Research Proposal

Title of study

Vibrational spectroscopic analysis to identify proteins (amino acids) biomarkers for detection

of pre-cancerous and cancerous head and neck lesions using Raman spectroscopy.

Researchers;

Dr. M.Shairaz Sadiq (principal investigator)

BDS, MCPS. PhD (oral medicine) student applicant at USM-Malaysia

Dr. Raja Azman Raja Awang

BDS, MClinDent , PhD main supervisor at USM

Dr. AB Rahman Rosalinda (co-supervisor)

BDS, MClinDent. Co-supervisor at USM

Dr. Qasim Saeed (research scientist)

BDS, PhD. Dean & Research incharge IOD-CMH-LMC, Pakistan

Dr. Salman Aziz Mian (field supervisor in Pakistan)

BDS, MSc, PhD. Biomaterial specialist, expert in Raman spectroscopy

Dr.Sabahat Javed Butt (facilitator in Pakistan)

MBBS, M.Phil. DCP. Gen pathologist, expert in histopathology &

immunohistochemistry

Introduction

Around the world oral cancer is becoming a dangerous and rising problem for mankind.

Along with pharyngeal cancers, they are ranked sixth as the most common type of cancers.

There are around 275,000 oral and 130,300 pharyngeal cancers diagnosed each year and

these figures do not include nasopharynx (Suarez et al., 2013). Developing countries account

for two third of the diagnosed cases. Survival rates for patients with this disease have not

improved in the past 30 years despite significant medical and surgical advances.

Oral premalignancies are traditionally classified as premalignant lesions such as Leukoplakia,

erythroplakia, palatal lesions of reverse smokers and premalignant conditions such as oral sub

mucous fibrosis, lichen planus (Warnakulasuriya et al., 2007). Rate of malignant

transformation vary partly according to the population, gender and tobacco use (Yardimci et

al., 2014).

Justification of study

Raman spectroscopy is fast becoming a technique of choice to gain "biochemical image" of a

sample by mapping its constituent chemical bonds and functional groups. The technique

offers a number of advantages as a diagnostic tool as very minimal sample preparation is

needed, only a small sample area is required and detection is rapid. As pre-cancerous lesions

and OSCC are clinically assessable lesions, this would permit the technique to be used non-

invasively to determine the level of dysplasia, detect OSCC and be used surgically for margin

detection. More importantly biopsies cannot be taken frequently for the monitoring of

suspected lesions. Therefore, to improve the prognosis it is of extreme importance that these

lesions are detected at early stages and treated accordingly.

Research questions

1. What are the differences in histopathological features of normal, precancerous and

cancerous oral mucosa?

2. What are the differences in immunohistochemistry features of normal, precancerous

and cancerous oral mucosa?

3. What are the differences in Raman spectroscopic features of normal, precancerous

and cancerous oral mucosa?

4. Is there specific proteins (amino acids) bio-molecules which can act as biomarkers for

early cancer diagnosis in order to improve prognosis?

Null hypothesis

1. There are no differences in histopathological features of normal, precancerous and

cancerous oral mucosa.

2. There are no differences in immunohistochemistry features of normal, precancerous

and cancerous oral mucosa.

3. There are no differences in Raman spectroscopic features of normal, precancerous and

cancerous oral mucosa.

4. There are no specific proteins (amino acids) bio-molecules which can act as

biomarkers for early cancer diagnosis in order to improve prognosis.

Objective

General objective

To determine proteins (amino acids) biomarkers for detection of pre-cancerous and cancerous

head and neck lesions using Raman spectroscopy.

Specific objectives

1. To compare histo-pathological (HP) findings of normal, pre-cancerous and

cancerous oral mucosa.

2. To compare immunohistochemistry (IHC) findings of normal, pre-cancerous and

cancerous oral mucosa.

3. To compare Raman spectroscopic (RS) findings of normal, pre-cancerous and

cancerous oral mucosa.

4. To define specific protein (amino acids) bio-molecules which can act as

biomarkers for early cancer diagnosis in order to improve prognosis.

