(i)Rationale/ gaps in existing knowledge,: Oral cancer, one of the top three common cancers in India is usually preceded by Oral potentially malignant disorders in nearly two thirds of the cases like Oral leukoplakia. OL has a high prevalence and malignant transformation rates(0.13% - 40.8%). There are no universal protocols for management in OL ranging from preventive, conservative, medical and surgical interventions. They all have limitations and high recurrence rates requiring regular follow up.

(ii)Novelty: Removal of pathological lesions are believed to reduce the risk of malignant transformation in OL. Surgical methods are associated with morbidity and high recurrence rates. Non -invasive methods (like Photodynamic therapy) that can remove the abnormal lesions would be preferred as they are cheaper, convenient, safe with less morbidity and better patient acceptability. They also have the advantage of repeatability with minimum morbidity and damage to adjacent normal tissues. Non -invasive medical management involves topical application of retinoids which are associated with systemic toxicity, recurrence and development of resistance.

(iii)Objectives,

Primary objective:

To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy and topical calcipotriol (0.005%) on clinical response after completion of treatment at 4 weeks from baseline during management of Oral leukoplakia

Secondary objectives:

To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy with topical Calcipotriol (0.005%) on lesion size, lesion roughness/ whiteness, and oral health quality of life after completion of treatment at 4 weeks and 12 weeks from baseline during management of Oral leukoplakia To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy with topical Calcipotriol (0.005%) on salivary molecular markers ( IL6, TNF-α, PCNA, Survivin and VEGF) after completion of treatment at 4 weeks from baseline To compare the adverse events, time to complete response and recurrence rates between the two groups

(iv)Methods,: An open label randomized clinical trial where Group A (n=83) will receive 4 sessions of Toludine blue mediated photodynamic therapy (Fluence 15J/cm2); GroupB (n=83) will receive twice daily tropical application of Calcipotriol (0.005%) over a period of 4 weeks. The clinical response, lesion thickness/ whiteness, lesion size /area, OHIP-14 scores, adverse events and recurrence rates will be compared with baseline at 4 weeks and 12 weeks after completion of therapy. Sterile PVA ophthalmic sponges will be used to evaluate molecular markers ( IL6, TNF-α, PCNA, Survivin VEGF) from treatment site of lesion at baseline and 4 weeks after completion of treatment by ELISA. The data will be analyzed statistically using Intention to treat and as per protocol analysis at significance levels of p<0.05.

(v)Expected outcome.: The study will validate and verify the effectiveness of the TB mediated PDT protocol in the management of OL as compared to other non invasive method of topical Calcipotriol in terms of clinical response ( complete/ partial/ none), lesion thickness/ whiteness, lesion size and it's effect on oral health quality of life. Adverse events if any and recurrence rates or malignant transformation rates will also help in assessment of safety of the protocol. The molecular markers will help to monitor and determine prognosis of the lessons following treatment.

Objectives

Primary Objectives:

3.1 To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy and topical calcipotriol (0.005%) on clinical response after completion of treatment at 4 weeks from baseline during management of Oral leukoplakia

Secondary Objectives:

3.2 To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy with topical Calcipotriol (0.005%) on lesion size, lesion roughness/ whiteness, and oral health quality of life after completion of treatment at 4 weeks and 12 weeks from baseline during management of Oral leukoplakia 3.3 To compare the effect of 2.5% Toluidine blue mediated topical photodynamic therapy with topical Calcipotriol (0.005%) on salivary molecular markers ( IL6, TNF-α, PCNA, Survivin and VEGF) after completion of treatment at 4 weeks from baseline 3.4 To compare the adverse events, time to complete response and recurrence rates between the two groups

Rationale:

Nearly two thirds of Oral Cancers are preceded by oral potentially malignant disorders like OL, caused by tobacco, alcohol and areca nut habits. OL has a high prevalence and malignant transformation rate ( 0.13%- 40.8%).There are no universal management protocols for OL. Preventive, conservative, medical and surgical interventions have several limitations and high recurrence rates. Non homogenous, dysplastic, large, mutifocal, proliferative recurrent and recalcitrant OL in subjects with co morbidities pose even more serious challenges in management for the oral physician who often resort to conservative measures with watchful observation. Patients with OL have to deal with unaesthetic appearance of lesion and fear of malignancy which affects their oral and psychological health.

