Introduction To Biomarkers

CHAPTER NO.
6 BIOMARKERS IN ORAL SUB-MUCOUS FIBROSIS
INTRODUCTION TO BIOMARKERS:
To raise new questions, new possibilities, to regard old problems from a new angle, requires creative
imagination and marks real advance in science’’. Albert Einstein.
During the past few years, extensive development has taken place in the field of biomarkers. It is a highly
complex field, and its multi-faceted applications exhibit the complexity of the science associated with the
biomarkers, and for the uninitiated, it is easy to get confused with the different terms used in different
contexts. The study of the biomarkers related to OSF is no exception.
Various researchers undertook the study of biomarkers of OSF to simplify our understanding of
etiopathology and malignant transformation of OSF. However, we have seemingly come full circle: from
overwhelming complexity to anticipated simplicity, back again to substantial complexity-a perspective that
was highlighted by a plethora of literature on biomarkers of OSF. 1039
Progress in our understanding of the molecular mechanisms underlying oral submucous fibrosis (OSF) has
to lead us in an increasing number of biomarkers that can be used to predict the behavior of OSF. However,
the number of clinically useful markers is pitifully small. Even though in recent years, the number of
molecular-based assays has increased, histopathology remains the gold standard for most diagnostic and
therapeutic decisions.
Immunohistochemistry (IHC) is a globally available tool that complements histopathological analysis by

detecting gene expression at the protein level. Advances in the understanding of OSF at the regulatory
protein expression level have resulted in the identification of some prognostic tumor biomarkers associated
with the clinical outcome of OSF, and knowledge of these cell and molecular tissue biomarkers can provide
information complementary to that which we can obtain from clinical examination and histopathological
studies. The utility of a given biomarker as a prognostic tool requires a carefully designed cohort
investigation, as well as the defined criteria to designate a tumor as negative or positive for a specific IHC
biomarker. Conversely, there is significant heterogeneity amongst experimental procedures, and the
literature usually gives no uniform recommendation for a particular primary antibody or defines a cut-off
for positivity. The recent guidelines proposed by REMARK should be considered to standardize future
studies to provide adequate measures of predictability and improve the quality of the prognostic
biomarkers studies examining OSF. 393.
The REMARK (Reporting Recommendations for Tumor Marker Prognostic Studies) guideline published by
Altman D.G. et al. 1087 includes a checklist which aims to improve the reporting of these types of studies.
The authors have expanded on the REMARK checklist to enhance its use and effectiveness through a better
understanding of the intent of each item and why the information is important to report. The paper
provides a comprehensive overview to educate on good reporting and provide a valuable reference of
issues to consider when designing, conducting and analyzing tumor marker studies and prognostic studies
in medicine in general.
REF: Altman DG, McShane LM, Sauerbrei W, Taube SE (2012) Reporting Recommendations for Tumor
Marker Prognostic Studies (REMARK): Explanation and Elaboration. PLoS Med 9 (5): e1001216.
doi:10.1371/journal.pmed.1001216 1087
Before the biomarkers are incorporated into routine clinical applications, it is essential that biomarkers
undergo rigorous analytical and clinical validation followed by clinical assessment since the biomarkers play
a critical role at all stages of the disease. 403
For a biomarker, to be clinically relevant, additional issues must also be addressed and biomarker must be
able to meet one of the following criteria:
(i) show specificity for a particular disease (diagnostic),
(ii) have prognostic value, and
(iii) correlate with disease activity.
(iv) be reasonably stable
(v) present in an easily accessible tissue
(vi) cost-effective to measure reproducibly on a large scale.
This then allows treatment efficacy to be assessed. 1199
One of the basic understanding of the study of the human genome was that by identifying molecular
defects that give rise to the spectrum of abnormalities that comprise pre-cancers/cancers and other human
diseases will lead to improved disease management by early diagnosis, development of the new
therapeutic strategies and enhanced prognosis .404
A variety of genetic and molecular signatures of precancerous lesions and OSCC identified as tumor
biomarkers. However, these biomarkers have, so far, not gained any use in routine diagnosis and risk
assessment of oral cancer. Malignant transformation or progression of OSF requires multiple genetic
alternations and involves a variety of stromal cells, such as macrophages, in the disease microenvironment.
Therefore, identification of molecular markers that can predict disease progression and techniques that can
readily use such biomarkers in routine practices will improve the management of these disorders. Although
the term “potentially malignant disorders” was recommended by the WHO to describe precancerous
lesions, the clinical and histological features alone cannot accurately predict whether these precancers of
the oral mucosa remain stable, regress or progress to malignancy. Recent progress in high-throughput
techniques, including the profiling of gene expression (microarray), detection of single-nucleotide changes
of preselected genes, miRNA analysis, and salivary proteomics, will greatly promote identification and
development of novel biomarkers. From the identification of a promising biomarker to its clinical use, there
is a long pathway involving many difficult hurdles, such as estimating the number of patients needed for
the validation phase and statistical validation, among others. This validation and qualification are
responsible for linking the promising biomarker with a biological process to clinical endpoints. 396,397,398
Genomic-based diagnostics can play a key role in creating a more efficient healthcare system by directing
patients toward beneficial therapies and away from therapies that pose a substantial risk or are unlikely to
improve outcomes for the patient.402
Biomarkers are used to identify the transformation in the physiology of a patient associated with the risk or
progression of a disease or the susceptibility of a disease to a given treatment. Biomarkers maintain a great
promise for personalized medicine since the information obtained from diagnostic markers or progression
can be used to adapt the therapy to the individual for highly effective intervention in the process of the
disease.
As Sanjit Mukherjee et al. 4 mentions “At present, one of the most significant challenges to oral
oncobiologists is to determine and identify the degree of tissue damage or stages of various precancerous
states of oral tissue and to detect the exact transition of healthy tissue to precancerous state. In this
current scenario with improved immunohistochemical techniques and morphometric analysis of
histopathological images may go hand in hand to provide better diagnosis and early detection of oral
cancer.” Moreover, upcoming novel biomarkers and imaging technologies will soon permit a more exact
and efficient assessment of disease diagnosis, disease progression, and disease regression. 4
The treatment options available at present for OSF are either conservative methods or more aggressive
surgical methods. Historically, the treating clinicians have labored to join the anatomic and morphologic
considerations of a patient’s disease (and patient preference and comorbidities) to select the appropriate
range of treatment options. The additional burden has been added for the already thwarted clinicians to
consider a new set of issues—the so-called ‘‘molecular determinants’’ of OSF.
Biomarkers are becoming a foundation for information-based medicine in determining who should be
treated, how and with what. For biomarkers to reach their full potential, alone or in combination, they
must undergo their development process. Biomarker development has lagged significantly behind
therapeutic growth because of scientific, economic and regulatory factors. 5 It is crucial to accelerate
biomarker development and to help biomarkers advance in parallel with therapeutics, but, they must be
tested with established therapies.6
The question arises here why do we need biomarkers? We need to have biomarkers since only 5% of the
Oral Premalignant Disorders ever progress to frank OSCC. The current clinicopathologic characteristics,
while associated with the risk of progression, do not have predictive accuracy. Biomarkers are utilized for
accurate identification of progressive and non-progressive lesions.
Since the development of the so-called omics technology, thousands of supposed biomarkers have been
identified and published, which have dramatically increased the opportunities to develop more effective
therapies. These developments have tremendous benefits for the patients and the health economy.
However, the path of transfer of biomarkers from the bench ( Laboratory ) to bedside ( Clinical Practice ) is
covered with plenty of obstacles. To put a potential biomarker into clinical practice, it needs to be
evaluated using an adequate number of specimens and be reproducible, specific and sensitive. In addition
to the lack of quality in biomarker validation, some other critical issues can be identified and should be
addressed. 7
Although the application of potential biomarkers in preclinical development is very advanced, only a
handful have passed the clinical trials and are already marketed successfully in the market. The reasons for
the pitfalls are multiple, including complex validation strategies and slow disease progression, requiring a
high number of well-stratified patients to undergo long-term treatment when conventional or related
diagnostic parameters are used endpoints. It is important to note that there is a notorious lack of sensitive
and specific substitute biomarkers for the progression or regression of the disease that would allow rapid
clinical detection of potential drug candidates. 7
However, given the urgent need for new drugs that positively impact the morbidity and mortality of
different conditions, the field of biomarkers is moving faster toward clinical translation. This development
is led by reflective preclinical validation, better study design and improved surrogate readings using
currently available methodologies. Also, upcoming biomarkers and imaging technologies will soon allow for
a more accurate and efficient assessment of disease diagnosis, illness progression, and disease regression. 7
Many diseases give rise to specific and appropriate changes in the chemical and biochemical profiles of
biological and tissue fluids before the development of clinical symptoms. These changes are often useful
biomarkers diagnostics and prognoses. The recognization of biomarkers that can be used for early
detection of cancer will result in more effective treatments, reduced suffering, and lower mortality rates.
An ideal screening test should be non-invasive with high sensitivity and specificity. The analysis of
proteomics and metabolomics of biological samples may reveal changes in the abundance levels of
metabolites and proteins that when validated and confirmed through clinical trials can function as clinical
tests for the detection Early diagnosis, monitoring of disease progression and prediction of therapeutic
response. While in the past decade the great strides have been made in the proteomics and metabolomics
research producing potential biomarkers for cancer, most identified biomarkers have not replaced existing
clinical trials. A search for scientific and medical literature indicates that many studies report the discovery
of potential biomarkers without proper validation and do not meet the above criteria. 8
In this chapter, we will be studying, in details, each biomarker associated with OSF and its malignant
transformation. Considering the malignant potential of OSF, we have added biomarkers associated with
OSCC in our discussions to make the picture complete. However, before we go into details of biomarkers,
we need to understand some of the basics of molecular biology and biomarkers.
HISTORY OF BIOMARKERS:
The first ever reported cancer biomarker was the light chain of immunoglobulin in the urine, identified in
75% of myeloma patients, in a study of 1848. The test for this marker is still used by clinicians today but
with the help of modern quantification techniques.
From 1930 to 1960, the researchers recognized several hormones, enzymes, and other proteins, whose
concentration changed in biological fluids from cancer patients. The modern era of monitoring the
malignant disease, however, began in the decade of 1960 with the discovery of alpha-fetoprotein and
carcinoembryonic antigen (CEA), which was facilitated by the introduction of immunologic techniques such
as the Radioimmunoassay.
In the year 1980s, the era of Hybridoma technology allowed the development of ovarian epithelial cancer
marker (CA) carbohydrate antigen. In 1980, the prostate-specific antigen (PSA [KLK3]), considered one of
the best cancer markers, was discovered.
Each era of biomarker discovery appears to be closely associated with the emergence of new and powerful
analytical technology. The past decade has witnessed impressive growth in the field of large-scale, high-
performance biology, which has contributed to an era of developing new technologies. The completion of
some genomic sequencing projects, the discovery of oncogenes and tumor suppressor genes, and the
recent advances in genomic and proteomic technologies, together with powerful bioinformatics tools, will
have a direct impact and essential in the way the search for cancer biomarkers is done. The earliest
discoveries of cancer biomarkers were based mainly on empirical observations, such as the overexpression
of CEA. Modern technologies can perform parallel analyses rather than in series, and can help identify
distinctive patterns and multiple markers rather than a single marker; Such strategies represent a central
component and a paradigm shift in the search for new biomarkers.
Notable landmarks in the history of Biomarkers Development are given in the following table.
HISTORICAL LANDMARKS IN THE DEVELOPMENT OF BIOMARKERS
YEAR FIGURES LANDMARKS
1847 Henry Bence Jones FRS (31 December 1813 – 20 April

