Clinical trial NCT04137627
Conditions: Oral Squamous Cell Carcinoma Neoadjuvant Chemotherapy
Keywords: Melatonin Locally Advanced Oral Squamous Cell Carcinoma Neoadjuvant Chemotherapy
Sponsor: Indonesia University
Start date: 2017-07-04; Completion date: 2018-12-18
Enrollment: 50 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Backgrounds

Squamous cell carcinoma of the oral cancer (OSCC) is the sixth most common malignancy. Surgery is the mainstay of treatment for oral cancers. In locally advanced and unresectable oral cancer, surgery presents challenges primarily because the head and neck region have many critical structures that can be damaged by tumor or treatment. Damage to the critical structures can result in significant structural, cosmetic and functional deficits that negatively impact quality of life.

Use of NC was found to achieve resectability in 39% of locally advanced unresectable oral cancers. Patil et al. reported response rate with the three drugs regimen (TPF) for NC was 32% and 27,37% for two drugs regimen (TP). The overall response rate in the TPF group was significantly higher than that in the PF group, both in the induction-chemotherapy phase and after locoregional therapy (33,3% vs 19,9%, p = 0,004). Chemoresistancy has become the challenge in OSCC treatment affecting tumor response to chemotherapy.

Hypoxic microenvironment found in OSCC is marked by the high expression of HIF-1α. CD44 and CD133 as a cancer stem cells marker in head and neck (HNSCC) and miR-210 known as hypoxamiR has been reported to contribute chemoresistancy. As hypoxia inarguably one of the main causes of chemoresistancy, it is agreeable to use melatonin as an antioxidant to reduce the hypoxic condition in tumor microenvironment. Melatonin, a potent endogenous antioxidant agent is proven to have an oncostatic effect, was given in expect to reduce the tumor hypoxic condition so that it would increase the tumor response on NC. Majority of the clinical study use oral melatonin given once daily in 20 mg dose as the minimal dose to yield anti-tumor effects.

The purpose of this study is to prove the effectiveness of melatonin to increase clinical response in locally advanced OSCC patients when treated with NC. The effect of melatonin in reducing tumor hypoxia will be seen through its effect in decreasing the gene expressions of HIF-1α, miR-210, CD44, and CD133.

Methods

Study Design

This study is a double blind, randomized clinical trial using placebo as comparison running from June 2017 to July 2018 . Locally advanced OSSC (stage IVA and IVB) patients that will receive NC were included in the study. Fifty patients treated at two centres (RSCM and RSKD) were randomly allocated into two arms. Twenty-five patients received melatonin combined with three regiment NC (Taxane, Cisplatin, and 5-FU) and the other received placebo with NC. However only 25 out of 50 patients had completed the study protocol (13 patients in melatonin arm and 12 in placebo arm)

Evaluation of Clinical Response

The clinical response were assessed by evaluating pre-treatment and post treatment MRI with the aid of RECIST 1.1. First, it is necessary to estimate the overall tumor burden at baseline (target and non-target lesion) and use this as a comparator for subsequent measurement. The tumor response then being determined according to the definition criteria according to RECIST 1.1, as follows: Complete response (CR) is the disappearance of all target lesions. Partial response (PR) means there is at least 30% decrement in the sum of diameters of target lesions, taking as reference the baseline sum diameters. Progressive disease (PD) means there is at least a 20% increment in the sum of diameters of target lesions or an absolute increment of at least 5 mm. Stable disease (SD) is when there is neither a sufficient shrinkage nor sufficient increment of target lesion. Patients who categorized as PR and CR undergone surgery while those with SD and PD undergone core biopsy.

Genes expression examination

The primer for HIF-1α miR210, CD44, and CD133 genes amplification was design using a Primer Quest Tool IDT software. The total sequence of each gene attained from GenBank data source: National Centre for Biotechnology Information (NCBI). The steps of gene expression examination are RNA isolation, cDNA synthesis, and absolute quantification qPCR. qPCR result was analyzed based on the gene expression concentration compare to the pre-determined standard curve (positive control) of each genes.

Statistical analysis

The data was analysed with statistics software SPSS 20. Saphiro Wilk was used to test data normal distribution. Data with normal distribution and with p \> 0,05 presented in mean +- standard deviation (SD). Data with abnormal data distribution presented in median (minimal and maximal value). The statistical difference of gene concentration level (numerical data) between melatonin and placebo was analysed using normality test of Saphiro Wilk. Data with normal distribution was tested using unpaired-T test, while data with abnormal distribution was tested using Mann Whitney. Statistically significant different stated as p \< 0,05.

