Title: Genetics of Oral Lichen Planus: Identifying prognosis values and novel therapeutic interventions

 Title: Genetics of Oral Lichen Planus: Identifying prognosis values and novel therapeutic interventions

 

First author: Dr. Dhruv Subramanian

Principal Investigator: Dr. Mitesh Shetty

Co-authors:

Dr. Achuth M Baliga, Dr. K. Srinivas,

Manipal Hospitals, Department of Dental, Medical Genetics and Oncology

Bengaluru, India, and Dr. Dhruv Dental Care (R &D India Dental)

Acknowledgment:

Dr. Somashekar, Dr. Rajashekar (Srivarshini), Dr. James Hartsfield Jr (University of Kentucky), Dr. Paula Bates (University of Louisville), Dr. Carlo Deep (Colgate)

 

BACKGROUND

Oral Lichen Planus (OLP) is an inflammatory condition affecting the mucosal mouth surface and is often co-diagnosed with xerostomia. OLP  affects  0.5% to 2% of the population. Treatments for OLP are usually palliative to control the inflammatory state of the disease, requiring life-long supportive care. When not controlled, OLP can lead to the formation of precancerous lesions which in some cases progress to oral cancer, making early preventative intervention extremely important. Previous studies have identified tctP (a tumor reversion gene or histamine release factor) to be an important biomarker for OLP. The uncontrolled expression of this gene has been observed to be an indicator of a poor prognosis among OLP patients with oral cancer. Understanding the genetic mechanisms associated with the TCTP pathway could lead to identifying potential targets for therapeutic intervention against OLP. These markers could serve as putative targets for targeted therapeutics such as small interfering RNA (siRNA) to knock down and modulate host genes that are associated with disease to control and alleviate OLP symptoms and help in oral cancer prevention.

 

OBJECTIVE:

Identify potential markers unique to OLP by regulating the affected genetic pathways important for the production of TCTP.

 

AIMS:

Specific Aim 1: Perform a differential expression study on healthy and disease tissues collected from the tooth-extracted wound and disease tissue using microarrays to identify potential markers differentially regulated to identify potential siRNA targets for OLP intervention. 

Specific Aim 2: Develop a clinical measure to identify OLP prognosis values based on tctP expression and potential novel gene markers previously identified to be unique among patients undergoing OLP treatment

 

RESEARCH DESIGN AND METHODS

Specific Aim 1: Perform a differential expression study on healthy and disease tissues collected from the tooth-extracted wound and disease tissue using microarrays to identify potential markers differentially regulated to identify potential siRNA targets for OLP intervention. 

 

Hypothesis: Differentially regulated genes in healthy and diseased tissues to identify genes up / down-regulated in diseased tissue as compared to healthy tissue

 

Rationale: Xiao et. al have seen that there is a differential gene expression profile of patients with oral lichen planus. Translationally Controlled Tumor Protein (TCTP) is known to be a tumor reversion protein. Recently, TCTP has been observed to work in prostate, lung, and colon cancer cell lines. An obvious choice would be to examine the role of TCTP in spontaneous regression, or generally for the treatment of OLP

 

Experimental Methods: Sample size 20 diseased and 20 normal tissues with 3-4 fold change. Normal tissues from extraction socket. Agilent microarray

 

Inclusion: OLP is usually bilateral, symmetric or asymmetric, located: in buccal mucosa, tongue, lips, and/or gingiva, showing fine white lines:  a lace-like network (known as Wickham’s striae). 3 OLP white  (reticular, popular, and plaque-like) and 3 OLP red  (erosive (ulcerated), atrophic (erythematous), and bullous. We will be focusing on selecting a reticular form to reduce variability in the study.

 

1.    30 years to 80 years

2.    Both genders

3.    no pre-existing medical comorbidities

4.    Satisfy diagnostic criteria

 

Exclusion:

 

1.    Pregnant and lactating women

2.    Patients with a known history of allergy to drugs

Study duration: 6 months to 1 year

Study Area: Dental and Oncology department with samples sent to Medical Genetics department of Manipal hospital, Old Airport Road, Bengaluru

Study population: Single-center prospective study that includes all OLP patients as per the inclusion & exclusion criteria registered at Manipal hospital..