Removal of the pathological OL lesions is believed to reduce the risk of malignancy. There are surgical, laser ablation, electrocautery, cryotherapy options available but they are associated with post operative complications like pain, swelling, infections, neuropathy, scarring and adversely affect oral function with high recurrence rates. These methods only address the physical abnormal area where instituted and do not bring about any beneficial change in the biology of the affected area. There is a need for non invasive methods of removing OL without the current limitations and which also bring about favourable molecular changes in the underlying and adjacent tissues to mitigate the risk of malignant transformation.

Photodynamic therapy (PDT) is a non invasive method of removing abnormal tissue using a Photosensitizer (PS) and approprite light activation which selectively targets abnormal cells and causes cytotoxicity and death. PDT also causes apoptosis, vascular disruption and has immuno modulatory effects to decrease the risk of malignancy. The most widely used and studied PS is 5 -aminolevulinic acid (5-ALA). 5 ALA has several limitations as it is very expensive, photosensitive, causes swelling, pain , burning sensation, allergic reaction on adjacent tissue , requires a dark room for application and long incubation hours ( 2 hours). Toludine Blue (TB) is a metachromatic dye which has been used as a vital stain in oral cancer screening as it preferentially stains mitotic and dysplastic cells. It can also be used as a PS as it gets activated by red light ( 620- 660nm) and has been used in PDT in a single case series of 15 OL lesions with 40% complete response. It is cheap, safe , less toxic and photosensitive, targets abnormal cells, simple application, requires less incubation time, no dark room required and no reported side effects. PDT has usually been tried with 5ALA as photosensitizer (Ps) and Laser light as activator. PDT with LED light source as activator is cheaper and more feasible in resource limited settings.

Topical Retinoids have been used as a non invasive method to remove lesions of OL due to their antikeratinocyte activity but have high recurrence rates, risk of systemic toxicity and development of resistance. Topical calcipotriol is a synthetic Vit D analogue which has been used in Psoriasis for its antiproliferative anti inflammatory anti angiogenic and immunomodulatory effects. There are only two studies that have used Calcipotriol in OL with complete response from 25%-80% with no side effects.

There is no RCT that have compared the effects of TB mediated PDT and topical calcipotriol in OL at present.

The levels of Pro- oncogenic cytokines ( IL6, TNF-α, PCNA, Survivin and VEGF) that promote inflammation, proliferation and angiogenesis are important prognostic biomarkers that can help to evaluate the benefits of any intervention in OL. There are no studies that have evaluated these cytokines after topical PDT or Calcipotriol therapy in OL.

Number of Patients:

Sample Size:In a systematic review and meta-analysis on PDT for Oral Leukoplakia by Zhang et al(2023) overall complete response was seen in 50% of patients.

While only 2 studies, mention results of topical Calcipotriol(0.005%) in oral leukoplakia with sample size of 20 in both study groups. In one RCT (Femanio et al 2001) which found a CR of 80% in OL after topical calcipotriol at 5 weeks. In another study(Galwash et al 2017) in which total response was 25% at 4weeks.

Due to lack and variability of data in available studies on response of topical calcipotriol(0.005%) in Oral Leukoplakia We can anticipate CR in 25% of patients in our study. Estimated sample size for two-sample comparison of proportions Test H0:P1=P2, where p1 is the proportion in population 1 and p2 is the proportion in population 2.

Assumption:

alpha=0.0500(two-sided) power=0.8000 p1=0.2500 p2=0.5000 n2/n1=1.00 Estimated required sample sizes: n1=66, n2=66 Final sample size was 83/ group considering 20% of patients as lost to follow up.

Total sample size: 166

Group A (n=83):Non-invasive PDT using LED of wavelength ranging from 630-660nm( Fluence 15 J/cm2) as light source and 2.5% TB as photosensitizer will be used in oral leukoplakia once a week for 4 weeks along with preventive and conventional therapy.

Group B (n=83):Topical application of Calcipotriol ointment 0.005% twice a day for 4 weeks along with preventive and conventional therapy

5.5 Study design An open label Randomized clinical trial

Block Randomization:

Block randomization with varying block size will be done using computer generated random numbers. Allocation ratio (1:1)

Allocation concealment:

Participants will be randomized using sequentially numbered, opaque sealed envelopes (SNOSE): the randomization group will be written on a paper and is kept in an opaque sealed envelope prepared with aluminium foil sheet and carbon sheet in each. The envelope will be labelled with a serial number and randomization code and will have an identifier of trial on its front. The intervention will be written on a paper inside the envelope and sealed by technician. Envelopes will be opened sequentially by the operator, and patients will be allotted a study arm as per the intervention mentioned inside the envelope. Used envelopes will be stored separately until the completion of trial.

