83% of the patients. [8] Steroids act by decreasing inflammation, decrease in fibroblastic proliferation and deposition of collagen. Hyaluronidase acts by break-down of the hyaluronic acid matrix, lowering the viscosity of the intercellular cementing substance and also decreasing collagen formation. This study was conducted to evaluate the possibility of using endogenous fluorophores present in the OSF mucosal sites to diagnose and to monitor the therapeutic response by analyzing their native fluorescence characteristics. [6],[12],[13],[14] The aims and objectives of this study were

To compare the autofluorescence spectra of the OSF with the normal mucosa. To compare the autofluorescence spectra of the OSF before and after treatment with intralesional dexamethasone and hyaluronidase. To compare the clinical improvement following treatment with the changes in the autofluorescence spectra. To prove that autofluorescence spectroscopy is a good method for diagnosis and assessment of treatment effectiveness in OSF.
Materials and Methods
The study was conducted at the Department of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, Chennai, and Division of Medical Physics and Lasers, Department of Physics, Anna University, Chennai, after obtaining approval. The study population included 20 patients who were seeking medical management for symptomatic OSF and 20 patients who had dental caries only without any oral mucosal diseases and oral habits were used as normal controls. Their ages ranged from 20 to 40 years, including both male and female. A positive history of chewing of the areca nut or one of its commercial preparations,
difficulty in swallowing and chewing and burning sensation on eating spicy foods and restricted mouth opening and changes in the oral mucous membrane, including the presence of palpable fibrotic bands, stiffness and blanching, were used to establish the diagnosis. Some OSF patients also had depapillation of the tongue and impaired tongue mobility. History of habits, including duration in months and frequency per day, duration of symptoms, oral mucosal sites of involvement, the maximal mouth opening, tongue protrusion and the severity of burning sensation were recorded in a structured proforma designed for this study. The severity of burning sensation was graded from 0 to 3 as no burning sensation, mild, moderate and severe. Mouth opening was measured as the interincisal distance from the mesioincisal edge of the upper left central incisor tooth to the mesioincisal edge of the lower left central incisor tooth. Measurement was made using a geometric divider and scale and was recorded in millimeters. The degree of tongue protrusion was recorded in units of 5 mm from the incisal edge of the lower teeth by viewing the protruded tongue from the lateral aspect of the head and measuring the distance from the mesial contact area of the lower central incisors to the tip of the protruded tongue. When the value was found to lie between units of 5 mm, it was adjusted to the higher limit. This was carried out to allow for the fine movement of the tip that did not permit an exact millimeter recording. The patients were graded clinically as follows: Grade I, only blanching of the oral mucosa without symptoms; grade II, grade I and burning sensation, dryness of the mouth, vesicles or ulcers in the mouth without tongue involvement; grade III, in addition to grade II, restriction of mouth opening; grade IV, in addition to grade III, palpable bands all over the mouth without tongue involvement; grade V, grade IV and tongue also involved; grade VI, OSF along with histopathologically proven oral cancer. Patients with systemic diseases like diabetes, hypertension, hepatic or renal diseases, pregnant and lactating mothers and any kind of allergy were excluded from the study. All the lesions were diagnosed based on history and clinical examination. Basic blood and urine investigations were carried out for all the patients. Autofluorescence spectroscopy Reassurance and counseling was given and informed consent was obtained from the patient. In vivo fluorescence emission spectra were obtained from each OSF buccal mucosal site and each normal buccal mucosal site of the control patients using a handheld optical fiber probe attached to a Fluoromax-2 spectrofluorometer (ISA Jobin Yvon - Spex, Edison, New Jersey, USA). A monochromator with a 150-W ozone-free Xenon lamp provided the excitation light [Figure 1] and [Figure 2]. The desired excitation wavelength and the emission spectrum were selected by a PC-controlled monochromator. The excitation light was guided to illuminate samples by one arm of a Y-type quartz fiber bundle and the emission fluorescence was collected by another arm of the fiber bundle and directed to the photomultiplier detector. The signal was then amplified and displayed on the computer monitor. All Fluoromax-2 functions are controlled by the DataMax software (Dover corporation (NYSE:DOV), Orlando, Florida, USA), which communicates between a PC- compatible computer and the Fluromax-2. The optical fiber probe was disinfected with 2.4% gluteraldehyde solution, rinsed with phosphate-buffered saline and wrapped with a transparent plastic membrane before each clinical use. Excitation-emission spectra In this study, ultraviolet light at 330 nm wavelength was used to excite each sample and the