1873) was an English physician and chemist.
In 1847, he described the Bence Jones protein, a globulin
protein found in blood and urine, suggestive of multiple
myeloma or Waldenström's macroglobulinemia.
(Wikipedia) 1400
REF: Jones HB (1848). "On a new substance occurring in

the urine of a patient with mollities
ossium." Philosophical Transactions of the Royal
Society. 138: 55–62. doi:10.1098/rstl.1848.0003
1954 Dr. Karmen's signal contribution to the medical field was
proving that cardiac damage, and the extent of it, could
be indirectly assessed by testing for specific enzymes
released from the heart when it is subjected to injury,
such as a heart attack. (Albert Einstein College of
Medicine)
REF:
1.KARMEN, A; WROBLEWSKI, F; LADUE, JS (January
1955). "Transaminase activity in human blood". The
Journal of Clinical Investigation. 34 (1): 126–
31. doi:10.1172/jci103055. PMC 438594. PMID 13221663
.
2. KARMEN, A (January 1955). "A note on the
spectrometric assay of glutamic-oxalaceticñnn
transaminase in human blood serum". The Journal of
Clinical Investigation. 34 (1):
131–. doi:10.1172/JCI103055. PMC 438594. PMID 132216
64
The The term “biomarker” started to appear in the literature

1960s in connection with metabolites and biochemical
abnormalities associated with several diseases. 1012
1967 Compared with other enzyme tests for myocardial

infarction diagnosis, creatine kinase has the advantage of
earlier increases, higher sensitivity, and higher specificity.
It invariably shows a rapid rise in serum in the early hours
after admission with chest pain from infarction. 1398
REF:
1] Rosalki SB. An improved procedure for serum creatine
phosphokinase determination. J Lab
ClinMed 1967;69:696-705.
2] Rosalki SB. Enzyme assays in diseases of the heart and
skeletal muscle. J Clin Pathol 1971;24(Suppl 4):60-70.
3] Collinson PO, Rosalki SB, Flather M, Wolman R, Evans
T. Early diagnosis of myocardial infarction by timed
sequential enzyme measurement. Ann Clin
Biochem1988;25:376-382
1971 Dr. Gold’s discovery of the CEA, along with the
description of alpha-fetoprotein at about the same time,
ushered in the modern era of human tumor marker
research along with the broad ramifications that this
work has had over the past five decades. (American
Association of Clinical Chemistry. 2004 EDWIN F.
ULLMAN AWARD)
REF:
1] Gold P, Goldenberg NA. The carcinoembryonic antigen

(CEA): past present, and future. Perspect Colon Rectal
Surg 1996;9:1–46.
2] Gold P, Freedman SO. Demonstration of tumor-specific
antigens in human colonic carcinomata by immunological
tolerance and absorption techniques. J Exp
Med 1965;121:439–62.
3]Gold P, Freedman SO. Specific carcinoembryonic
antigens of the human digestive system. J Exp
Med 1965;122:467–81.
1987 , Troponin I as a biomarker for myocardial infarction

(Cummins et al. 1987). Troponin T was discovered by the
German physician Hugo A. Katus at the University of
Heidelberg. He also developed the troponin T assay. 1398
REF:
1] Katus HA, Looser S, Hallermayer K, Remppis A,

Scheffold T, Borgya A, et al. Development and in vitro
characterization of a new immunoassay of cardiac
troponin T. Clin Chem 1992;38:386 –93
Early Accelerator mass spectrometry used for the analysis of