Literature review

Epidemiology

Oral cancer accounts for approximately 3% of all malignancies in USA and up to 30 -40% in

India (Suarez et al., 2013). Lichen planus (LP) is a chronic inflammatory disease of the skin,

mucous membranes and nails. Oral lichen planus (OLP) is a chronic disease, with unknown

etiology, where the autoimmune mechanisms take a particular place. The overall prevalence

of lichen planus in the general population is about 0.1–4.0%. Malignant transformation rate

of oral keratosis (leukoplakia) ranges from 0.06 to 0.3 % in India and in Sweden it reaches

around 3.6 % (ESPINOCELULAR and CANCERIZÁVEIS, 2002). Erythroplakia are a lot

rare and carry over 90% risk of malignant transformation (ESPINOCELULAR and

CANCERIZÁVEIS, 2002) .

DIAGNOSIS

Early detection of OLP could be of great help to the patients as it has 5 – 16 % malignant

transformation chances. Early diagnosis of OSCC can achieve better prognosis and improved

quality of life for patients despite of advances in surgical techniques, reconstruction

procedures, radiation and medical oncology (Omar, 2013). Visual inspection of the oral

cavity is currently the first line of screening for pre-cancer and cancer yet which may vary in

results of multiple observers (Shin et al., 2010). Any mucosal alterations must be observed by

the dentist as a potential cancerous lesion which may help in diagnosing oral cancers at early

stages. Histopathological evaluation of surgically removed biopsies is considered to be the

gold standard for diagnosis and surveillance of oral cancers (Messadi, 2013).

Histopathology;

Histopathology refers to the microscopic examination of tissue in order to study the

manifestations of disease. Specifically, in clinical medicine, histopathology refers to the

examination of a biopsy or surgical specimen by a pathologist, after the specimen has been

processed and histological sections have been placed onto glass slides. In

contrast, cytopathology examines free cells or tissue fragments.

Immunohistochemistry;

Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g. proteins) in

cells of a tissue section by exploiting the principle of antibodies binding specifically to

antigens in biological tissues.

Biopsy;

Biopsy is a microscopic examination of tissue removed from a living body to analyze the

presence, cause, or extent of a disease.

Limitations of biopsies:

These surgical interventions can be performed by scalpel tissue biopsy, punch tissue biopsy

or fine needle aspiration (FNA) (Kumaraswamy et al., 2012). Although histopathological

evaluation is currently the best tool for cancer diagnosis it is associated with surgical

interventions and therefore have their own limitations. These techniques are invasive which

cause anxiety for the patients along with pain and discomfort. Pre-malignant or dysplastic

lesions need surveillance in order to diagnose cancer at an early stage. More importantly

biopsies cannot be taken frequently for the monitoring of suspected lesions.

Need for non-invasive and real time detection:

A non-invasive and real time procedure which can detect tissue changes can resolve these

issues and facilitate consistent screening. Furthermore, the other threat for the patients is the

risk of post-operative metastasis and recurrence. Therefore, to improve the prognosis it is of

extreme importance that these lesions are detected at early stages and treated accordingly.

RAMAN SPECTROSCOPY

Raman spectroscopy (RS), which is a noninvasive technique, can be employed to detect pre-

cancerous conditions / lesions and cancer at early stages leading to an improved prognosis.

RS is fast becoming a powerful analytical tool to identify biochemical changes related to

carcinogenesis and recognize biomolecules associated with cancer. In 1928, the phenomenon

of inelastic scattering of light was first discovered by Chandrashekhara Venkata Raman who

was an Indian physicist, known as the Raman Effect. It elaborates the shift in wavelength of a

small fraction of radiation scattered by molecules which differs in frequency of that of the

incident beam (Krishnan and Shankar, 1981). The shift in the wavelength relies on the

chemical structure of the molecules which are accountable for scattering of incident light.

The scattered light provides information about molecular vibrations which provide statistics

regarding structure, shape, electronic environment and bonding of the molecules, therefore

resulting in qualitative and quantitative analysis of each compound (Kneipp et al., 1999). RS

is the most precise and sensitive of all the optical techniques but with tremendously weak

signals, in the order of one trillionth of the incident beam.