resulting emission spectra were recorded from 350 to 600 nm in 1 nm increments. During measurement, the probe was gently touched on the surface of the normal or lesional buccal mucosal site. The spectrum at each site was obtained by averaging three spectra measured from three adjacent sites (2 mm apart) of the same lesion. Treatment protocol Then, patients of the study group were subjected to a treatment protocol of intralesional injection of a combination of dexamethasone sodium phosphate 4 mg/ml and hyaluronidase 1500 IU twice a week for 6 weeks. They were also given antioxidants and micronutrients supplementation and were taught muscle-stretching exercises. At each visit, following topical application of lignocaine 2%, 1500 IU of hyaluronidase was dissolved in 2.0 ml of dexamethasone sodium phosphate in a 2 ml disposable syringe. The drugs were injected at multiple sites submucosally by means of a gauge 24 needle, not more than 0.2 ml solution per site. The patient was evaluated at each visit during the treatment period of 6 weeks. A clinical examination was carried out at every visit and the parameters of maximal mouth opening, tongue protrusion and burning sensation were recorded. Emission spectra after treatment After 6 weeks, the fluorescence emission spectra of the treated site were recorded as before. There were no adverse effects throughout the study. Statistical analysis After normalization, the mean ( SD) fluorescence intensities at 385 5 nm (I 385 5 nm ) and 440 5 nm (I 440 5 nm ) emission peaks and the mean ratio of I 440 5 nm /I 385 5 nm were calculated for the normal oral mucosa and OSF mucosa before and after treatment. The differences in the mean values of I 385 5 nm , I 440 5 nm and ratio of I 440 5 nm /I 385 5 nm between the normal oral mucosa and the pre treatment OSF mucosa and between the normal oral mucosa and the post treatment OSF were assessed for statistical significance by unpaired Student's t- test. The differences in the mean values of I 385 5 nm , I 440 5 nm and ratio of I 440 5 nm /I 385 5 nm between the pre treatment OSF and the post treatment OSF were assessed for statistical significance by paired Student's t-test. The mean ( SD) values of maximal mouth opening, tongue protrusion and burning sensation of the pre treatment OSF group were compared with that of the post treatment OSF group using paired Student's t-test. A P-value of less than 0.05 was considered to be statistically significant. Results
Comparison of autofluorescence characteristics of the normal oral mucosa and the OSF mucosa All the spectra were normalized by dividing the intensity of each wavelength by the maximum intensity of the emission spectrum to avoid the intensity variations due to different excitation
light power, fluorescence collection efficiency as well as interpatient variations. The normalized fluorescence spectra of the normal oral mucosa and the OSF mucosa are shown in Figure 3. The fluorescence spectrum of OSF had an intense fluorescence emission at 385 nm, with a secondary emission peak at 440 nm, compared with that of the normal oral mucosa. In order to find out whether any considerable statistical significance exists in the spectral signature between normal oral mucosa and OSF mucosa, three different ratio parameters, such as R1 = I 440 5 nm /I 385 5 nm [Table 1] , R2 = I 420 5 nm /I 370 5 nm and R3 = I 470 5 nm /I 450 5 nm were introduced. They were subjected to Unpaired Student's t-test. In this context, the ratio value R1 is considered for further analysis and discussion. When the mean ( SD) fluorescence intensities at 385 5 nm (I 385 5 nm) and 440 5 nm (I 440 5 nm ) emission peaks and the mean ratio of I 440 5 nm /I 385 5 nm were compared between the groups, the OSF group had a significantly higher mean value of I 385 5 nm , a significantly lower mean value of I 440 5 nm and a significantly lower mean ratio of I 440 5 nm /I ** ) [Table 1]. The scattered graph is plotted 385 5 nm than the normal oral mucosa (P < 0.001 between the intensity ratios of I 440 5 nm /I 385 5 nm and the number of subjects. From the scatter plot, it was observed that there is a distinct variation in the distribution of R1 value between the normal oral mucosa and the OSF mucosa [Figure 4]. It is found that if the ratio value R1 is greater than 0.96, the tissue is normal oral mucosa, whereas at values lesser than 0.96, the tissue is OSF mucosa. Based on the R-value 0.96, the diseased persons are classified with 90% sensitivity and normal oral mucosa with 80% specificity. Comparison of normal oral mucosa and pre treated and post treated OSF mucosa In this context, the post treated OSF cases were subjected to measurement of fluorescence at identical conditions, i.e. as