1990s biological samples for biomarkers. Accelerator mass
spectrometry (AMS) is a form of mass spectrometry that
accelerates ions to extraordinarily high kinetic
energies before mass analysis. Accelerator mass
spectrometry is widely used in biomedical research.
(Wikipedia) 1400
REF:
1] Brown, K.; Dingley, K. H.; Turteltaub, K. W.
(2005). Accelerator mass spectrometry for biomedical
research. Methods in Enzymology. 402. Pp. 423–
443. doi:10.1016/S0076-6879(05)02014-8.
2] Vogel, J. S. (2005). "Accelerator mass spectrometry for
quantitative in vivo tracing." BioTechniques. 38 (S6):
S25–S29. doi:10.2144/05386SU04.
3] Palmblad, M.; Buchholz, B. A.; Hillegonds, D. J.; Vogel,
J. S. (2005). "Neuroscience and accelerator mass
spectrometry." Journal of Mass Spectrometry. 40 (2):
154–159. Bibcode:2005JMSp...40..154P. do
i:10.1002/jms.734
1995 Applications of proteomics for discovery of biomarkers

and use in molecular diagnostics.1012 Proteomics is the
large-scale study of proteins. Marc R. Wilkins is an
Australian scientist who is credited with defining the
concept of the proteome. Wilkins is a Professor in the
School of Biotechnology and Biomolecular Sciences at
the University of New South Wales, Sydney
(Wikipedia)1401
1999 The emergence of metabolomics for the study of

biomarkers. Metabolomics is the scientific study of
chemical processes involving metabolites, the small
molecule intermediates, and products of metabolism.
The term "metabolic profile" was introduced by
Horning, et al. in 1971 after they demonstrated that gas
chromatography-mass spectrometry (GC-MS) could be
used to measure compounds present in human urine and
tissue extracts. (Wikipedia)
2000 Sequencing of the human genome completed opening

the way for the discovery of gene biomarkers. 1012 The
Human Genome Project was a 15-year-long, publicly
funded project initiated in 1990 to determine the DNA
sequence of the entire euchromatic human genome
within 15 years
2005 Discovery and application of biomarkers become a
significant activity in the biotechnology and
biopharmaceutical industries. 1012
REF: 1081] Sanjay Reddy B, Madhavi Reddy B, and Shyam NDVN. 2010. “Tumor Markers in Oral
Neoplasia.” IJDA. Vol. 2. http://rep.nacd.in/ijda/pdf/2.1.103.pdf.
DEFINITION OF BIOMARKERS:
In simplest terms “Biomarkers (short for biological markers) are biological measures of a biological state.”
Hulka and his colleagues1 have defined biomarkers as “cellular, biochemical or molecular alterations that
are measurable in biological media such as human tissues, cells, or fluids.” More recently, the definition has
been broadened to include “biological characteristics that can be objectively measured and evaluated as an
indicator of normal biological processes, pathogenic processes, or pharmacological responses to a
therapeutic intervention”.2
The ‘biomarker,’ has been defined in various ways. The classic definition, as given by S. Naylor 2, is that “A
biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a
physiological as well as a pathological process or pharmacological response to a therapeutic
intervention.”Biomarkers help not only in disease diagnosis but also in tracking progression, regression,
and outcome after intervention.2
The National Research Council - USA (1989), defined a biomarker as “a change induced by a contaminant in
the biochemical or cellular components of a process, structure or function that can be measured in a
biological system.” A primary concept of this approach is based on the intercorrelability of the effects of a
contaminant at different levels of structural organization (enzyme activity, cells, tissues, organism a.o.). The
homeostatic responses to the chemical injury are potential biomarkers. 1014
According to Yasser M. Moustafa and Rania E. Morsi89 “Biomarkers are naturally occurring, omnipresent
and stable complexes that are objectively measured and evaluated as an indicator of a particular state. The
biomarkers are useful in many scientific fields; medicine, cell biology, exposure assessment, astrobiology,
geology, and petroleum industry.” 89
According to the National Cancer Institute, a biomarker is “a biological molecule found in blood, other body
fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease,” such as
cancer. 1075
A tumor marker is defined as a substance present in or produced by a tumor or by the tumor’s host in
response to the tumor’s presence that can be used to differentiate a tumor from the healthy tissue or to
determine the presence of cancer-based on measurement in the blood or secretions. 1076
Tumor markers can also be defined as “specific novel or structurally altered cellular macromolecules or
temporarily spatially or quantitatively altered normal molecules that are associated with malignant (and in
some cases benign) neoplastic cells. 1077
Cellular products that are abnormally elaborated by malignancies that can be detected in various body
fluids and on the surface of the cancer cells. 1078
Tumor markers can also be broadly defined as “biological or molecular attributes of tumor cells that
distinguish them from healthy cells. 1079
CLASSIFICATION OF BIOMARKERS:
Biomarkers are classified in various ways.
[1] There are two major types of biomarkers: biomarkers of exposure, which are used in risk prediction,
and biomarkers of disease, which are used in screening and diagnosis and monitoring of disease
progression.
1) Biomarkers of Exposure Or Antecedent Biomarkers
2) Biomarkers of Disease
[2] Biomarkers can also be categorized as pharmacodynamic, prognostic or predictive 8

1) Pharmacodynamic biomarkers indicate the outcome of the interaction between a drug
and a target, including both therapeutic and adverse effects.
2) Prognostic biomarkers are defined as markers indicating the probable course of a disease in a non-
treated person; They can also be described as markers suggesting the probable outcome of illness
regardless of the treatment
3) Predictive biomarkers propose the population of patients who are likely to respond to a particular
treatment.
The Biomarkers and Surrogate End Point Working Group 90 has defined a classification system that can be
used for biomarkers 91:
1. Type 0 consists of disease natural history biomarkers that correlate with clinical indices;
2. Type I tracks the effects of intervention associated with drug mechanism of action;3. Type II consists of
surrogate endpoints that predict clinical benefit.
The biomarkers have a full spectrum analysis. Biomarkers can take many different forms. Some of the types
of biomarkers are :
1] proteins or peptides (e.g., prostate-specific antigen as an indicator of increased risk for prostate cancer),
2] antibodies (e.g., anti-citrullinated protein antibodies for rheumatoid arthritis),
3]cell types (e.g., white blood cell counts in infection or cancer),
4]metabolites (e.g., phenylalanine in the urine of newborns with phenylketonuria),
5] lipids (e.g., cholesterol and other lipid levels in cardiovascular disease),
6] hormones (e.g., thyroid stimulating a hormone in Hashimoto's Disease),
7] enzyme levels (e.g., various hepatic enzymes for liver cancer),
8] physiological states such as blood pressure or fever,
9] imaging studies of particular organs or organ systems (e.g., neural degeneration in Parkinson's Disease).
10] A biomarker is introduced into a patient to assess the functions of the internal organ systems such as
radioactive iodine used to measure thyroid function.
An ideal tumor marker theoretically should have the following criteria: 1080
1. It should be highly sensitive and should have low false negatives.
2. It should be highly specific and should have low false positive.
3. It should have high positive and negative predictive value.
4. 100% accuracy in differentiating between healthy individuals and tumor patients.
5. It should be able to differentiate between neoplastic and non-neoplastic disease and show a positive
correlation with tumor volume and extent.
6. It should predict early recurrence and have prognostic value.
7. It should be clinically sensitive, i.e., detectable at an early stage of the tumor.
8. Its levels should be preceding the neoplastic process so that it should be useful for screening early
cancer.
9. It should be either a universal marker for all types of malignancies or specific to a kind of malignancy.
10. It should be easily assayable and be able to indicate all changes in cancer patients receiving
treatment.
REF: 1080] Malati, T. 2007. “TUMOUR MARKERS : AN OVERVIEW.” Indian Journal of Clinical Biochemistry.
2007 / 22 (2) 17-31
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3453798/pdf/12291_2008_Article_BF02913308.pdf.
Malati’s classification: 1080