When biological tissues are analyzed by RS, there are four components which contribute to

the Raman spectra; these are nucleic acids (DNA and RNA), proteins (amino acids, iso-

enzymes, immunoglobulin, hormones and keratins), water and lipids (cell membranes).

Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of

amino acid residues. An essential amino acid or indispensable amino acid is an amino

acid that cannot be synthesized by the organism, and thus must be supplied in its diet. The

nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, tryptophan,

methionine, leucine, isoleucine, lysine, and histidine (i.e., F V T W M L I K H).

Six other amino acids are considered conditionally essential in the human diet, meaning their

synthesis can be limited under special pathophysiological conditions, such as prematurity in

the infant or individuals in severe catabolic distress. These six are arginine, cysteine, glycine,

glutamine, proline, and tyrosine (i.e. R C G Q P Y). Five amino acids are dispensable in

humans, meaning they can be synthesized in the body. These five are alanine, aspartic

acid, asparagine, glutamic acid and serine (i.e., A D N E S).

MOLECULAR ALTERATIONS IN CANCER & SPECTROSCOPY

The general features of neoplastic cells are specific changes in nucleic acid, protein, lipid,

and carbohydrate quantities and/or conformations (ur Rehman et al., 2012). Proteins, lipids,

and nucleic acids are the marker molecules, which may be indicative of neoplasia, and

changes in these molecules might be expected. Not surprisingly, Raman spectroscopies, due

to their fingerprint character, can be used to describe and recognize the changes that take

place in cancer cells. Several studies have elucidated the spectral features of cancer cells and

their various components such as collagen. The Raman spectrum of a sample corresponds to

the characteristic molecular groups in the sample, and the technique can give detailed

information on changes in structure and composition of the cellular molecules (Mahadevan-

Jansen and Richards-Kortum, 1996) and can provide important diagnostic information. These

features can potentially be used as diagnostic parameters to identify malignant tumors.

Several research groups have employed RS in various studies related to cancer and its early

detection.

PRINCIPLES AND MECHANISM OF RAMAN SPECTROSCOPY:

The exposure of a molecule to a monochromatic light leads to elastic and inelastic scattering

of light. There is no change observed in the photon frequency or wavelength in elastic

scattering. On the other hand, inelastic scattering is referred to as there is shift in photon

frequency as a result of excitation of molecular vibrations during which energy may be

gained or lost by photons (Kaur H.S., 2006).

Figure 4 Principles of RS. Adapted from (Swinson et al., 2006)

The four major components of a Raman spectrometer are light source, monochromator,

sample holder and detector. The stability of the instrument, sufficient resolution and high

signal-to-noise ratio may affect the analysis on Raman spectra.

Figure 6 Diagram demonstrating the mechanism of RS.

Formalin-fixed paraffin-embedded (FFPE) material presents a readily available resource in

the study of various biomarkers. There has been interest in whether the storage period has

significant effect on the extracted macromolecules. In a study, it’s investigated if the storage

period had an effect on the quantity/quality of the extracted nucleic acids and proteins. The

study systematically examined the quality/quantity of genomic DNA, total RNA, and total

protein in the FFPE blocks of malignant tumors of lung, thyroid, and salivary gland that had

been stored over several years. Results show that there is no significant difference between

macromolecules extracted from blocks stored over 11–12 years, 5–7 years, or 1–2 years in

comparison to the current year blocks (Kokkat et al., 2013).

Materials and method

Study design

The design of the study will be descriptive retrospective study.

Study population and sample

References population

Formalin-fixed paraffin-embedded specimens will be taken from CMH Lahore medical

college pathology department of Pakistan population.

Source of population

The study group will be comprised of archived specimen data of precancerous and cancerous

condition from CMH pathology department in Pakistan from 2000 to 2015.

