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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
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