in the case of measuring the pre treated OSF condition. The post treated OSF mucosa had a lower mean value of I 385 5 nm and I 440 5 nm and a higher mean ratio of I 440 5 nm /I 385 5 nm compared with that of the normal oral mucosa [Table 2],[Figure 5]. The fluorescence emission spectra of the pre- and post treated OSF groups are shown in Figure 6. The average fluorescence spectrum of the post treated OSF mucosa had a lesser intensity at around 385 nm and a higher intensity at around 440 nm than that of the pre treated OSF mucosa, thereby mimicking the normal oral mucosa [Table 3]. Scatter plot drawn for I 440 5 nm /I 385 5 nm for the pre- and post treated cases showed R factor of 0.96 to separated the pre treated from the post treated cases [Figure 7]. If R-value is < 0.96, the tissues are pre treated OSF mucosa and for R-value > 0.96, the tissues are post treated OSF mucosa. Thus, it is interestingly observed that the ratio values of the post treated OSF group have enhanced to greater than 0.96, as in the case of normal subjects, indicating the response of treatment and the transformation of the OSF toward normalcy. Comparison of treatment response between autofluorescence and conventional clinical methods In order to verify whether the autofluorescence technique is comparable with that of conventional clinical methods to monitor the treatment response, the pre- and post OSF patients were subjected to measurement of maximal mouth opening, tongue protrusion and the severity of burning sensation [Figure 8]. The mean SD of these three parameters was also estimated [Table 4].
The mean ( SD) maximal mouth opening of the post treated OSF group was significantly higher than that of the pre treated OSF group (P < 0.001 ** ). The mean ( SD) tongue protrusion of the post treated OSF patients was significantly higher than that of the pre treated oral submucous fibrosis patients (P < 0.001 ** ) [Figure 6]. The mean ( SD) severity of burning sensation of the post treated OSF patients was significantly lower than that of the pre treated OSF patients (P < 0.001 ** ). All the three clinical parameters showed high statistical significance, with P < 0.001, as in the case of classification of the pre treated oral submucous fibrosis mucosa from the post treated OSF mucosa, using the autofluorescence technique. Further, from the scattered plot, it is verified that the ratio value 0.96 is greater for both the normal oral mucosa and the post treated OSF mucosa. Of the 20 treated patients, only one patient's R-value was < 0.96, indicating that autofluorescence may also be effectively used to monitor the therapeutic response. Discussion
In this study, we found that with ultraviolet light at 330 nm excitation, the spectra of the OSF mucosa had an intense fluorescence emission at 385 nm and a secondary emission peak at 440 nm with that of the normal oral mucosa. In addition, the pre treated OSF mucosa had a significantly higher mean value of I 385 5 nm , a significantly lower mean value of I 440 5 nm and a significantly lower mean ratio of I 440 5 nm /I 385 5 nm than the normal oral mucosa (P < 0.001). Previous studies have shown that at 330 nm excitation, the 380 and 460 nm emission peaks of the autofluorescence spectra for oral mucosal tissues reflect the collagen content in the subepithelial connective tissue and the NADH content in the epithelial cells, respectively. [7],[10],[12] Therefore, the increased subepithelial collagen content could explain why the pre treated OSF mucosa had a significantly higher intensity of the 385 nm emission peak of the autofluorescence spectra than the normal oral mucosa and the post treated OSF mucosa. Furthermore, compared with the normal oral epithelium, the markedly atrophic OSF epithelium allowed more excitation energy to penetrate into the subepithelial connective tissue and more collagen- derived emission fluorescence to go through. This factor also contributed to the high intensity of the 385 nm emission peak of the autofluorescence spectra of the OSF mucosa. In addition, the markedly atrophic OSF epithelium might contain less amount of NADH than the normal oral epithelium. The fibrosis-induced reduction of blood vessels in the lamina propria might also diminish the metabolic rate of the oral epithelial cells, which, in turn, results in a lower NADH content in the OSF epithelium. Therefore, the OSF mucosa had a significantly lower intensity of the 440 nm emission peak than the normal oral mucosal sites. This suggests that autofluorescence spectroscopy can detect the increased amount of collagen in the subepithelial connective tissue and the decreased amount of NADH in the epithelium. The monochromatic light signal penetrates only about 500 m deep into the tissue. Thus, this technique is capable of analyzing only the most superficial portions of the oral lesions. [9] In this study, no significant differences in the pattern of the 385 and 440 nm emission band was found between the normal oral mucosa and the post treated OSF mucosa. Steroids are well known to act as antiinflammatory agents for prevention or suppression of the fibroproductive inflammation found in the OSF mucosa thus ameliorating this fibrocollagenous condition. [5] Tsai et al. had suggested that in addition to the known mechanisms, a