Sampling technique: Biopsy of sampling OLP patients as per criteria

Clinical protocol:

1.    Identify OLP as per the diagnostic criteria mentioned above

2.    Incisional biopsy with BP blade of the affected area

3.    Specimen sent for investigations in formalin

4.    3-0’ vicryl sutures to suture biopsy site

5.    Post-operative care instructions

Specific Aim 2: Develop a clinical measure to identify OLP prognosis values based on TCTP expression and potential novel gene markers previously identified to be unique among patients undergoing OLP treatment

Hypothesis: TCTP and other genes differentially regulated in healthy and diseased tissues indicative of good or bad prognosis which have been identified. Therefore, enabling us to identify poor prognosis OLP and genes up or downregulated, which might help identify a treatment protocol for such cases.

 

Rationale: the amount of TCTP in the cancer cell lines seems to vary according to microenvironmental conditions as observed in hypoxic-acclimatized hepatocarcinoma cells, which showed a 2.4-fold increased expression compared to normoxic cells  

 

Experimental Methods: Comparative study. Follow up with patients after 4 months to see, whether healed with conventional treatment or not. Therefore, indicative of good or bad prognosis based on the 3-4 fold change of the gene expression to see if, the data correlates

RESULT AND ALTERNATE APPROACHES

Commercial purpose: Silence with a local rinse that has anti-sense RNA to TCTP transcript.

Basic Work Principle

The new Agilent’s SurePrint G3 Human gene expression v3 microarray features long non-coding RNA

(lncRNA) probes:   cover the catalog of lncRNA from the LNCipedia 2.1 database & contains

updated mRNA probes. The Agilent 2-Color Microarray-based Gene Expression Analysis uses cyanine 3& cyanine 5-labeled targets to measure gene expression in experimental & control samples.

Sample input, Processing, and Data Analysis

Sample Input Type: Total RNA

Sample Input Range: 10ng-200ng of Total RNA

METHODOLOGY:

Total RNA Extraction (From Tissue)

RNeasy Mini Kit (Qiagen) will be used for the extraction & purification of total RNA from tissue samples.

RNA storage

RNA Purified would be stored at −80 to −65°C in RNAse-free water.

Quantify, Purity, and Integrity of RNA

The conct. (quantification) of RNA would be determined by Qubit 3 Fluorometer.

The purity and integrity of RNA will be determined by NanoDrop2000.

Two-Color Microarray-Based Gene Expression Analysis (With Control)

Labeling of Experimental and Control RNA (Cyanine 3 for Control/Cyanine 5 for Sample)

STEPS:

1. Template Total RNA (10-100 ng) with RNA Spike in (Spike A  for control and Spike B  for Sample)

2. cDNA (Complementary DNA) Synthesis using Reverse Transcriptase Enzyme

3. cRNA (Complementary RNA) synthesis and labeling using Cyanine 3-CTP / Cyanine-5 CTP by using T7

RNA polymerase

4. Purification of the labeled amplified cRNA

5. Quantification of labeled cRNA using NanoDrop

6. Pooling of the Experimental sample with the respective control sample

7. Fragmentation of labeled and pooled cRNA at 60 0 for 30 minutes using the Fragmentation mix.

8. Loading on the Array slide and 17-hour of hybridization (65ºC)

9. Buffer Wash of Array Slide

10. Scan using Agilent Sure Scan Microarray Scanner

11. Feature Extraction to check the quality of the image generated and QC files for each experimental

sample(Amplification above typically a 100-fold from total RNA to cRNA with  this kit)

Data Analysis will be done with the help of GeneSpring GX 9.0 software. Comparison analysis (patients vs. controls arrays) Xiao et. al (Providing the results of the study)

I expect to observe there is greater than a 3-4 fold change in the TCTP gene expression in the healthy and diseased tissue which can be targeted. There might be a higher expression of TCTP in poor prognosis patients. Alternatively, other potential biomarker genes may also, be seen, which might correlate to poor prognosis in OLP patients.

BUDGET: $10,000 or 6-8 lakhs rupees for the cost of reagents and laboratory supplies

REFERENCE

1.      Yamamoto, K., Hanada, R., Kikuchi, A., Ichikawa, M., Aihara, T., Oguma, E., Moritani, T., s Shimanuki, Y., Tanimura, M., and Hayashi, Y. (1998) Spontaneous regression of localized neuroblastoma detected by mass screening, Journal of clinical oncology : official journal of the American Society of Clinical Oncology 16, 1265-1269.