Ethical approval

The ethical approval for the research will be sought from ethical review boards; committee of

USM, Malaysia and from institute of dentistry, combined military hospital Lahore medical

college, Lahore Pakistan where the study is going to be conducted.

Sample Size Calculation

Sample size will be calculated using calculator (+/- 300 specimens, 100 in each group)

Inclusion criteria

Any sufficient soft tissue biopsy

Specimen of any age

Specimen of any sex

Specimen of head & neck region

Exclusion criteria

Autolyzed biopsy specimen

Data collection

Specimens will be obtained from hospital laboratory within the period of 2000 – 2015.

Statistical analysis;

Using SPSS 22

FFPE

Group Anormal

histo-pathological examination - cellular

structure

immuno-histologica exam - antibodies

raman spectroscopy-biological analysis of

nucleic acids, proteins, water,lipids

Group Bprecancerous

histo-pathological examination - cellular

structure

immuno-histologica exam - antibodies

raman spectroscopy-biological analysis of

nucleic acids, protiens, water, lipids

Group Ccancerous

histo-pathologicalimmuno-histological raman spectroscopic

analysis

raman spectroscopy- biological analysis of

nucleic acids, proteins, water, lipids

immuno-histologica exam - antibodies

References

ESPINOCELULAR, B. P. P. D. C. & CANCERIZÁVEIS, B. E. L. (2002). Biomarkers to predict oral squamous cell carcinoma in precancerous stages. Rev Fac Odontol Bauru, 10(3), 63-67.

Kneipp, K., Kneipp, H., Itzkan, I., Dasari, R. R. & Feld, M. S. (1999). Ultrasensitive chemical analysis by Raman spectroscopy. Chemical reviews, 99(10), 2957-2976.

Kokkat, T. J., Patel, M. S., McGarvey, D., LiVolsi, V. A. & Baloch, Z. W. (2013). Archived formalin-fixed paraffin-embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein. Biopreservation and biobanking, 11(2), 101-106.

Krishnan, R. & Shankar, R. (1981). Raman effect: History of the discovery. Journal of Raman Spectroscopy, 10(1), 1-8.

Kumaraswamy, K. L., Vidhya, M., Rao, P. K. & Mukunda, A. (2012). Oral biopsy: oral pathologist's perspective. J Cancer Res Ther, 8(2), 192-198. doi: 10.4103/0973-1482.98969

Mahadevan-Jansen, A. & Richards-Kortum, R. R. (1996). Raman spectroscopy for the detection of cancers and precancers. Journal of Biomedical Optics, 1(1), 31-70.

Messadi, D. V. (2013). Diagnostic aids for detection of oral precancerous conditions. International Journal of Oral Science, 5(2), 59-65. doi: 10.1038/ijos.2013.24

Omar, E. A. (2013). The outline of prognosis and new advances in diagnosis of oral squamous cell carcinoma (OSCC): Review of the literature. Journal of Oral Oncology, 2013.

Shin, D., Vigneswaran, N., Gillenwater, A. & Richards-Kortum, R. (2010). Advances in fluorescence imaging techniques to detect oral cancer and its precursors. Future oncology (London, England), 6(7), 1143-1154. doi: 10.2217/fon.10.79

Suarez, E., Gonzalez, L., Diaz-Toro, E. C., Calo, W. A., Bermudez, F. & Ortiz, A. P. (2013). Incidence of oral cavity and pharyngeal cancers by anatomical sites in population-based registries in Puerto Rico and the United States of America. P R Health Sci J, 32(4), 175-181.

ur Rehman, I., Movasaghi, Z. & Rehman, S. (2012). Vibrational spectroscopy for tissue analysis: CRC Press.

Warnakulasuriya, S., Johnson, N. W. & van der Waal, I. (2007). Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med, 36(10), 575-580. doi: 10.1111/j.1600-0714.2007.00582.x

Yardimci, G., Kutlubay, Z., Engin, B. & Tuzun, Y. (2014). Precancerous lesions of oral mucosa. World Journal of Clinical Cases : WJCC, 2(12), 866-872. doi: 10.12998/wjcc.v2.i12.866