1. Oncofetal antigens (e.g., alpha-fetoprotein [AFP], carcinoembryonic antigen [CEA], pancreatic oncofetal
antigen, and fetal sulfoglycoprotein)
2. Tumor-associated antigens/cancer antigens, for example, CA125, CA19-9, CA15-3, CA72-4, and CA50
3. Hormones, for example, beta human chorionic gonadotropin, calcitonin, and placental lactogen
4. Hormone receptors (e.g., estrogen and progesterone receptors)
5. Enzymes and isoenzymes (e.g., prostate-specific antigen [PSA], prostatic acid phosphatase [PAP],
neuron-specific enolase [NSE], glycosyl transferases, placental alkaline phosphatase [PALP], terminal
deoxy nucleotidyl transferase, lysozyme, alpha amylase)
6. Serum and tissue proteins (beta-2 microglobulinmonoclonal immunoglobulin/para proteins, glial
fibrillary acidic protein [GFAP], protein S-100, ferritin, and fibrinogen degradation products)
7. Other biomolecules, for example, polyamines
REF: 1080] Malati, T. 2007. “TUMOUR MARKERS : AN OVERVIEW.” Indian Journal of Clinical Biochemistry.
2007 / 22 (2) 17-31
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3453798/pdf/12291_2008_Article_BF02913308.pdf.
REF: 1081] Sanjay Reddy B 1, Madhavi Reddy B 2, and Shyam NDVN. 2010. “Tumor Markers in Oral
IMMUNOHISTOLOGICAL MARKERS MARKERS FOR TUMOUR PROGNOSTICATION: 1081
1. Markers to predict response to therapy: A.] Oestrogen and progesterone receptors. B.] Androgen
receptors. C.] Steroid-regulated proteins- Cathepsin D. and pS2 D.] c-erbB-2 Gene
2. Markers to monitor drug resistance: - P-glycoprotein (a transmembrane protein)12,13 - c-erbB-2
3. Growth factors and receptors: Epidermal growth factor receptors, Erb-2 oncoprotein, insulin, and
insulin-like growth factor receptors, transforming growth factor - receptors, fibroblast growth factor
receptors and the somatostatin receptors.
4. Tumour angiogenesis: Microvascular density is an independent marker of prognostic relevance.
5. Tumour growth fraction: Ki 67 ANTIBODY
Proliferating Cell Nuclear Antigen (PCNA) & P27 KiP1 Gene

6. Tumour suppressor genes: p53 tumour suppressor gene and Retinoblastoma susceptibility suppressor
gene
7. Anti-apoptosis genes: bcl-2
8. Nm23 Anti-metastasis gene: The nm23 gene family was initially identified in a murine melanoma cell
line, and nm23-HI was found to be transcribed at a 10-fold higher rate in cells of lower metastatic
potential.
9. DNA Repair genes- microsatellite instability (MSI): The human genome is punctuated with an
enormous number of short repetitive nucleotide sequences known as microsatellites. They are less
likely to be associated with lymphatic and distant metastasis, and the improved prognosis applies even
they are stratified by stage.
10. Miscellaneous markers: K-ras and c-myc oncogenes, transforming factors TGF-a, TGG-b, adhesion
proteins E-cadherin and CD 44, and the matrix metalloproteases and inhibitors and others.
Schliephake’s classification: 1082

[A] Tumor growth markers
a. Epithelial growth factor (EGF)
b. Cyclin
c. Nuclear cell proliferation antigens
d. Argyrophilic nucleolar organizer region (AgNORs)
e. S-phase kinase-interacting protein 2
f. HSP 27 and 70 (heat shock protein)
g. Telomerase.
[B] Markers of tumor suppression and antitumor response
a. Retinoblastoma protein (pRb)
b. Cyclin-dependent kinase inhibitors
c. p53
d. bax
e. Fas/FasL.
[C} Angiogenesis markers
a. Vascular endothelial growth factor/receptor
b. Platelet-derived endothelial cell growth factor
c. FGFs.
[D] Markers of tumor invasion and metastatic potential
a. Matrix metalloproteases
b. Cathepsins
c. Cadherins and catenins
d. Desmoplakin.
[E] Cell surface markers
a. Carbohydrates
b. Histocompatibility antigen
c. CD57 antigen.
[F} Intracellular markers
a. Cytokeratins.
[G] Markers of anomalous keratinization
a. Filaggrin
b. Involucrin
c. Desmosomal proteins
d. Intercellular substance antigen
e. Nuclear analysis.
[H] Arachidonic acid products
a. Prostaglandin E2
b. Hydroxyeicosatetraenoic acid
c. Leukotriene B4
d. Enzymes
e. Glutathione S-transferase.
Classification of Speight and Morgan: 1082

• Proliferative markers: PCNA, Ki67, BrU, histones, and AgNORs
• Genetic markers: Ploidy
• Oncogene: C-myc
• Tumor suppressor markers: P53 mutations
• Cytokines
• Blood group antigens
• Integrin extracellular matrix ligands.
Waxman’s classification: 1084

1. Oncofetal antigens
a. AFP
b. CEA.
2. Hormone catecholamines
a. Calcitonin
b. B-hCG.
3. Glycoproteins
a. CA125
b. CA15-3
c. CA19-9
d. CA72-4
e. PSA.
4. Metabolites
a. Vanillylmandelic acid
b. Hydroxy indoleacetic acid.
5. Tumor-associated antigen
a. Viral antigens – Polyoma, SV40
b. MHC-related antigens – H-2k antigen
c. Enzymes – PAP, NSE, PLAP
d. Oncogene products – c-myc, c-erbB2
e. Cytogenetic products – Philadelphia.
6. Tumor-associated markers
a. Proteins – Immunoglobulins, b-2M
b. Enzymes – Lactate dehydrogenase, alkaline phosphatase, pteridines, pterins
c. Acute-phase proteins – C-reactive protein, ferritin d. Inflammatory markers – Erythrocyte
sedimentation rate (ESR), viscosity.
7. Ultrastructural components
a. Intermediate filament components – Desmin, vimentin AFPCEA, ESR, NSE, PAP, PLAP, PSA, Simian
virus.
Manikantan et al’s.Classification: 1079

1. Epithelial markers
a. Cytokeratins
b. Epithelial membrane antigen

c. Oncofetal antigens
i. AFP
ii. CEA.
d. Desmoplakin.
2. Mesenchymal markers
a. Muscle antigens
i. Desmin
ii. Actin
iii. Myoglobin
iv. Myosin.
b. Vasculator antigens
i. CD34
ii. CD31.
c. Neural antigens
iii. S100
iv. NSE
v. GFAP
vi. Synaptophysin
vii. Nerve growth factor receptors.
3. Prognostic markers
a. Cell adhesion molecules
i. Cadherins
ii. Integrins
iii. Selectins.
b. Proliferative markers
i. PCNA
ii. Ki67
iii. AgNORs.
4. Biochemical markers
a. Enzymes and isoenzymes
i. PAP
ii. PSA
iii. PALP.
iv. Lysozyme.
b. Protein
i. Ferritin
ii. Glycoprotein
iii. Beta-protein
iv. Immunoglobulins.
c. Hormone receptors
i. Estrogen receptor
ii. Progesterone receptor
d. Epithelial markers
REF: 1079] Manikantan, NS, Dhanya Balakrishnan, Ad Manoj Kumar, and Brijesh Shetty. n.d. “Tumor
Markers: At a Glance. Oral and Maxillofacial Pathology Journal” 5 (1): 437–40. Accessed September 13,
2018. https://doi.org/10.5005/jp-journals-10037-1006.
BIOMARKERS CLASSIFICATION 1117
Three main categories (US-NCI):
1] Prognostic biomarkers
2] Predictive biomarkers
3] Pharmacodynamic biomarkers
1] Prognostic biomarkers
A] Correlate with clinical outcome and allow prediction of the natural course of cancer
C] Guide the therapeutic decision
They include:
1. Biological progression markers
2. Risk biomarkers (or screening biomarkers)
A. Prognostic biomarkers
1.Biological progression markers
Measures of tumor activity (CEA, alphaFP, PSA, CA-125, hCG)
2. Risk biomarkers (or screening biomarkers)

Describe the risk of cancer occurrence or cancer progression because they are implicated in
neoplastic progression and include:
A] Carcinogen exposure
B] Genetic predisposition (e.g., BRCA1/2 mutations)
C]Over expression of genes (e.g., PTEN, BCR-ABL, HER-2/neu, RAS, AKT)
D] Pharmacogenomicparameters (genetic polymorphisms, DNA methylation, aneuploidy)
E] Environmental factors and lifestyle (e.g., HPV or HBV infection, tobacco use)
F] Multifactorial risk models (e.g., Gail model, OncotypeDX, MammaPrint)
B. Predictive biomarkers
Correlate with the probability of clinical response to treatment:

