RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE – II

PROFORMA FOR THE REGISTRATION

OF

SUBJECT FOR DISSERTATION.

BY

Dr. Shalini . S

1ST YEAR MDS.

DEPARTMENT OF ORAL & MAXILLOFACIAL PATHOLOGY

2012.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. /

NAME OF THE CANDIDATE AND ADDRESS:

/ DR. SHALINI. S
POST GRADUATE STUDENT,
DEPARTMENT OF ORAL AND
MAXILLOFACIAL PATHOLOGY,
KRISHNADEVARAYA COLLEGE OF
DENTAL SCIENCES AND HOSPITAL,
KRISHNADEVARAYANAGAR,
HUNASAMARANAHALLI, VIA
YELAHANKA, BANGALORE 562157
2. /
NAME OF THE INSTITUTION:
/ KRISHNADEVARAYA COLLEGE OF
DENTAL SCIENCES AND HOSPITAL,
KRISHNADEVARAYANAGAR,
HUNASAMARANAHALLI, VIA
YELAHANKA, BANGALORE 562157
3. /

COURSE OF THE STUDY AND SUBJECT:

/ MASTER OF DENTAL SURGERY IN
ORAL AND MAXILLOFACIAL
PATHOLOGY.
4. / DATE OF ADMISSION TO COURSE / 25 MAY 2012
5. / TITLE OF THE TOPIC
“ THE SALIVARY ANALYSIS OF TELOMERASE LEVELS IN ORAL SQUAMOUS CELL CARCINOMA ”
6. /