Intervention

Conventional non-invasive management (Usual care):

All subjects in the two groups will receive the same standard conventional non- invasive management advised for OL as per current scientific evidence.

• Brief behavioral Tobacco, areca nut and alcohol habit cessation counseling as per WHO 5As and 5Rs technique at baseline and each follow up.
• Oral prophylaxis
• Removal of oral irritational factors like sharp teeth, appliances or prosthesis, impacted buccoverted third molars, parafunctional habits
• Oral hygiene maintenance instructions
• Control of systemic conditions (Anemia, Diabetes, hypertension, thyroid disorders) by specialist referral
• Removal of all predisposing factors for oral candida infection
• Advocacy for safe sexual practices
• Nutrition and diet counseling( seasonal and regional food rich in nutrients, vitamins, antioxidants avoidance of spicy /sour/ hot foods and drinks)
• Regular surveillance for malignant transformation Investigations: Orthopantomogram, Complete hemogram, Liver function tests, Renal Function tests, Lipid profile, Parathyroid hormone, Serum Ca2+, Serum Vit D3 level, Serum electrolytes level Site selection of Oral leukoplakia in trial In subjects with multiple areas of involvement, the non homogeneous and larger lesions will be selected for treatment and assessment.

Photodynamic Therapy Protocol:

Protocol for preparation and application of 2.5% TB as Ps:

The protocol foresees the application of 2.5% TB, prepared following standard procedures and adapting the proportions as stated by Epstein et al., on the lesions, using a cotton applicator tips, with a margin of 3mm from the surrounding normal tissue and leaving it to sit for 5min, avoiding any contact with the saliva using cotton rolls, cheek retractor and suction apparatus Protocol for LED Photodynamic therapy in Oral Leukoplakia patients Equipment LED light source Wavelength 630-660nm Peak Power 0.04 W/cm2 Fluence 15 J/cm 2 Spot size 0.66cm2 Distance Intraorally: At oral mucosal surface(Contact mode) Mode Continuous, circular motion, overlapping, in clockwise concentric manner with LED handpiece perpendicular to surface Duration of cycle 360 seconds over 1 cm2 area of diagnosed OL lesion. Cycles per sitting One cycle per sitting No. of sittings Four, One sitting / week ( @ Day 0, 1 week, 2 week, 3 week Area of intervention Intraoral over OL lesion ( non homogenous and larger lesion given preference in subjects with multiple site involvement ) Treatment time 360sec/6 minutes for OL lesion of size 1cm2/12 min for 2cm2 Dosimetry and calibration of LED device Every month using power meter and spectrometer

Protocol for application of topical Calcipotriol(0.005%) ointment in Oral Leukoplakia patients:

All patients will be shown the site of OL lesion and demonstration for application of topical Calcipotriol ointment(0.005%) will be given to them. Patients are advised to apply Cal ointment twice daily for 4 weeks after meals.

Steps in application of topical Calcipotriol(0.005%) ointment:

Dry the lesion site with sterile gauze Apply the agent as a ointment with a cotton bud Leave the ointment for at least 30 minutes Avoid swallowing the drug Avoid drinking/ eating/ spitting for at least 30 minutes after drug application Reiteration of the protocol to subject with physical demonstation at each follow-up visit

Training of operators:

The operator and the assistant, giving and assisting intraoral PDT therapy will be trained in the protocol as per study.

Therapeutic monitoring of PDT:

Site of application of PDT will be evaluated continuously for any discomfort, exacerbation or increase in size of lesion, ulceration, erythema, burning sensation, pain (increase in NRS scores NRS≥8), or develop reaction to Ps during therapy and during follow up.

The following options will be considered during PDT therapy:

Stop/ Defer the treatment. Subjects will be interviewed to know any adverse effects they might be feeling after initiation of treatment with PDT.

Therapeutic monitoring ofCalcipotriol(0.005%)ointment:

Site of application of Calcipotriol will be evaluated continuously for any discomfort, exacerbation or increase in size of lesion, ulceration, erythema, burning sensation, pain (increase in NRS scores-difference of NRS≥5 from previous visit), or develop any reaction to Calcipotriol(0.005%)during application and during follow up.

The following options will be considered during Calcipotriol(0.005%) therapy:

Stop/ Defer its application. Subjects will be interviewed to know any adverse effects they might be feeling after application of Calcipotriol(0.005%).

Protocol Deviation : If subjects have exacerbation or increase in size of lesion, ulceration, erythema, burning sensation, pain (increase in NRS scores-difference of NRS≥5 from previous visit), or develop reaction to Ps /Calcipotriol (0.005%) they will be withdrawn from the study.