1] Amplification of HER-2 gene in breast cancer for treatment with anti-HER-2
2] ER/PgR expression in breast cancer for hormone therapy
3] Presence of Philadelphia chromosome (BCR-ABL fusion gene) in leukemia for anti-BCR-ABL
4] Mutations of EGFR in lung tumors for EGFR inhibitors
5] Extended RAS mutation wildtype(exons 2, 3 and 4 of KRAS and NRAS) in colorectal cancer for
EGFR monoclonal antibodies
6] BRAF V600E mutations in melanoma for BRAF inhibitors
7] EML4/ALK translocations in lung cancers for ALK inhibitors
C. Pharmacodynamic biomarkers
Correlated to biological and clinical effects of the drug on the tumor
It is measured in tumor biopsies or healthy tissue including platelet-rich plasma, hair follicles,
circulating tumor cells, saliva, or urine.
They could be:

1] Lower expression or activity of a molecular target (e.g., EGFR expression)
2] Decrease in phosphorylation signal of target (e.g. phosphoSer473 AKT)
3] Induction of metabolism (e.g., interference with cytochrome P-450)
4] Interference with cell growth processes (e.g., Ki-67, BCL-2, cytokeratins)
5] Vascularization and metabolism (imaging biomarkers)
“Proximal” and “Distal” biomarkers
1] Proximal: reflect the immediate effect of the drug on its target (e.g., decrease in a protein
substrate of phosphorylation downstream the target kinase such as the phosphorylation of the AKT
substrate PRAS40 to reflect the effect of an AKT inhibitor on AKT)
2] Distal: reflect the effect of the drug downstream the molecular target (e.g., Ki-67 expression for a
reduction in proliferation)
BIOMARKERS AND HUMAN BIOMONITORING (WHO 1118)
“The Matrix” (The body fluid or tissue to be tested)
1] Blood
2] Urine
3] Breast milk
4] Expelled air
5] Hair
6] Nails
7] Saliva
8] Teeth
9] Meconium
9] Amniotic fluid
10] Adipose tissue
11] Other tissues and fluids
BIOMARKERS AND HUMAN BIOMONITORING
(Human biomonitoring is a scientific technique for assessing human exposures to environmental

agents and their effects, based on sampling and analysis of an individual's tissues and fluids)
ADVANTAGES:
1] Confirms absorption into the human body
2] Measures integrated exposure
3] Very low-level exposures detectable
4] Helps to test and validate exposure models
5] Helps to follow exposure trends
6] Helps to evaluate public health interventions
DISADVANTAGES:
1] Does not define sources, pathways or duration of exposure
2] Cannot define the toxic dose
3] Susceptible to inferior or unscrupulous analytical laboratories
4] Lack of meaningful reference levels
5] Lack of toxicological and epidemiological information about the vast majority of environmental
chemicals
•
Disease Diagnostic Biomarkers to

biomarkers Translation
biomarkers: type 0 detect drug effects:
biomarkers biomarker
Molecular
type I biomarkers
diagnostics, e.g.,
CA-125 for
Clues to ovarian cancer
pathomechanism
of a disease A biomarker that can
Efficacy biomarker:
Pattern diagnosis, be applied in both a
indicator of beneficial
e.g., serum preclinical and a
Diagnostic protein biomarker effect of a drug
biomarkers: early
clinical setting
pattern diagnosis
detection of of ovarian cancer
disease
Prognostic
biomarker for
Biomarkers as Mechanism
links between biomarker: reports a
prognosis or diagnostics and
outcome of disease
theraPattern downstream effect of
diagnosis, e.g., a drug
Tracking disease serum protein
progression over time biomarker
pattern diagnosis
of ovarian
cancerpeutics Toxicity biomarker:
reports toxicological
effect of a drug in an
in vitro or an in vivo
system
Biomarkers as Biomarkers
Predictive
Valid for drug
surrogate end discovery
biomarkers
points in clinical biomarkers:
trials: type II validated in Target
biomarker:
Biomarker
associated
biomarkers clinical trials reports with a risk for
interaction of disease as a
the drug with candidate for a
its target screening test
As a substitute Disease
measure for biomarkers as
clinical outcome, targets for drug To predict disease
e.g., cholesterol discovery at presymptomatic
levels in statin stage:
autoantibodies
therapy
To predict the
In vivo imaging as effect of a drug
on disease
end point: MRI of
multiple sclerosis
lesions in To predict the
interferon toxicity of a
drug
therapy
CLINICAL BIOMARKERS: CATEGORIES AND TYPES
REF: Drucker and Krapfenbauer: Pitfalls and limitations in translation from biomarker discovery to clinical
utility in predictive and personalized medicine. The EPMA Journal 2013 4:7 doi:10.1186/1878-5085-4-7 4
Term Application
Predisposition biomarker To identify predisposition to a disease, e.g., genetic
Screening biomarkers To identify those suffering from a disease
Staging biomarker To determine the stage of progression of the disease
Prediction biomarker To predict the course of the disease
Prognostic biomarker To assess disease progression and outcome
Recurrence monitoring biomarkers To identify the recurrence of the disease
The terminology of biomarkers of disease

relevant to clinical development
REF:1012] Jain, Kewal. (2010). The Handbook of Biomarkers Regulatory Issues. 443-457. DOI: 10.1007/978-
1-60761-685-6_10. Springer, NY.
APPLICATION AND LIMITATIONS OF BIOMARKERS:
In oncology, biomarkers have many potential uses. These are
1] risk assessment,
2] Screening,
3] Differential diagnosis,
4] Determination of prognosis,
5] Prediction of response to treatment, and
6] Monitoring of progression of the disease.
According to Richard Mayeux3 "The biomarkers provide an aggressive and dynamic approach to
understanding the spectrum of various diseases with utilization in observational and analytical
epidemiology, randomized clinical trials, Research and diagnosis, and prognosis of the disease process.
Defined as alterations in tissue components or body fluids, these markers offer the means for the
homogeneous classification of disease and the risk factors and can extend our base information on the
pathogenesis of an underlying illness. Biomarkers can also reflect the entire disease spectrum from the first
manifestations to the final stages.” 3
Biomarkers are any measurable characteristics of an organism that indicate a particular physiological state.
In medicine, biomarkers are often compounds isolated from serum, urine, or other fluids that can be used
as an indicator of the presence or severity of a particular disease state. Biomarkers can also be used to
assess the effectiveness of specific therapies in ameliorating the effects of a disease. By using readily
obtained and assayed biomarkers to monitor a patient's reaction to a particular drug, it is possible to
determine whether treatment is sufficient for that individual by measuring drug response rate or toxic
effects associated with the drug. This information could eventually lead to earlier detection of adverse drug
response, reducing the number of expensive laboratory tests – and possibly other medical interventions –
necessary to adjust the proper dosage of a drug.
Biomarkers are essential for the diagnosis, prognosis and monitoring severity of the disease. Biomarkers
play an indispensable role in advancing new pharmacological therapies through the discovery of "drug
targets." In addition to identifying drug targets, biomarkers have the potential to accelerate the
development of new disease therapies by using "progression" markers to delineate the development and
course of a disease. Researchers can use changes in progression markers to understand whether and how a
new therapy is slowing down successfully – or even reversing – the disease process. The results of studies
such as these will allow researchers to focus efforts and resources on the most effective therapies, reducing
time and cost to bring a new treatment to the market and eventually to the patient.
According to Schulte 4 contributions of valid biomarkers in Clinical Research are as follows:
● Delineation of events between exposure and disease

