Download Advanced diagnostic methods in diagnosis of oral cancers

TITLE
Advanced diagnostic methods in diagnosis of oral cancers – A Review
AUTHORS
(1) Dr. Vishnudas Dinesh Prabhu, M.D.S , Professor, Department of Oral Pathology &
Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore –
Karnataka.
(2) Dr. Shakil M. (M.D.S), Post Graduate student, Dept of Department of Oral Pathology &
Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore –
Karnataka.
(3) Dr. Soniya Adyanthaya.
M.D.S, Assistant Professor, Dept of Department of
Oral
Pathology & Microbiology, Yenepoya Dental College & Hospital, Yenepoya University,
Mangalore – Karnataka.
(4) Dr. Maji Jose. M.D.S, Professor and Head, Dept of Department of Oral Pathology &
Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore –
Karnataka.
Corresponding Author:
Dr. Vishnudas Dinesh Prabhu
Professor
Department of Oral Pathology & Microbiology
Yenepoya Dental College & Hospital,
Yenepoya University, Mangalore – Karnataka.
E-mail : [email protected]
ABSTRACT:
Advances in the analysis of DNA, RNA, and proteins in tissues have permitted improved
biologic insights into neoplastic diseases including oral cancers and also many
developmental, inflammatory, metabolic, and infectious diseases. Molecular diagnostic
methods as an investigative tool in the field of histopathology have evolved into the most
widespread, diagnostically useful techniques in the assessment of disease states. It is likely
that, in the future, these methods will increasingly enter into the day-to-day diagnosis and
management of patients. The pathologist will continue to play a fundamental role in diagnosis
and will likely be in a pivotal position to guide the implementation and interpretation of these
tests as they move from the research laboratory into diagnostic pathology.
Advanced
molecular diagnostic methods and their application to diagnostic pathology of oral cancers
are reviewed in this article.
KEY WORDS; CANCER, DIAGNOSIS, IMMUNOHISTOCHEMISTRY, TUMOR
MARKERS, MOLECULAR ONCOLOGY
INTRODUCTION
The diagnosis of cancer relies primarily on invasive tissue biopsy, as noninvasive diagnostic
tests are generally insufficient to define a disease process of cancer. The conventional
histopathology based on light microscopy, however, has recently been complemented with
ultrastructure, immunohistochemistry and molecular diagnostics. Even though the biopsy
study is fundamental, it is a diagnostic method with limited sensitivity where one of the most
important features is the subjective interpretation of the examining pathologist. Changes
occur at the molecular level before they are seen under the microscope and before clinical
changes occur. These issues underline the importance of discovering and developing new
diagnostic methods, improving the existing ones and discovering new therapeutics targets for
oral neoplastic diseases.
1,2,3,4
In recent decades, we have seen a dramatic switch from
histopathological to molecular methods of disease diagnosis. Strategies have been developed
whereby, from single tissue sample, different techniques can be performed in order to fully
characterize molecular phenotype and genotype of tumors. Immunohistochemistry,
cytogenetics, analysis of DNA content and molecular genetic assays which have been added
as valuable adjuncts to light microscopy in oral cancer diagnosis are discussed.
IMMUNOHISTOCHEMISTRY
Immunocytochemistry is a technique for identifying cellular or tissue constituents (antigens)
by means of antigen-antibody interactions, the site of antibody binding being identified either
by direct labeling of the antibody, or by use of a secondary labeling method. The selection of
antibodies for immunohistochemical testing is made on the basis of their tumor specificity
and the likelihood that they will react with the tumor under evaluation.
IHC has greatly reduced the number of unclassified tumors and has been of major assistance
in defining metastatic tumors of unknown primary site. It is of great value in the diagnosis of
undifferentiated tumors where light microscopy is unable to discern diagnostic features.
Few interpretations are: Expression of cytokeratins strongly suggests an epithelial origin;
leukocyte common antigen (LCA) is evidence of lymphoid origin while expression of S-100
protein and HMB 45 is characteristic of malignant melanoma.5 Immunohistochemistry can
help in the diagnosis of undifferentiated tumors which include poorly differentiated
carcinoma, , amelanotic melanoma or, less commonly sarcoma. Presence of neural markers
like neuron specific enolase and synaptophysin are suggestive of neuroectodermal tumors,
and the markers of skeletal muscle differentiation, desmin and myoglobin, are indicative of
rhabdomyosarcoma. 5 The interpretation in IHC should be taken care with strict adherence to
laboratory practice. A panel of antibodies is generally recommended to characterize a
diagnostic problem.