When/If subjects develop changes suspicious of malignancy (erosion, ulceration, induration, exophytic growth) during follow up they will undergo incisional biopsy to rule out malignant changes and managed as per institutional protocol for oral malignant lesions by referral to IRCH.

5.6 Dosages of drug All subjects in the three groups will receive the same standard conventional non- invasive management advised for OL as per current scientific evidence. Topical PDT will use Toludine Blue 2.5% as Photosensitizer for PDT and Calcipotriol ointment (0.005%) will be used.

5.7Duration of treatment The PDT and topical Calcipotriol therapy will be given as per protocol mentioned above, Since OL have risk of malignant transformation all the subjects in study group will be kept under regular follow- up as per standard protocol

5.8.Investigation specifically related to projects

Study Procedure:

A structured questionnaire will be used to record past medical history, history of present illness and symptoms etc. followed by clinical / physical examination of the subjects to diagnose Oral Leukoplakia as according to Van der wall classification of OL.

Clinical Evaluation:

• Extraoral examination: For ulcer, abscess, sinus tract, fistula, scars, paranasal sinusitis, palsies, tics, dystonias, facial asymmetry, nasal obstruction
• Facial swelling: site, size, colour, consistency, contour, tenderness
• Cervical lymph node: site, size, consistency, contour, tenderness
• Intraoral Examination:

History of white lesion Location,Onset, Color, Texture, Induration, Progress, Burning sensation, Trismus, Dysphagia, Poor oral hygiene, Duration(months), Pain (NRS 0-10), Burning sensation(NRS 0-10), Bleeding, Swelling, Xerostomia, Sharp teeth, Past treatments for OL History of oral habits: Type, Quantity, Frequency/day, duration in months, its current status and quit period.

All subjects will receive brief behavioral counseling for habit cessation as per national guidelines and departmental protocol Intraoral examination of other tissues: Mucogingival tissues, Teeth, Periodontium, Occlusion

Sample collection for pro-oncogenic salivary cytokines IL6, TNF-α, PCNA, Survivin and VEGF pre and post therapy:

Four Sterile PVA ophthalmic sponges (Merocel) will be used to collect samples of oral secretions for four cytokine study from the oral buccal mucosa This is a non- invasive method and makes it site -specific which is an advantage over other serum and saliva estimations. This procedure will be done for pre and post therapy during follow up. The subjects will be asked to abstain from eating, drinking and rinsing the mouth at least 2 hours prior to sampling. The sponges will be pre-wet with sterile normal saline, kept in contact with the lesion/ buccal mucosa without rubbing or movement for 1 minute and then immediately stored in sterile containers at -80 °C till further analysis by ELISA. A photographic and clinical record of the sample site will be kept with the patient proforma for future reference.

Quantification of pro-oncogenic salivary cytokines ( IL6, TNF-α, PCNA, Survivin and VEGF) levels by ELISA (pg/ng per ml) The ophthalmic sponges (Merocel ) will be thawed at room temperature for 10 min. Sponges will then be inserted into a micro centrifuge tube containing a 0.2 μm filter (SpinX centrifuge tube), equilibrated by adding 300 μl of extraction buffer and incubated for 30 min at 4 °C, followed by centrifugation at 4 °C for 30 min at 14,000 rpm. After centrifugation, the resultant supernatant will be collected for the quantification of( IL6, TNF-α, PCNA, Survivin and VEGF) levels by ELISA method with commercially available kits. The supernatant will be stored at -80 °C until use.

A monoclonal antibody against the antigens ( IL6, TNF-α, PCNA, Survivn and VEGF) has been pre-coated onto the wells of the microtiter strips provided. Antigens present in the sample or standard will be incubated with the plates to allow binding of antigens to the antibody. This is followed by the addition of a primary monoclonal anti-( IL6, TNF-α, PCNA, Survivin and VEGF) antibody respectively conjugated to biotin in respective microtiter plates. An avidin-HRP conjugated antibody specific for primary antibody will then be added to the wells. After incubation and following a wash, to remove any unbound antibody enzyme reagent, a TMB one-step substrate reagent reactive with HRP will be added to the wells. The color development will be terminated by adding acid and absorbance was measured at 450 nm. A reference curve will be obtained by plotting the different concentrations of standard samples versus absorbance and levels of the antigens in samples tested will be calculated by its standard plot.