● Establishment of dose-response
● Identification of early events in the natural history
● Identification of mechanisms by which exposure and disease are related
● Reduction in misclassification of exposures or risk factors and disease
● Establishment of variability and effect modification
● Enhanced individual and group risk assessments
REF: Gouse Mohiddin Shaik. The Introduction to Biomarkers. Slideshare 1013
According to Thariani Rahber et al. 402 Genomic-based diagnostics can play a crucial role in creating a more
efficient healthcare system by directing patients toward beneficial therapies and away from treatments
that pose a substantial risk or are unlikely to improve outcomes for the patient. The authors have outlined
how the value provided by diagnostics is closely linked to a range of factors including magnitude of health
outcome improvement, avoiding the adverse effect, diagnostic parameters, the process of care, resource
utilization, and costs.
According to Wilson, Jennifer L, and Russ B Altman. 1705 Precision medicine evolved because of the
understanding that human disease is molecularly driven and is highly variable across patients. This
understanding has made biomarkers, a different class of biological measurements, more relevant for
disease diagnosis, monitoring, and selection of treatment strategy. Biomarkers’ impact on precision
medicine can be seen in cancer, pharmacogenomics, and safety. The successes in these cases suggest many
more applications for biomarkers and a more significant impact for precision medicine across the spectrum
of human disease. The authors assess the status of biomarker-guided medical practice by analyzing themes
for biomarker discovery, reviewing the impact of these markers in the clinic, and highlight future and
ongoing challenges for biomarker discovery. This work is timely and relevant, as the molecular, quantitative
approach of precision medicine is spreading to many disease indications.1705
Richard Mayeux 3 believe that Biomarkers provide a dynamic and powerful approach to understanding the
spectrum of diseases with applications in observational and analytic epidemiology, randomized clinical
trials, screening and diagnosis, and prognosis. Defined as alterations in the constituents of tissues or body
fluids, these markers offer the means for homogeneous classification of a disease and risk factors, and the
can extend our base information about the underlying pathogenesis of the disease. Biomarkers can also
reflect the entire spectrum of disease from the earliest manifestations to the final stages. This brief review
describes the significant uses of biomarkers in clinical investigation. Careful assessment of the validity of
biomarkers is required concerning the stage of the disease. Causes of variability in the measurement of
biomarkers range from the individual to the laboratory.3
Various components must be integrated to study the molecular basis of human pre-cancer and cancer.
Several host factors contribute to tumorigenesis in humans, including diet, environmental factors,
polymorphisms and mutations in susceptibility genes, age and immunity. Cells undergo genomic changes
(DNA mutations and repair, methylation, amplification, deletions, and rearrangements), leading to
tumorigenesis. Tumor development also depends on factors in the microenvironment — some of these are
produced locally, whereas others are generated systemically (growth factors, infiltrating cells and
cytokines). Reciprocal interactions between the premalignant and malignant cells, stromal cells,
extracellular matrix components, many inflammatory cells and a range of soluble mediators, therefore,
contribute to tumor development and progression. Once tumor samples are obtained, genomic,
transcriptomic and proteomic tools can be used to profile specific compartments.3
PITFALLS AND FAILURES IN BIOMARKERS IDENTIFICATION
REF: Drucker Elisabeth and Krapfenbauer Kurt: Pitfalls and limitations in translation from biomarker
discovery to clinical utility in predictive and personalized medicine. The EPMA Journal 2013 4:7
doi:10.1186/1878-5085-4-7 4
REF: Global Oral Cancer Forum. Henry Schein Cares.
Biomarkers have a significant role to play during the biological process of development of oral submucous
fibrosis from the normal oral mucosa and subsequent malignant transformation of OSF and complications
associated with this transformation. The whole biological behavior of OSF can be summarized into the
following four stages:
1] Development of OSF: Biomarkers involved in the conversion of normal oral mucosa into the fibrosed oral
mucosa.
2] Malignant transformation of OSF: Biomarkers involved in the malignant transformation of OSF into Oral
Sq. Cell Carcinoma.
3] Development and progress of OSCC: Biomarkers engaged in the development of frank OSCC and its
further growth.
4] Metastasis of OSCC: Spread of OSCC to local and distant tissues.
Each of these stages shows distinct histopathological alterations and biomarkers associated with these
alterations. Different mechanisms and molecules are proposed in the pathogenesis of OSMF, to name a
few, growth factors and inflammatory cytokines, reactive oxygen species, matrix metalloproteinases -
tissue inhibitor of metalloproteinases, copper - lysyl oxidase enzyme and genetic polymorphism. The
progressive, irreversible nature and morbidities associated with OSMF have made combating the disease
challenge. Current research is heading toward unveiling the pathogenic mechanisms contributing to fibrosis
and thus formulating definitive treatment strategies. 9
Traditionally, histopathology has been used to identify morphologic changes associated with an in vivo
diagnosis, evaluation of response to therapy, primary research, and nonclinical safety assessment. There is
often a strong correlation between specific histopathologic findings and clinical signs, and therefore
histopathology is used in nonclinical biomarker qualification studies to establish correlative and temporal
relationships between the biomarker and morphologic changes. Since the visible physiological and
morphological modifications must also correlate with global alterations of the molecular profiles of gene
transcription, it is necessary first to understand physiological and morphologic changes taking place during
the development of OSF and the malignant transformation of OSF, before correlating the biomarkers
associated with these changes.
STAGE 1: ( NOM TO OSF ):
In the simplest terms, OSF is primarily a disease of collagen metabolism disorder, in which four significant
events occur which ultimately complete hyalinization of subepithelial tissues and fibrosis.
A. Inflammation of juxta-epithelial tissues
B. Increased Collagen Production
C. Cross-linking of Collagen Fibers.
D. Decreased collaged destruction.
Fibrosis is every so often defined as a wound-healing response that has gone out of control. Oral
submucous fibrosis is considered as a response to constant mechanical and chemical insults on oral mucosa
by constant chewing of betel-nut and tobacco products. Such an Injury to cells and tissues leads to a series
of events that contain the damage and initiate the healing process which is either regeneration and repair
of the damaged tissues. Restoration of the damaged tissue takes place either by scar tissue formation or
fibrosis. 1047
The extracellular matrix (ECM) forms the complex and dynamic 3D environment that defines tissue
structure and function. Matrix molecules provide much of the microenvironmental plasticity that dictates
cell behavior, and their dysregulated expression is associated with a wide range of diseases including
cancers, and inflammatory and fibrotic conditions. The ECM is a network of proteins and sugars that lies
around and between the cells in every tissue of our bodies (Figure 1). Once thought to exist merely as
structural support for cells, we now know that the matrix does much more than this. The cells and their
matrix are always in active communication; the ECM provides environmental information to the cells, and
cells respond by modifying their behavior to cope with changing conditions in the tissue. The ECM is not a
fixed structure, but is remarkable dynamic.1045
To understand the process of fibrosis, it is necessary first to know ECM. After realizing the changes that
occur in the ECM, we can develop a clinically-translated model for conditions such as aging and fibrosis. The
common denominator of fibroproliferative diseases is a dysregulated tissue remodeling that leads to the
excessive and abnormal accumulation of extracellular matrix components (ECM), thus generating an ECM
with different structural and signaling properties in the affected tissues. 1046
EXTRACELLULAR MATRIX- ECM:
The extracellular matrix has been defined as structural support for cells and a source of mechanical and
biochemical, environmental cues that actively signal to determine cell phenotype. The potential role of the
ECM in regulating inflammation is now recognized. Fibrosis is an abnormal or excessive accumulation of the
extracellular matrix (ECM). ECM has traditionally been seen as a scaffold to support cell adhesion and
migration, although it is much more complicated than what is assumed.
An incredible variety of stimuli make the ECM undergo remodeling in fibrotic diseases, including OSF and
although our understanding of the fibrosis mechanisms is increasing at an exciting rate, there is still a lot to
learn. Intensified ECM deposition is a rational response to injury; the sustained deposition of ECM,
however, leads to its thickening, which can affect common tissue characteristics and may lead to eventual
organ failure. 1048
ECM components are essential for wound healing as they provide the framework for cell migration,
maintain the correct polarity of cells for the reassembly of multilayer structures, and participate in the
formation of new blood vessels (angiogenesis). Also, ECM cells (fibroblasts, macrophages, and other types
of cells) produce growth factors, cytokines, and chemokines that are critical to regeneration and repair. 1047
The renovation and restoration of tissues depend not only on the activity of the soluble factors but also on
the interactions between the cells and the components of the extracellular matrix (ECM). ECM regulates
growth, proliferation, movement, and differentiation of the cells that live within it. It is perpetually
remodeling, and its synthesis and degradation accompanies morphogenesis, regeneration, wound healing,
chronic fibrous processes, tumor invasion, and metastasis. ECM sequesters water, providing turgor to soft