Detailed description:

Arms: arm 1: The group received standard treatment with the oral administration of Melatonin arm 2: The group received standard treatment with the oral administration of Placebo
Interventions: intervention 1: Melatonin 20 MG Oral Capsule intervention 2: Placebo oral capsule
Intervention descriptions: intervention 1: The administration of Melatonin 20 mg in addition to neoadjuvant chemotherapy to observe the antioxidant and onco-static effect. intervention 2: The administration of placebo capsule in addition to neoadjuvant chemotherapy

Protocol documents:
Prot_000.pdf: 
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Study Protocol 
 
Official Title 
 
: Melatonin Effect in Combination with Neoadjuvant Chemotherapy to 
Clinical Response in Locally Advanced Oral Squamous Cell Carcinoma 
Unique ID 
 
: MLTOSCC 
NCT Number  
: NCT04137627 
Document Date 
: 28/10/2019 
 
1. Objectives 
a. General Objectives 
• To determine the role of melatonin on locally advanced oral squamous cell 
carcinoma to neoadjuvant chemotherapy through its oncostatic and antioxidant 
properties 
b. Specific Objectives 
• To analyze effect of melatonin administration on locally advanced oral squamous cell 
carcinoma after neoadjuvant chemotherapy to clinical response compared to 
placebo 
• To analyze effect of melatonin administration on locally advanced oral squamous cell 
carcinoma after neoadjuvant chemotherapy to HIF-1α expression compared to 
placebo 
• To analyze effect of melatonin administration on locally advanced oral squamous cell 
carcinoma after neoadjuvant chemotherapy to miR-210 expression compared to 
placebo 
• To analyze effect of melatonin administration on locally advanced oral squamous cell 
carcinoma after neoadjuvant chemotherapy to CD44 expression compared to 
placebo 
• To analyze effect of melatonin administration on locally advanced oral squamous cell 
carcinoma after neoadjuvant chemotherapy to CD133 expression compared to 
placebo 
 
 


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2. Design 
This study used a parallel randomized controlled trial design with placebo as comparison. 
This study is conducted in patients with locally advanced oral squamous cell carcinoma that 
are receiving neoadjuvant chemotherapy and 20 mg per day of melatonin or placebo. 
 
3. Methods 
a. Location and Period of Study 
This study is conducted in surgical oncology clinic of dr. Cipto Mangunkusumo National 
Central General Hospital (RSCM), Dharmais Cancer Hospital (RSKD). Histopathology 
examination is conducted in Anatomical Pathology Department of FKUI-RSCM. DNA/RNA 
examination is performed in Integrated Laboratory of FKUI-Jakarta. Period of study is from 
June 2017 to July 2018 
b. Population and Sample 
Target population are patients with locally advanced oral squamous cell carcinoma (OSCC) 
(stage IVA and IVB) and accessible population are patients with locally advanced OSCC on 
surgical oncology clinic of RSCM and RSKD that are planned to receive neoadjuvant 
chemotherapy (NC).  
Study sample are patients with locally advanced oral squamous cell carcinoma (OSCC) that 
are planned to receive NC in surgical oncology clinic of RSCM, RSKD, Persahabatan General 
Hospital (RSP), and Fatmawati General Hospital (RSF) that fulfilled inclusion and exclusion 
criteria. 
c. Study Criteria 
c.1. Inclusion Criteria 
• Stage IVA and IVB locally advanced OSCC proven by histopathology examination 
• Subject planned to receive NC 
• Subject had never undergone any definitive surgery or never received NC 
• Subject consent to participate by signing informed consent 
• Subject with Karnofsky score ≥ 50 
c.2. Exclusion Criteria 
• Subject not eligible for chemotherapy 
 


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c.3. Dropout Criteria 
• Subject loss to follow up or passed away 
• Subject refused to continue therapy 
d. Sample Size 
Formula for two unpaired mean hypothesis test with numeric variable 
n1 = n2 = 2 {S (Zα + Zβ ) / (X1 – X2) }2 
- 
Type I error = 5%, unidirectional hypothesis, therefore Zα = 1,645 
- 
Power of study = 80% and type II error = 20%, therefore Zβ = 0,84 
S value is calculated by standard deviation of combination of two group means.  
Calculated by formula for standard deviation for two group means: 
σ = √∑(x1 - µ)2/n 
σ= √ (n1 - 1) S1
2 + (n2 - 1) S 2/(n1 - 1) + (n2 - 1) 
Difference in mRNA expression mean to investigator clinical judgement 25% 
Combined standard deviation: 
σ= 20.8 
Inserted into formula: 
n= 2 {20,8 (1,645 + 0.84)/(64 - 48)}2 
n1 = n2 = 21 
n = n1 + n2 + 10% = 46 
Minimal sample size needed for this study is 42 subjects, added by 10% chance of dropout, 
therefore sample size becomes 46 subjects. 
e. Study Variable 
Independent variable is oral melatonin administration while dependent variable is clinical 
response, HIF-1α expression, miR-210 expression, CD44 and CD133 gene expression. 
f. Sampling Method 
Sampling method in this study is by consecutive sampling 
g. Sample Allocation 
Allocation for study subjects are done by block randomization. Concealment is done by 
giving serial number on drug preparations. 
 