BRIEF RESUME OF INTENDED WORK

6.1. NEED FOR THE STUDY: -

Oral cancer (OC) is a major health hazard encountered in various parts of our country. Approximately 80,000 new cases are diagnosed annually, the cause of which is mainly attributed to different forms of tobacco consumption.
•  Unlike cancers in inaccessible areas, Oral cancer is located in the oral cavity; hence the direct contact between the saliva and oral cancerous lesion makes the measurement of tumour markers in saliva an attractive alternate to the serum and tissue based biomarkers.
•  Monitoring of saliva is a real alternative compared to plasma because saliva lacks "the drama of blood, the sincerity of sweat and the emotional appeal of tears" (Mandel, 1990).
•  Abraham J.E. et al.states that Advantages of Saliva include lower overall cost, lower infection risk, increased patient convenience, acceptability, compliance and uptake.”
•  Viltrop T. et al states“Saliva collection is a painless procedure with no risk of disease transmission and no requirements for specialized medical personnel. Also, saliva collection allows wider population sampling without consuming much time.”
Various Tumour markers in saliva for Oral Squamous Cell Carcinoma such as Telomerase, p53, p16 and many more are of research interest in the recent years.
•  Telomeres are specialized DNA protein structures at the ends of eukaryotic chromosomes, the DNA of human telomere consist of a conservative array of tandem repeat of Guanine rich DNA sequence (TTAGGG) with a length from a few hundred bp to approximately 30kb with a mean of 15 kb.
•  Telomerase – A ribo nucleoprotein complex that displays reverse transcriptase activity that is capable of extending telomeres via the addition of telomere repeat units.
•  Normal somatic cells (except the germ cells and some hemopoietic stem cells) don’t express the telomerase activity but almost 85% of the tumour tissues express this activity.
•  Apart from its role in cellular senescence, telomerase reactivation might be a prerequisite for the development of malignant tumour cells from somatic cells.
•  The tumour progression is considered to be characterized with the telomerase reactivation and the telomerase activity can be one of the didynamic criteria on cell malignant change.
Alterations in saliva Telomerase levels have been reported to occur in oral cancerous lesions when compared with normal subjects (2).
In accordance with these findings we aim to investigate the potential clinical usefulness of Telomerase levels expressed in saliva in oral squamous cell carcinoma patients.
6.2. REVIEW OF LITERATURE:-
Oral cancer has emerged as an alarming public health problem with increasing incidence and mortality rates all over the world.
Therefore, the implementation of newer screening and early detection approaches are of utmost importance which could reduce the morbidity and mortality associated with this disease. Sensitive and specific biomarkers for oral cancer are likely to be most effective for screening, diagnosis, staging and follow-up for this dreaded malignancy.
The direct contact between saliva and oral cancer lesion makes the measurement of tumor markers in saliva an attractive alternative to serum and tissue testing. The DNA, RNA and protein molecules can be conveniently obtained from saliva as they are in contact with the living cancer cells.
Thus, salivary biomarkers, a non-invasive alternative to serum and tissue based biomarkers may be an effective modality for early diagnosis, prognostication and monitoring post therapy
status. In the current post-genomic era, various technologies provide opportunities for high-thoroughput approaches to genomics and proteomics; which have been used to evaluate altered expressions of gene and protein targets in saliva of oral cancer patients. The emerging field of
Salivary biomarkers has great potential to prove its clinical significance to combat oral cancer. (9)
Protein compositions, or the proteomes, found in human salivary and plasma fluids are compared from recent experimental work by many laboratories, a catalogue of 2290 proteins found in whole saliva has been compiled. This list of salivary proteins is comparable with the 2698 proteins found in plasma. Nearly 40% of the proteins that have been suggested to be candidate markers for diseases such as cancer, cardiovascular disease, and stroke can be found in whole saliva. These comparisons and correlations should encourage researchers to consider the use of saliva to discover new protein markers of disease and as a diagnostic non-proximal fluid to detect early signs of disease throughout the body.(10)
A study on the telomerase activity in premalignant and malignant lesions of human oral mucosa in 1997 by Kannan et al has demonstrated a strong association between carcinogenesis and reactivation of telomerase in various human tumors. In addition, the level of telomerase activity have an inverse correlation with the treatment response in the early stage tumors.(8)
A study by Zhong LP, Chen GF, Xu ZF, Zhang X, Ping FY, Zhao SF et al (2005), showed that the telomerase activity assayed by the telomerase PCR-ELISA method, was detected positively in 75.0% of patients with oral squamous cell carcinoma. But it was positive in only 6.67% of normal persons. These results suggest that the detection of telomerase activity in saliva could be used as an adjuvant marker for oral squamous cell carcinoma. (2)
6.3. AIMS AND OBJECTIVES OF THE STUDY:-
1)  To study the expression and variations of the levels of Telomerase in SALIVA, in control (normal) subjects and Oral squamous cell carcinoma patients.
2)  To investigate the clinical usefulness of Telomerase in SALIVA, in diagnosing Oral squamous cell carcinoma.
7. /
MATERIALS & METHODS
7.1. SOURCE OF DATA:
The study shall mainly consist of subjects (Total Number= 60) divided into 2 groups, namely the control(healthy) subjects and OSCC cases as follows:-
1)  Controls or Healthy individuals, n=30
·  Age: 20-60 yrs, with no history of any major illness in the recent past like viral infections, allergies, hypertension, diabetes, kidney problems, hypercholesterolemia etc. and no periodontal diseases in the oral cavity.
2)  Oral squamous cell carcinoma, n= 30
·  Patients who have been histopathologically diagnosed as Oral squamous cell Carcinoma proven but untreated.
-  A detailed case history is taken for all the patients suffering from Oral Squamous cell Carcinoma and Clinically, TNM staging of malignant disease is determined.
According to TNM classification of malignant tumors, International Union against Cancer, Springer, Berlin, 1992.
-  Subjects are briefed about the procedure and a Written Consent is obtained from all the individuals who are participating in the study.
7.2  METHOD OF COLLECTION OF DATA:
INCLUSION CRITERIA
Control:
Saliva of normal healthy individuals of 20-60 years age group, with no localized illness or history of systemic illness , renal problems, hypertension, TB, cystic fibrosis, diabetes, viral diseases like HIV etc and absence of acute or chronic infections or ulcers in the oral cavity or any allergies or periodontal problems .
Oral Squamous cell Carcinoma :
Saliva from individuals of age group 20-60 yrs clinically and histopathologically diagnosed as Oral squamous cell carcinoma and who is not undergoing any treatment .
EXCLUSION CRITERIA
Controls:
·  Individuals < 20 years or > 60years of age.
·  People with known history of with localized illness or systemic illness and absence of acute or chronic infections or ulcers in the oral cavity or any allergies, renal problems, hypertension, diabetes , renal problems, cystic fibrosis, TB, viral diseases like HIV, etc or periodontal problems
Oral Squamous cell Carcinoma:
·  Individuals < 20 years or > 60years of age
·  Oral Squamous cell Carcinoma patients who are undergoing treatment and recurrent cases of OSCC.
·  OSCC patients with known history of localized illness or systemic illness and absence of acute or chronic infections or ulcers in the oral cavity or any allergies, renal problems, hypertension, diabetes , renal problems, cystic fibrosis, TB, viral diseases like HIV, etc or periodontal problems.
SAMPLE COLLECTION:
A Written consent will be taken from the all the people who are participating in this study
-  Patient was seated in a dental chair in upright position.
-  3ml of resting saliva was collected into a sterile test tube.
-  The 3ml saliva is diluted into 15 ml fluid with sterile saline at once.
-  The 15ml fluid was centrifuged at 3300*g for 15 min at 40C, the supernatant was discarded.
-  Centrifuged cells were resuspended with sterile saline in 1.5ml Eppendorf tube.
-  Then, centrifuged at 1200*g for 10 min at 40C .
-  Supernatant was discarded, leaving cells in Eppendorf tube.
-  Stored at -200 C.
Reagents and Techniques used :
-  The collected sample was subjected to testing on addition of Lysis Reagent.
-  The protein content of each supernatant was measured by Bradford assay.
The samples were then subjected to ELISA PCR Assay using Telomerase ELISA PCR Assay Kit.
Assay procedure:
PCR: PCR (polymerase chain reaction) is a method to analyze a short sequence ofDNA(orRNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. PCR is highly efficient so that untold numbers of copies can be made of the DNA within few hours. Moreover, PCR uses the same molecules that nature uses for copying DNA.