TUMOR MARKERS
Tumor markers are biologic or biochemical substances produced by tumors and secreted into
blood, urine, other body fluids or body tissues of some patients with certain types of cancer in
higher than normal amounts. A tumor marker may be produced by tumor itself, or by the
body in response to the presence of cancer or certain non-cancerous conditions. Tumor
markers can be detected by various methods including antigen-antibody based techniques.6
Tumor marker levels are not elevated in every person with cancer- especially in the early
stage of disease. The field of tumor markers is ever expanding with many new candidate
markers either in clinical use or under active evaluation. Usually multiple tumor markers are
associated with individual malignancies; vice versa individual tumor markers may be
associated with various malignancies. Thus, the use of multiple markers based on the
combination pattern for the selected malignancy will improve sensitivity and specificity of
the detection.7
MOLECULAR ONCOLOGY
At a molecular level, a cancer cell may be distinguished from its normal counterpart by
abnormalities in structure or expression of certain genes. These abnormalities, directly or
indirectly, result in disturbance of cell cycle regulation and induce dysregulated growth in
cancer cells. Molecular oncology studies the alterations in genetic and biochemical processes
at the molecular level. It helps in establishing a definitive diagnosis and classification of
tumors based on the recognition of complex profiles ('finger-prints') or unique molecular
alteration that occur in specific tumor types. The changes can be studied on chromosomes,
DNA or RNA.
CHROMOSOME ANALYSIS
Chromosome abnormalities are frequently found in malignant cells. Many abnormalities may
be specific to tumor types. In hematological malignancies, individual abnormalities can be
easily analyzed on bone marrow aspirate samples. Conventional cytogenetic analysis is a
fundamental method in the study of small round cell tumours. It reveals translocations and
other structural abnormalities as well as numerical aberrations.8
Detection of chromosome abnormalities has traditionally been performed through banding
analysis of metaphase chromosome for solid tissue biopsies. Fluorescence in situ
hybridization (FISH) technique is applicable to interphase cells and is more sensitive
compared to conventional cytogenetics. It involves hybridization of conjugated probes to
chromosomes, and visualization of the probe by fluorescent microscopy. FISH seems to be
emerging as a major focus for development of molecular tests for subclassification and
prognostication in lymphomas.9 It can readily identify gains or losses of whole chromosomes
or chromosome arms. Several genetic translocations associated with common subtypes of
lymphoma can be conveniently detected by FISH.10
Comparative genomic hybridization (CGH) is a newly described method that globally assays
for chromosomal gains and losses in genomic complement. Comparative genomic
hybridization (CGH) enables the screening of the whole genome and reveals DNA sequence
copy number changes (losses, gains and high-level amplications).11 CGH has shown to be
especially useful in obtaining cytogenetic data of solid tumours which often fail to produce
mitotic cells in cell culture or in which the cytogenetic complexity prevents any real
interpretation of the karyotype. Recurrent alterations in a specific tumour type have led to the
identification of target oncogenes12 or tumour suppressor genes.13 Fresh, fresh frozen or
paraffin-embedded tumour tissue samples can be used. Normal cell contamination should be
avoided and the proportion of tumour cells should be high in order to ensure the highest
possible sensitivity of CGH.14 The molecular cytogenetic techniques like FISH and CGH are
increasingly been used in addition to conventional cytogenetics to properly discern various
chromosomal abnormalities in tumor samples.
Genetic analysis in oral cancer/ Molecular genetic assays:
Malignancy is considered as a process caused by the accumulation of multiple genetic
alterations, which affect the cell cycle as well as normal cell differentiation. These alterations
are mainly acquired (somatic) although some of them may be inherited and when they
activate protooncogenes, inactivate tumour suppressor genes or affect enzymes, which repair
DNA, they could lead to a malignant transformation. Most of the oral cavity carcinogens are
chemical (tobacco), physical (radiation) and infectious (Human papilloma virus, Candida)
mutagenic agents that may cause changes in gene and chromosome structure by point
mutations, deletions, insertions and rearrangements. However, some of these changes may
occur spontaneously. These genetic alterations, which occur during carcinogenesis, can be
used as targets for detecting tumour cells in clinical samples.2,4,15 Not all mutations in cancer
genes are apparent at cytogenetic level, so it has become increasingly important to identify
genes themselves, and relevant changes within their structure. Genes and their encoded
proteins, may serve as markers of lesions with high risk of progression to malignant
disease—or they may be predictive of patient response to treatment and survival. 16-20 The
gene alterations can be broadly classified into following:
Oncogenes: normal cell proteins that become abnormally activated.
Tumor suppressor genes: normal antiproliferative genes or their products that becomes
inactivated.
DNA repair genes: become inactivated, causing accumulation of potentially damaging
mutations.