 
 


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h. Protocol 
h.1 OSCC  
On all subjects will be performed anamnesis, physical examination, and supporting 
examination, which is complete blood examination, MRI of oral cavity, chest x-ray, liver 
ultrasonography, and biopsy 
h.2 Supporting Examination 
h.2.1 Biopsy 
Biopsy will be performed by a surgeon in outpatient clinic by open biopsy (incisional biopsy) 
or core biopsy with local or general anesthesia 
h.2.2 Blood Examination 
Blood sample is taken from peripheral blood vessel before receiving treatment, and 
receiving NC 
h.2.3 Radiological Examination 
On all subjects will be performed MRI of oral cavity to evaluate organ that is involved and 
extension of tumor cells. Chest x-ray and liver ultrasonography is performed to evaluate 
distant metastasis 
h.3 Patient selection 
Potential subject identification uses list from locally advanced OSCC patients that are 
planned to receive chemotherapy, done by surgeon on duty in outpatient clinic. The 
investigator will then explain and hand out informed consent regarding purpose and flow of 
the study. After understanding and agreeing to participate, study subjects is asked to sign 
the informed consent. Subjects who wishes to participate will receive a copy of informed 
consent, while subjects who doesn’t participate will undergo procedure according to 
hospital standard. 
h.4 Randomization 
If the subjects fulfilled inclusion and exclusion criteria, study is continued by randomization 
procedure done by a third party so the investigator or researcher doesn’t know the drug 
that were given. Every OSCC patients received chemotherapy and melatonin or placebo 
with randomization pattern that was computerized, split into two arms which is 
intervention arm and control arm. Intervention arm will receive capsule containing 20 mg 
melatonin, while control arm will receive capsule containing placebo. Capsule containing 
melatonin or placebo is distributed to OSCC patients seven days prior to chemotherapy, 


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initiated until NC administration is completed (three cycles). If the subjects didn’t fulfill 
inclusion criteria, subjects will be excluded from the study, however will still receive therapy 
according to hospital protocol. 
h.5 Administration of Melatonin and Placebo 
1. Intervention arm will receive NC and 20 mg melatonin per day. Melatonin is taken seven 
days prior to chemotherapy. Then melatonin is taken along with NC until three cycles. 
Melatonin is taken at night 
2. Control arm will receive NC and placebo. Placebo is taken seven days prior to 
chemotherapy. Then plcebo is taken along with NC until three cycles. Placebo is taken at 
night 
Subjects will receive melatonin on intervention arm and placebo on control arm as much as 
74 capsule (a week before NC is initiated and for 1 initial cycle) and then 1 week after first 
NC patient will be given 50 capsule for second cycle, and then the same goes for third cycle. 
Patient will be assessed of their adherence by investigator phone call to the subjects twice a 
week. 
h.6 Neoadjuvant Chemotherapy 
Neoadjuvant chemotherapy that is given are regimen of docetaxel 75 mg/m2 on first day; 
cisplatin 80-100 mg/m2 or carboplatin on first day, with consideration if the patient has the 
Karnofsky score of 50-70 will be given cisplatin 80 mg/m2, however if the score is above 70 
will be given cisplatin 100 mg/m2; and 5-FU 1000mg/m2 since day 1-5. Docetaxel and 
cisplatin will be administered simultaneously on day one, while 5FU will be given in day 1-5. 
Chemotherapy is given every three weeks. Two weeks prior to chemotherapy, the patient is 
asked for outpatient visit to be examined for blood sample for chemotherapy preparation. If 
laboratory results didn’t fulfill requirements for chemotherapy, the patient will be observed 
for improvement of condition. Melatonin and placebo will still be administered when the 
patient is in observation for improvement. 
h.7 Evaluation of Neoadjuvant Chemotherapy 
Two weeks after third NC, physical examination and supporting examination 
(Oropharyngeal MRI, Chest X-ray, and Liver USG) is performed. Tumor size will be measured 
after NC administration. Patients with PR and CR will undergo surgery. Surgery will be 
performed one week after evaluation of response continued by tissue and blood 