ELISA : The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture.

STATISTICAL ANALYSIS
-Statistical analysis is done using SPSS statistical software ( Version 10 )
7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR OTHER ANIMALS?
-  YES. Detailed history and oral examination of the individuals, collection of saliva for salivary analysis needs to be done.
7.4. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
- YES. A copy of the same has been enclosed.
EXPECTED RESULTS:-
Salivary levels of Telomerase activity may be increased in patients with oral squamous cell carcinoma when compared with controls (normal subjects) (2,8 )
8. / LIST OF REFERENCES:-
1)  Brad W. Neville, DDS;Terry A. Day, M. WHO collaborating centre for oral precancerous lesions, definition of leukoplakia and related lesions
an aid to studies on oral precancer. Oral Surg: 1978; 46 : 518
.
2)  Zhong LP, Chen GF, Xu ZF, Zhang X, Ping FY, Zhao SF. Detection of telomerase activity in Saliva from Oral Squamous cell Carcinoma Patients.
Int J Oral Maxillofac Surg: 2005; 34: 566-70.
3)  Kim NW .Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266: 2011-2015
4)  Sumida T, Sogowa K, Hamakawa H, Sugita A, Tanioka H, Ueda N. Detection of Telomerase activity in oral lesions.
J Oral Pathol Med 1998 ; 27:111-115
5) Yao H,Wu B,Wu Q, Zhang X.
Detection and effect of telomerase activity in oral mucosal squamous cell carcinoma.
Chin J Stomatol 2000; 35:269-270
6) Curran AJ, St.Denis K, Irish J,Gulane PJ, Macmillan C, Kamel Reid S.
Telomerase activity in oral squamous cell carcinoma.
Arch Otolaryngol Head neck surg 1998 ;124: 784-788
7) Chang LY,Lin SC, Chang CS, Wong YK, Hu YC, Chang KW.
Telomerase activity and in situ telomerase RNA expression in Oral carcinogenesis.
J Oral Pathol Med 1999; 28: 389-396
8) S Kannan, H Tahara, H Yokozaki, et al. Telomerase activity in premalignant
and malignant lesions of human oral mucosa.
Cancer Epidemiol Biomarkers Prev 1997; 6 :413-420.
9) Franky D. Shah • Rasheedunnisa Begum • Bhairavi N. Vajaria • Kinjal R. Patel •
Jayendra B. Patel • Shilin N. Shukla • Prabhudas S. Patel;
A Review on Salivary Genomics and Proteomics Biomarkers in Oral Cancer
Ind J Clin Biochem Oct-Dec 2011 ; 26(4):326–334
10) J.A. Loo,1,2W. Yan,1P. Ramachandran,1andD.T. Wong3,*
Comparitive human salivary and Plasma Proteomes
J Dent Res.2010 October ;89(10) : 1016–1023.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARKS OF THE GUIDE:
11. / NAME AND DESIGNATION OF
11.1 GUIDE
11.2. SIGNATURE: / DR. RADHIKA. M. B
PROFESSOR AND H.O.D
DEPARTMENT OF ORAL AND
MAXILLOFACIAL PATHOLOGY
KRISHNADEVARAYA COLLEGE OF
DENTAL SCIENCES, BANGALORE
11.3. CO-GUIDE:
11.4. SIGNATURE: / DR. PAREMALA K
READER
DEPARTMENT OF ORAL AND
MAXILLOFACIAL PATHOLOGY
KRISHNADEVARAYA COLLEGE OF
DENTAL SCIENCES, BANGALORE
12.1 REMARKS OF THE
PRINCIPAL:
12.2  SIGNATURE OF THE
PRINCIPAL:

KRISHNADEVARAYA COLLEGE OF DENTAL SCIENCES AND HOSPITAL HUNASAMARANAHALLI, BANGALORE-562157

ETHICAL CLEARANCE FOR DISSERTATION STUDY

Ethical clearance for dissertation study “ The Salivary analysis of Telomerase levels in Oral Squamous cell Carcinoma ” by DR. SHALINI .S, Postgraduate student in the Department of Oral and Maxillofacial Pathology, under the guidance of DR. RADHIKA .M. B , Prof & Head, Department of Oral and Maxillofacial Pathology, Krishnadevaraya College of Dental Sciences and Hospital, under Rajiv Gandhi University of Health Sciences, Karnataka.

Ethical committee meeting was held on 06/11/2012, in the Boardroom of Krishnadevaraya College of Dental Sciences & Hospital.

The Members discussed all the ethical issues involved

1.  Review of literature and remarks of previous studies.

2.  Risks and expected beneficial effects.

3.  Cost factor.

4.  Supervision by Senior Staff members.

5.  ICMR guidelines for Research Studies.

The members were satisfied regarding all the above Ethical issues concerned and ethical clearance was granted for the Dissertation Study.