Regulators of apoptosis: inactivation of pro-apoptotic genes or activation of anti-apoptotic
genes promotes cell survival.5
Overexpression of the cell cycle–associated oncoproteins cyclin D1 and MDM2, as well as
underexpression of the tumor suppressor proteins p53, p16, and p27, may be important tumor
markers.16-20 In some oral cancers the antiapoptotic proteins Bcl-X and Bcl-2 are
overexpressed.21,22 Moreover, expression of the proapoptotic protein Bax has been positively
correlated with increased sensitivity to chemotherapeutic agents in head and neck
carcinomas.23 Growth factor receptors like EGFR and HER-2/neu are oncogenes that encode
transmembrane receptors that are believed to increase cell cycle, cell motility, and
angiogenesis and are overexpressed in many types of malignancy, including head and neck
carcinomas.24-28 Squamous cell carcinomas that overexpress EGFR are associated with a
poorer outcome than those that do not overexpress this receptor.29,30 Proteins involved in
signal transduction (ie, the
process whereby interactions between a receptor on the cell surface and its ligand are
transmitted to the nucleus of the cell), such as ras (GTP-binding proteins), and nuclear
regulatory proteins, such as myc (transcriptional activator proteins), are also abnormally
expressed in oral cancers. Mutations in the tumour suppressor gene p53 are the most frequent
genetic alterations in human cancer and show a variable frequency in oral cancer. 26
2. Epigenetic alterations, Loss of hetrozygosity and Microsatellite instability
The applicability of other molecular markers such as epigenetic alterations (hypermethylation
of promoter regions) and genomic instability such as loss of hetrozygosity (LOH) and
microsatellite instability (MSI) has also been studied.31,32 The main epigenetic modification
in tumours is methylation and it seems that the changes in the methylation patterns can play
an important role in tumorigenesis. These epigenetic alterations are often associated with the
loss of genetic expression and their occurrence seems to be essential for the multiple
necessary genetic events. So malignant progression takes place because these alterations can
inactivate DNA repairing genes. Rosas et al. detected abnormal hypermethylation patterns in
p16, MGMT and DAP-K genes in smears of patients suffering from head and neck cancer by
methylation specific Polymerase Chain Reaction (PCR).31 Huang et al. used PCR techniques
to amplify DNA from exfoliated cytology samples and found that 66% of the tumours studied
showed LOH at one position in the p53 sequence, while 55% showed LOH at some other
location.33 PCR analyses have also been used for the detection of microsatellite markers, i.e.
short repetitive DNA sequences. Microsatellite regions are distributed along the genome and
have been widely and satisfactorily used as molecular markers for carcinogenesis. Alterations
in these regions have been used as clonal markers and for detecting tumoral cells among
normal cells.34,35 Several studies have demonstrated these by using microsatellite markers that
alterations in certain regions of chromosomes 3p, 9p, 17p and 18q are associated with the
development of head and neck squamous cell carcinomas. 36,37
Analysis of DNA content:
Molecular detection methods involve the analysis of nucleotide sequences within nucleic acid
to detect the presence of malignant cells in fluids and tissues. DNA is, in general, stable in
tissues and cells after removal. RNA, on the other hand, is unstable and highly degradable by
endogenous RNAses released by lysosomes or dead cells. The analysis is usually done on
total cellular DNA using southern blot (SB) procedure or polymerase chain reaction (PCR) in
which regions of DNA are amplified and more easily identified. PCR-based methods can be
used to detect gene rearrangements and translocation-derived fusion genes associated with
Ewing tumours and lymphomas.8 Messenger RNA (mRNA) detection of genes and their
products is done by the techniques like northern blot, reverse transcription-PCR (RT-PCR)
and in situ hybridization.
A relatively newer technique like micro-array methods allows measurement of differential
expression of all the genes including those of low abundance. Microarrays exploit the unique
feature of single-stranded DNA to hybridize to complementary DNA sequences, thereby
permitting sequence specific identification of DNA and allow us to simultaneously monitor
the transcription of thousands of genes in parallel.38,39 In oral cancer it has been used to
classify & predict the biologic process underlying this malignancy. Premalignant &
malignant oral lesions contain genetic changes that are present prior to phenotypic &
morphologic changes. Identification of gene expression profiles will allow clinicians to
differentiate harmless white lesions from pre-cancerous lesions or from very early cancer.37,38
Conclusion:
A good understanding of molecular biology of head & neck cancer along with an improved
understanding of the various advanced diagnostic techniques will definitely contribute to
early detection & future therapeutic improvements. Molecular genetic tests may shape the
new insights into the pathogenesis of oral cancers, diagnostic approach, and may lead to new
therapeutic options. We are still at an early stage of the integration of molecular genetic tests
into diagnosis of oral cancer. The future for implementation of molecular genetic testing to
benefit patients with head and neck cancer is bright, with methods either established already
or on the brink of readiness for application in diagnostic laboratories. Professionals in the
field of histopathology must now work hard to ensure that clinical needs and technical
effectiveness are the major factors in determining which tests are brought into standard use
over the next few years.
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