Adverse events Oral pain: The numeric rating scale (NRS) is a pain screening tool, commonly used to assess pain severity at that moment in time using a 0-10 scale, with zero meaning "no pain" and 10 meaning "the worst pain imaginable" . Patient is asked to circle the number between 0 and 10 that fits best to pain intensity.In many clinical settings, pain is assessed regularly in outpatient care using the NRS; these scores are recorded in the patient's electronic health record (EHR), allowing researchers and clinicians to track pain intensity over time(70).An initial NRS score will be taken on enrolment into the study. Subsequent NRS scores, as well as the patient's perception of the change in their pain from the last measurement (i.e., "a lot less," "little less," "about the same," "little more," or "a lot more") will be recorded at baseline and at every visit of PDT/Calcipotriol protocol and subsequent follow-up at day 7,day 14, day21, day 28 and day120 post treatment. Each NRS score will be made on a new, unmarked scale. Patients will not be allowed to review their previous scores.

Oral burning sensation: The same procedure with NRS scale will be followed for Oral burning sensation assessment Allergic Reaction: All the subjects will be asked to report immediately any local/ systemic allergic reaction arising due to the interventions like redness and swelling due to photosensitizer Toludine Blue , mucosal or skin reactions due to calcipotriol to the investigator at any time during the study. The interventions will be withdrawn and remedial interventions will be started immediately and the adverse event will be recorded and reported as per institutional protocol.

Malignant change: Any change in lesion at treatment site or other areas of the oral cavity at any time of the trial, which is suspicious / obvious for malignant change ( ulceration, induration) will undergo biopsy and withdrawn from study after confirmation Recurrence: Any reappearance of lesion at the site of treatment and assessment vii. Patient diary: Recording of Pain, burning sensation, habit status, size of lesion( increase / decrease/ disappearance/ recurrence) allergy, redness, swelling, and missed medications) All the subjects will be asked to record the frequency, duration and exacerbating factors of pain and burning sensation. Status of habit ,its current use, frequency of consumption, quit period will also be recorded in the chart provided to the patients. Patient will be guided to do self-examination of lesion and any increase or decrease in size will be noted in the chart provided in the diary. Baselines scores will be recorded pre- randomization to prevent bias in assessment by investigator. Scores will be recorded at each session, post treatment at 1 week, 2 weeks, 3 weeks 4 weeks and 12 weeks by same investigator for both the groups.

viii. Withdrawal Criteria: If any patient withdraws consent after treatment is initiated/ there is an allergic reaction/ malignant change/ exacerbation of pain and burning sensation that is not controlled by anesthetics, analgesics, antiseptics, ( NRS ≥ 8) then the subject will be withdrawn from the study.

Statistical Analysis:

Data will be entered in an Excel sheet as per prepared proforma Baseline characteristics will be compared between the groups using chi-square test(categorial variables) and unpaired t-test/Wilcoxon-ranked test(continuous variables). Both intention-to-treat and per-protocol analysis will be carried out for the primary outcome. The primary outcome, rate of complete response, will be compared between the groups using z-test for two-proportions. Fishers Exact test will be used for analysis of adverse events. Complete response and time to recurrence will be compared between the groups using Kaplan- Meier curve followed by log- rank test. Results will be presented as Difference in means/proportions with 95% confidence interval and p<0.05% will be considered statistically significant.

Inclusion Criteria:

• All diagnosed cases of Oral Leukoplakia with size ≥ 2 cm2 (Van der Waal classification ≥ L2) reporting to the department of OMR, CDER and willing to participate in the study will be included
• Subjects clinically and histologically diagnosed as Oral Leukoplakia.
• Subjects > 18 years of age.
• Subjects willing to participate in the study and to complete the treatment sessions.

Exclusion Criteria:

• History of any topical and/or systemic therapy for Oral Leukoplakia other than general management in last 2 months.
• Subjects with current or past history of malignancy.
• Subjects with any lesion suspicious of malignancy requiring biopsy.
• Subjects with severe dysplasia and carcinoma in situ on biopsy of oral lesion
• Subjects with history of hyperphotosensitivity (genetic disorders, dermatological conditions, drug induced, systemic conditions)
• Subjects with calcium, Vit D metabolic and parathyroid disorders
• Pregnant and lactating females.

The study methodology, outcomes/ results will be published in peer reviewed scientific and academic journals.

3 years after publication of results

Any academic/ scientific enquiry about methodology and results of the trial from researchers in the field can be accessed from the information in the publication or on reasonable request via email (shalinigupta@hotmail.com) to principal investigator

The outcome assessors for the molecular biomarkers will be masked to the identity of the groups