2.      Role of TCTP in Cell Biological and Disease Processes by Ulrich-Axel Bommer and Toshiaki Kawakami

3.      Oral lichen planus: An overview R. Jayasri Krupaa, S. Leena Sankari, K. M. K. Masthan, and E. Rajesh

4.      Histamine H4 receptor in oral lichen planus. A Salem 1, A Al-Samadi, V Stegajev, H Stark, R Häyrinen-Immonen, M Ainola, J Hietanen, Y T Konttinen

5.      Histamine metabolism and transport are deranged in human keratinocytes in oral lichen planus. A Salem, S Rozov, A Al-Samadi, V Stegajev, D Listyarifah, V-P Kouri, X Han, D Nordström, J Hagström, and K K Eklund

6.      HL-A antigens in lichen planus N J Lowe, A G Cudworth, and J C Woodrow

7.      Gene Expression Profiling of Lichen Planus Reflects CXCL9þ-Mediated Inflammation and Distinguishes this Disease from Atopic Dermatitis and Psoriasis Joerg Wenzel, Bettina Peters, Sabine Zahn, Michael Birth, and Kay Hofmann

8.      Microbial Community Analysis of Saliva and Biopsies in Patients With Oral Lichen Planus. Xuewei Wang, Zhibai Zhao, Nan Tang, Yuping Zhao, Juanyong Xu, Liuyang Li, Ling Qian, Junfeng Zhang, and Yuan Fan

9.      Topical application of siRNA targeting cutaneous dendritic cells in allergic skin disease. Miyuki Azuma , Patcharee Ritprajak, Masaaki Hashiguchi

10.  Ultra-low Input Total RNA Gene Expression Analysis Using the Agilent SurePrint G3 Human Gene Expression Microarray Makoto Ogino, PhD Research Fellow, Drug Discovery Resrearch, Astellas Pharma, Inc. Yayoi Fukuoka and Allant Kodaka, Agilent Technologies Japan, Ltd.

11.  Differential gene expression profiles of whole lesions from patients with oral lichen planus  Xiao-An Tao,Chun-Yang Li,Juan Xia,Xi Yang,Xiao-Hua Chen,Yu-Tao Jian,Bin Cheng

12.  Genomic Analysis of Oral Lichen Planus and Related Oral Microbiome Pathogens . Evelyn F. Zhong, Andrea Chang, Andres Stucky, Xuelian Chen, Tarun Mundluru, Mohammad Khalifeh and Parish P. Sedghizadeh

13.  Upregulation of TCTP expression in human skin squamous cell carcinoma increases tumor cell viability through anti-apoptotic action of the protein DI Wu, Ze Guo, Wei Min, Bingrong Zhou, Mingna Li, Wei Li, Dan Luo

14.  Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer Virginie Baylot, Maria Katsogiannou, Claudia Andrieu, David Taieb, Julie Acunzo, Sophie Giusiano, Ladan Fazli, Martin Gleave, Carmen Garrido, and Palma Rocchi

15.  High tctp expression as prognostic factor in different cancer types. Nicolas Fischer, Mohamed E.M. Saeed, Elena Lippe, Wilfried Roth, and Thomas Efferth

16.  Tumor reversion: a dream or a reality. Avantika Tripathi, Anjali Kashyap , Greesham Tripathi, Joni Yadav, Rakhi Bibban, Nikita Aggarwal, Kulbhushan Thakur , Arun Chhokar, Mohit Jadli, and Ashok Kumar Sah

17.  Oral symptoms and salivary finding in oral lichen planus, oral lichenoid lesions, and stomatitis. Kristine Roe Larsen, Jeanne Duus Johanssen, Jesper Reibel, Claus Zachariae, Kasper Rosing, and Anne Marie Lynge Pedersen

18.  An association between oral lichen planus and a persistently dry mouth. Angus Colquhoun and Martin Ferguson.

19. A study of role of tacrolimus in the treatment of lichen planus. Runuk Singh, Sonal Vahanwala

20. Diagnostic Criteria of Oral Lichen Planus: A Narrative Review Doina Iulia Rotaru,¹ Diana Sofineti,² Sorana D. Bolboacă,³ and Adriana E. Bulboacă⁴

21. Bahram Arezi, Gene Expression Profiling and Validation Using Agilent SurePrint G3 Gene Expression

Arrays, Application Note

22. SurePrint V3 Microarrays DataSheet 5991-5943EN

23. G4140-90050_GeneExpression_TwoColor_6.9, Two-Color Microarray-Based Gene Expression

Analysis

24. RNeasy® Mini Handbook, Oct-19