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examination for qRT-PCR. Patients with SD and PD will be performed secondary biopsy and 
blood exam for qRT-PCR.  
h.8 Gene Expression Examination 
h.8.1 Primer Design and gBlocks Gene Fragment Primer Design Synthesis 
Primer design for CD44, CD133, miR-210, and HIF-1α gene amplification is performed using 
Primer Quest Tool IDT software. Sequence information of each gene is obtained from 
National Centre for Biotechnology Information (NCBI) database. 
Creation of gBlocks gene fragments 
Measurement of mRNA expression of the 4 gene that is analyzed is performed using qRT-
PCR absolute quantification. Therefore, a standard curve for template amplification (gene 
fragment) result is needed with various known concentration. Gene fragments that is used 
is obtained by designing gBlocks gene fragment with various concentration, starting from 
100 ng/mL that contain HIF-1α, CD44, CD133, miR-210 gene fragments (each sized <150 
bp). Sequence selection for each gene and gBlocks synthesis is done by gBlocks Gene 
Fragments IDT (integrated DNA technologies).  
h.8.2 RNA Isolation 
Sample used to analyze mRNA expression of CD44, CD133, miR-210, and HIF-1α gene is 
obtained from incisional biopsy or core biopsy and blood examination. Tissue sample is stored 
in 1.5 ml microcentrifuge tube containing RNAlater RNA Stabilization Reagent in -800C 
temperature until RNA isolation is performed. Total RNA isolation uses QIAamp RNA Blood 
Mini Kit (Qiagen). Protocol of RNA isolation is according to insert manual kit. Purity and 
concentration of RNA as result of isolation is measured using NanoDrop 2000 (Thermo 
Scientific). Result of the measurement is used to determine RNA template volume according 
to calculation of final concentration that will be used in cDNA synthesis process. 
h.8.3 cDNA Synthesis 
cDNA synthesis is performed using QuantiTect® SYBR® green PCR kit (Qiagen). Protocol 
of cDNA synthesis is done according to manual insert kit. Before cDNA synthesis is 
performed, normalization is done by measuring final concentration of each sample. Final 
concentration of each blood sample is 1 ng/20 µL while final concentration of tissue sample is 
100 ng/20 µL. 
 
 
 


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h.8.4 Two-Step Quantitative PCR (qPCR) 
Gene amplification is done using two-step qRT-PCR absolute quantification. Standard curve 
creation and mRNA expression quantification of each sample gene is done using QuantiTect® 
SYBR® green PCR kit (Qiagen) with details as described below. All reagents in the kit is 
deposited in ice box, and then waited until perfectly thawed. Reagent mix is prepared by 
adding QuantiTect SYBR Green PCR Master Mix of 12.5 µL, forward primer (10 µM), 0,75 
µL, primer reverse (10 µM) 0,75 µL, and RNase-free water 9 µL until volume reaches 23 µL 
and then homogenized by up-down pipetting and then continued by adding cDNA of 2 µL, 
and then the mixture is homogenized. 
qPCR Profile Phases 
1. PCR initial activation step, in 950 C temperature for 15 minutes, once. 
2. PCR cycle of 40 times consisting of 15 seconds denaturation at 940 C, continued by 
annealing at 57-630 C for 30 seconds, and extension at 720C  
3. Melt curve, melted in 72-950 C, raised by 0.50 C in each top phases. Wait for 5 seconds for 
each top phases. 
 

SAP_001.pdf: 
Statistical Analysis Plan (SAP) 
 
Official Title 
 
: Melatonin Effect in Combination with Neoadjuvant Chemotherapy to 
Clinical Response in Locally Advanced Oral Squamous Cell Carcinoma 
Unique ID 
 
: MLTOSCC 
NCT Number  
: NCT04137627 
Document Date 
: 28/10/2019 
 
1. Statistical Analysis 
 
mRNA expression concentration of each sample from each gene is converted based on 
standard curve result. Data of study result includes HIF-1α concentration, miR-210 
expression, CD44 and CD133 mRNA expression before and after therapy both on 
intervention arm and control arm.  
 
After the study is completed, we performed editing, coding, and inputting. Data is shown in 
tables and figures. Pre and post treatment data of control and intervention group is analyzed 
by statistical analysis tests.  
 
Normality of data distribution is evaluated using Saphiro-Wilk test, for p value of greater than 
0.05, normally distributed data is shown in Mean ± Standard Deviation (SD) while for non-
normally distributed data are shown in Median (Range) 
 
Difference between intervention and control arm (2 groups numeric) is analyzed by Saphiro-
Wilk test for normality. For normally distributed data will be analyzed using independent t-
test. For non-normally distributed data will be analyzed using Mann-Whitney test. Analysis is 
performed using IBM© SPSS® version 20. Difference is considered significant if p value is 
lesser than 0.05.