corticosteroid-induced increase in collagen degradation could result from enhanced collagen phagocytosis by the fibroblast. [11] Hyaluarnidase degrades the hyaluaronic acid matrix actively, promoting lysis of the fibrinous coagulum. [4] It is due to the above actions that the collagen content of the OSF mucosa had reduced and the atrophic oral epithelium had healed. Hence, following the treatment, there is a similar fluorescence characteristic as in the case of the normal oral mucosa. The improvement in mouth opening, tongue protrusion and decrease in the burning sensation when compared with the initial stage was highly significant and correlated well with the autofluorescence characteristics. This again is attributed to treatment efficacy, resulting in collagen degradation and decrease in the inflammation. Conclusion
In summary, the change in the fluorescence emission spectrum for both the normal and the OSF mucosa before and after treatment can be explained by analyzing the changes in the fluorescence intensity of endogenous fluorophores. This study adds evidence that high collagen and low NADH characterize the OSF mucosa. It is also obvious from this study that autofluorescence spectroscopy can monitor the therapeutic response and characterize the tissue transformation following treatment. Furthermore, investigations with a large group of experimental subjects will also be useful for the development of a statistical database and a user-friendly diagnostic algorithm that could facilitate early detection. Autofluorescence spectroscopy being a non invasive and easily applicable tool for the detection of the alterations in the structural and biochemical composition of cells may reduce the patient morbidity and, therefore, is of great clinical importance. From this study, we conclude that autofluorescence spectroscopy provides valuable information for the diagnosis and also for monitoring the therapeutic response in OSF. However, results of this study on autofluorescence characteristics in the diagnosis and therapeutic monitoring in OSF has to be validated with more studies involving large samples and longer follow-up. Acknowledgments
We express our sincere thanks to our Principal, the faculties in the Departments of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, and Mr. Sivabalan, Research Scholar, Department of Medical Physics, Anna University, for their valuable suggestions, kind help and encouragement in the present study.
References 1. Udenfriend S. Proteins and peptides. In: Horecker B, Marmur J, Kaplan N, Scheraga HA, editors. Fluorescence assay in biology and medicin. Molecular biology. London: Academic Press: 1969;11:248-83. Izatt RM, Christensen JJ. Heats of proton ionization, pK and related thermodynamic
2.
3. 4. 5. 6.
7.
8.
9.
10.
11.
12. 13.
14.
quantities. In: Fasman GD, editor. Handbook of biochemistry and molecular biology. 3 rd ed. Cleveland: CRC Press; 1975;205-10. Fasman GD (Ed.). Handbook of biochemistry and molecular biology. 3 rd ed. Cleveland: CRC press; 1975; 205-10. Kakar PK, Puri RK, Venkatachalam VP. Oral submucous fibrosis - treatment with hyalase. J Laryngol Otol 1985;99:57-9. Gupta D, Sharma SC. OSF - A new treatment regimen. J Oral Maxillofac Surg. 1988;46:830-3. Glasgold R, Glasgold M, Savage H, Pinto J, Alfano R, Schantz S. Tissue auto-fluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis. Cancer Lett 1994;82:33-41. Bottiroli G, Croce AC, Locatelli D, Marchesini R, Pignoli E, Tomatis S, et al. Natural fluorescence of normal and neoplastic human colon: A comprehensive 'ex-vivo' study. Lasers Surg Med 1995;16:48-60. Lai DR, Chen HR, Lin LM, Huang YL, Tsai CC. Clinical evaluation of different treatment methods for OSF. A 10-year experience with 150 cases. J Oral Pathol Med 1995;24:4026. Gillenwater A, Jacob R, Richards-Kortum R. Fluorescence spectroscopy: A technique with potential to improve the early detection of aero digestive tract neoplasia. Head and Neck 1998; 20:556-62. Chen CT, Chiang HK, Chow SN, Wang CY, Lee YS, Tsai JC, et al. Auto- fluorescence in normal and malignant human oral tissues and in DMBA-induced hamster buccal pouch carcinogenesis. J Oral Pathol Med 1998;27:470-4. Tsai CC, Ma RH, Shieh TY. Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for OSF. J Oral Pathol Med 1999;28:59- 63. Chen HM, Wang CY, Chen CT, Yang H, Kuo YS, Lan WH, et al. Auto- fluorescence spectra of OSF. J Oral Pathol Med 2003;32:337-43. Tsai T, Chen HM, Wang CY, Tsai JC, Chen CT, Chiang CP. In vivo autofluorescence spectroscopy of oral premalignant and malignant lesions. Distortion of fluorescence intensity by submucous fibrosis. Lasers Surg Med 2003;33:40-7. Wang CY, Tsai T, Chen HM, Chen CT, Chiang CP. PLS-ANN based classification model for OSF and oral carcinogenesis. Lasers Surg Med 2003;32:318-26.

SMF

Uploaded by

Copyright:

Available Formats

You might also like

SMF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

SMF

Uploaded by

Copyright:

Available Formats

Introduction

Dosing Interactions Contraindications Precautions

4 mg IV/IM (suggested in studies)

Triamcinolone acetonide (Aristocort, Kenaject)

Dosing Interactions Contraindications Precautions

Betamethasone valerate (Diprosone)

Dosing Interactions Contraindications Precautions

Suggested dose: 0.05% topically q6h for 3 wk

Hyaluronidase (Wydase Injection)

Dosing Interactions Contraindications Precautions

150 U added to vehicle solution and administered SC/ID

Interferon gamma (Actimmune)

Dosing Interactions Contraindications Precautions

Dosing Interactions Contraindications Precautions

16 mg/d PO (suggested in study)

Dosing Interactions Contraindications Precautions

400 mg tid as coated, sustained-release tab

No known indicated pediatric dosage in relation to oral submucous fibrosis

Ensure nutritional intake is not compromised postoperatively.

Further Outpatient Care

Discovery of Novel Biomarkers in Oral Submucous Fibrosis by Microarray Analysis

Oral submucous fibrosis

Novel biomarker Microarray analysis

Previous SectionNext Section

Materials and Methods

Isolation of RNA from Tissue Samples

Microarray Experiments and Data Mining

5-ACGTGGTCTTGGACACAAC-3, 121 bp;

5-TCATAGTCCTCTGGGATGGT-3, reverse forward 55bp;

AGGGTCTTGAGATTGAGCTG-3, reverse 5-CTCACTATCAGCTCGCACAT-3, product sizes 161 bp;

In this window In a new window

In this window In a new window

View larger version:

In this page In a new window

Download as PowerPoint Slide

mRNA Expression of the Five Genes by RT-PCR

View larger version:

In this page In a new window

Download as PowerPoint Slide

Evaluation of Each Protein Expression by Western Blotting

View larger version:

In this page In a new window

Download as PowerPoint Slide

Expression of CXCL9 Protein

Expression of KRT19 Protein

Expression of CYP 3A5 Protein

Correlation between the Expression and Clinical Pathologic Variables in OSF

In this window In a new window

Disclosure of Potential Conflicts of Interest

squamous cell carcinoma. J Oral Pathol Med 2007;36:437.

Rollins BJ. Chemokines. Blood 1997;90:90928.

Date of Submission Date of Decision Date of Acceptance Date of Web Publication

17-Jun-2008 02-Feb-2009 03-Mar2009 30-Oct-2009

Materials and Methods

You might also like