tissues, and minerals that give rigidity to the bone, but it does much more than fill spaces around cells to
maintain tissue structure.
Its various functions include: 1402
1] Mechanical support for cell anchorage and cell migration, and maintenance of cell polarity.
2] Control of cell growth. ECM components can regulate cell proliferation by signaling through cellular
receptors of the integrin family.
3] Maintenance of cell differentiation. The type of ECM proteins can affect the degree of differentiation of
the cells in the tissue, also acting mainly via cell surface integrins.
4] Scaffolding for tissue renewal. Maintenance of the healthy tissue structure requires a basement
membrane or a stromal scaffold. The integrity of the basal layer or stroma of the parenchymal cells is
critical for the organized regeneration of the tissues. It is particularly remarkable that although labile and
stable cells are capable of recovery, injury to these tissues results in the restitution of the typical structure
only if the ECM is not damaged. The interruption of these structures leads to the deposition of the collagen
and the scar formation.
5] Establishment of tissue microenvironments. The basal layer serves as a boundary between the
epithelium and the underlying connective tissue.
6] Storage and presentation of regulatory molecules. For example, growth factors such as FGF and HGF are
secreted and stored in the ECM in some tissues. This allows rapid deployment of growth factors after local
injuries, or during regeneration.
Summary of ECM functions in development.
The ECM is multi-functional and can influence multiple biochemical and mechanical processes
simultaneously. This figure illustrates different functional states of the ECM and their biological contexts.
The five categories are not mutually exclusive. When interpreting ECM loss-of-function phenotypes, one
should consider that multiple processes may be compromised thus specific roles of individual ECM
components are difficult to glean. A couple of essential properties of ECM are not illustrated in this cartoon.
First, ECMs are highly dynamic and can be modified by the cells that come into contact with them creating a
bi-directional mode of cell-matrix communication. Second, ECM–ECM interactions vary the chemical and
mechanical composition of the extracellular microenvironment.
REF: Rozario, T., and D.W. DeSimone, The extracellular matrix in development and morphogenesis: a
dynamic view. Dev Biol, 2010. 341(1): p. 126-40. http://dx.doi.org/10.1016/j.ydbio.2009.10.026 1015
The ECM is composed of three groups of macromolecules:
1] fibrous structural proteins, such as collagens and elastins that provide tensile strength and recoil;
2] adhesive glycoproteins that connect the matrix elements and cells; and
3] proteoglycans and hyaluronan that give resilience and lubrication.
These molecules assemble to form two primary forms of ECM: interstitial matrix and basement
membranes.
The interstitial matrix is found in the spaces between epithelial, endothelial and smooth muscle cells, as
well as in the connective tissue. It consists mainly of fibrillary and non-fibrillar collagen, elastin, fibronectin,
Proteoglycans, and Hyaluronan. Basement membranes are closely associated with cell surfaces and consist
of nonfibrillar collagen (mostly type IV), laminin, heparin sulfate, and proteoglycans.
If the tissue insult is severe or chronic, like in OSF, and results in damage of both the parenchymal cells and
the stromal frame of the tissue, healing can be achieved by deposition of the collagen and other
components of the ECM, causing the formation of a scar. In contrast to regeneration involving restitution of
tissue components, the repair is a fibroproliferative response that "patches" instead of restoring tissue.
However, if the damage persists, inflammation becomes chronic, leading to excess deposition of the
connective tissue known as fibrosis. In most healing processes, a combination of repair and regeneration is
produced. The relative contributions of repair and restoration are influenced by the following factors: 1047
(1) The proliferative capacity of the cells of the tissue;
(2) The integrity of the extracellular matrix; and
(3) The resolution or chronicity of the injury and inflammation
Repair by connective tissue deposition includes the following essential features:
1. inflammation
2. angiogenesis,
3. migration and proliferation of fibroblasts,
4. scar formation
5. remodeling of the connective tissue.
The term fibrosis is more broadly used to denote the excessive deposition of collagen and other ECM
components in tissue. The persistence of the insults leads to chronic inflammation, which is associated with
the proliferation and activation of macrophages and lymphocytes, and the production of a plethora of
inflammatory and fibrogenic factors and growth cytokines.1015
REF: 1402] Upik A. Miskad. Tissue Renewal, Regeneration, and Repair. Ch.3 2008
ECM STRUCTURE
REF: Susan Aungier and Kim Midwood. The extracellular matrix: a new dimension in disease diagnosis and
treatment. Beyond the cell. August 2016 © Biochemical Society 1045
REF: Morten A. Karsdal, Tina Manon-Jensen, Federica Genovese, X Jacob H. Kristensen, Mette J. Nielsen,
Jannie Marie B. Sand, Niels-Ulrik B. Hansen, Anne-Christine Bay-Jensen, Cecilie L. Badger, Aleksander Krag,
Andy Blanchard, Henrik Krarup, Diana J. Leeming, and Detlef Schuppan. Novel insights into the function
and dynamics of the extracellular matrix in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 308: G807–
G830, 2015.doi:10.1152/ajpgi.00447.2014 1046
REF: Wynn, T.A., Common, and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J
Clin Invest, 2007. 117(3): p. 524-9. 1044
STAGE TWO: CONVERTING OSF INTO FRANK OSCC:
For decades tumors have been recognized as “wounds that do not heal.” Besides the commonalities that
tumors and injured tissues share, the process of wound healing also portrays similar characteristics with
chronic fibrosis. According to Rybinski, B et al. 1048 It is clear that the same cell types, as well as soluble and
matrix elements that drive wound healing (including regeneration) via distinct signaling pathways, also fuel
chronic fibrosis and tumor progression.
REF: Rybinski, B., Franco-Barraza, J., & Cukierman, E. (2014). The wound healing, chronic fibrosis, and
cancer progression triad. Physiological Genomics, 46(7), 223–244.
http://doi.org/10.1152/physiolgenomics.00158.2013 1048
According to Mohit Sharma et al. 1706, the genesis of oral submucous fibrosis is still unclear despite
the superfluity of literature. Considering, oral submucous fibrosis as an over-healing wound explains
both pathogenesis and malignant transformation. Certainly, abnormalities in coagulation and
fibrinolytic system are a common denominator in the profibrotic milieu and associated malignancy.
The highly interconnected nature of science creates significant artificiality in identifying discrete topics
around which discussions of both physiological and pathological tissue repair revolve. By necessity,
however, science is often performed in easily digestible ‘bite-sized chunks’ that allow us to answer specific
questions using reductionist approaches and it is through this type of investigation that we hope to better
understand the processes that define a physiological response to injury and the myriad ways in which it can
go awry. Perhaps the simplest (and most conventional) way to approach this topic is, as the title suggests,
to divide the discussion among the concepts of inflammation (comprising the influx of inflammatory cells
and mediators that orchestrate the initial response to tissue injury), wound healing (dealing with the
physiological steps necessary to restore normal tissue structure and function), and fibrosis (concerning the
pathophysiological response following injury that leads to ineffective and/or inappropriate tissue repair).
To do so, however, invites the acknowledgement that the distinction between physiology and pathology is
blurry, defined by matters of degree; enough inflammation and repair can heal a wound and restore tissue
integrity and function, whereas excessive inflammation and/or repair (even if accomplished by
physiological mechanisms) may lead to tissue dysfunction. Thus, while inflammation and fibrosis may occur
due to abnormal processes, they may also be construed as ‘too much of a good thing’. In this year’s Annual
Review Issue, we begin to tackle some of these issues, all the while recognizing that further work in these
arenas will be necessary to advance our knowledge for purposes of appreciating the true underpinnings of
tissue homeostasis, injury repair, and fibrosis.1708
Increasingly, researchers in the fields of inflammation, wound healing, and fibrosis are recognizing that
extracellular influences exist to alter the rate and efficacy of wound healing. For instance, it is becoming
widely recognized that the ECM is much more than just scaffolding upon and in which cells reside, but
rather a complex admixture of proteins that provide spatial and temporal context for cells. The importance
of the ECM in directing such cellular functions is reflected in the recent coining of the term ‘matrisome’ to
include ECM and ECM-related molecules that together direct cell function. 1708
Can we then consider the origin of Oral Submucous Fibrosis as a process of wound healing that has
gone wrong? Successful wound repair relies upon a crucial balance between the yin of matrix
deposition and the yang of matrix degradation. Regenerative processes are favored when the matrix
composition and architecture are unaltered. Thus, wounds that do not heal may reflect excess
proteinase activity, decreased matrix accumulation, or altered matrix assembly. Conversely, fibrosis
and scarring may result from reduced proteinase activity or increased matrix accumulation. Whereas
the formation of new collagen during repair is required for increased strength of the healing site,
chronic fibrosis is a significant component of diseases that involve chronic injury.
We will review the wound healing process briefly.
Repair by connective tissue deposition includes the following basic features: 1402
1] inflammation
2] angiogenesis,
3] migration and proliferation of fibroblasts,
4] scar formation
5] connective tissue remodeling
Three fundamental cellular mechanisms are necessary for wound healing:
• Cellular migration
• Extracellular matrix organization and remodeling
• Cell proliferation
MIGRATION OF CELLS INITIATES REPAIR:

Cells That Migrate to the Wound:
Migratory Cells Contents and Function
1] platelets and basophils. cytokines, chemoattractants, proteases, and

mediators of inflammation, which together
(1) control vascular delivery,
(2) degrade damaged tissue, and
(3) initiate the repair cascade
2] Mast cells Heparin. Angiogenesis
3] Resident macrophages
4] Leukocytes Focal adhesions
5] Polymorphonuclear leukocytes degrade and destroy nonviable tissue
6] Macrophages phagocytose debris and orchestrate the

developing granulation tissue by the release of
cytokines and chemo-attractants.
7] Fibroblasts, myofibroblasts, pericytes, and fibroplasia, synthesis of connective tissue

smooth muscle cells matrix, tissue remodeling, wound contraction and
wound strength.
8] Endothelial cells Angiogenesis. Exchange of gases, the delivery of

nutrients, and the influx of inflammatory
cells
9] Epithelial cells penetrate the provisional matrix,

moreover, migrate upon stromal collagen, which is
coated with plasma glycoproteins, fibrinogen, and
fibronectin
10] Stem cells A renewable source of epidermal and dermal cells

capable of differentiation, proliferation, and
migration.Angiogenesis
EXTRACELLULAR MATRIX SUSTAINS THE REPAIR PROCESS:
• Basement membrane
• Provisional matrix
• Connective tissue (interstitial matrix or stroma)
1] Basement Membrane 1] Epithelium, adipocytes, muscle cells, Schwann

cells, and capillary endothelium produce
basement membranes.
2] Basement membranes are constructed from
extracellular matrix molecules, including collagen
IV, laminin, entactin/ nidogen, and perlecan, a
heparan sulfate proteoglycan
3] Basement membranes act as filters, cellular
anchors, and a surface for migrating epidermal
cells after injury
2] Provisional Matrix 1] the temporary extracellular organizations of

plasma-derived matrix proteins and tissue-
derived components that accumulate at sites of
injury (e.g., hyaluronan, tenascin, and fibronectin).
2] Plasma-derived provisional matrix proteins
include fibrinogen, fibronectin, and vitronectin.
These proteins become insoluble by binding to the
stromal matrix and by forming cross-links
3] Stromal (Connective Tissue) Matrix 1] Connective tissue forms a continuum between

tissue elements such as epithelia, nerves, and
blood vessels and provides physical protection by
conferring resistance to compression or stretching
2] contains both extracellular matrix elements
and individual cells that synthesize the matrix. The
cells are primarily of mesenchymal origin and
include fibroblasts, myofibroblasts, adipocytes,
chondrocytes, osteocytes, and endothelial
cells. Bone marrow-derived cells (e.g., mast cells,
macrophages, and transient leukocytes) also
populate connective tissue.
Cell migrations during the repair: 1707
(1) Leukocytes attach to, and migrate between, capillary endothelial cells, penetrate the basement
membrane, and enter the matrix.
(2) Capillary endothelial cells, released from the basement membrane, migrate through the matrix to form
new capillaries.
(3) Pericytes detach from endothelial cells and their basement membranes to migrate into the matrix.
(4) Fibroblasts become bipolar and migrate through the matrix to the site of injury.
(5) Epithelial keratinocytes detach from neighboring cells and basement membranes and migrate between
the scab and the wound along with the provisional matrix of the dermis. FGF fibroblast growth factor; VEGF
vascular endothelial growth factor.
Summary of the healing process: 1707
The initial phase of the repair reaction, which typically begins with hemorrhage into the tissues.
(1) A fibrin clot forms and fills the gap created by the wound. Fibronectin in the extravasated plasma is
cross-linked to fibrin, collagen, and other extracellular matrix components by the action of
transglutaminases. This cross-linking provides a provisional mechanical stabilization of the wound (0–4
hours).
(2) Macrophages recruited to the wound area, process cell remnants and damaged the extracellular
matrix. The binding of fibronectin to cell membranes, collagens, proteoglycans, DNA, and bacteria
(opsonization) facilitates phagocytosis by these macrophages and contributes to the removal of debris (1–3
days).
(3) Fibronectin, cell debris, and bacterial products are chemoattractants for a variety of cells that are
recruited to the wound site (2–4 days). The intermediate phase of the repair reaction.
(4) As a new extracellular matrix is deposited at the wound site, the initial fibrin clot is lysed by a
combination of extracellular proteolytic enzymes and phagocytosis (2–4 days).
(5) Concurrent with fibrin removal, there is deposition of a temporary matrix formed by proteoglycans,
glycoproteins, and type III collagen (2–5 days).
(6) The final phase of the repair reaction. Eventually, the temporary matrix is removed by a combination of
extracellular and intracellular digestion, and the definitive matrix, rich in type I collagen, is deposited (5
days–weeks).
Granulation tissue:
1] Granulation tissue has two significant components: cells and proliferating capillaries. The cells are mostly
fibroblasts, myofibroblasts, and macrophages. The macrophages are derived from monocytes and
macrophages. The fibroblasts and myofibroblasts derive from mesenchymal stem cells, and the capillaries
arise from adjacent vessels by the division of the lining endothelial cells (detail), in a process termed
angiogenesis. Endothelial cells put out cell extensions, called pseudopodia, that grow toward the wound
site. Cytoplasmic growth enlarges the pseudopodia, and eventually, the cells divide. Vacuoles formed in the
daughter cells eventually fuse to create a new lumen. The entire process continues until the sprout
encounters another capillary, with which it will connect. At its peak, granulation tissue is the most richly
vascularized tissue in the body.
2] Once the repair has been achieved, most of the newly formed capillaries are obliterated and then
reabsorbed, leaving a pale avascular scar.
3] A photomicrograph of granulation tissue shows thin-walled vessels (arrows) embedded in a loose

connective tissue matrix containing mesenchymal cells and occasional inflammatory cells.
Fibroblasts and collagen fibers. Electron micrographs. A. Chick embryo fibroblast (F) lying between collagen
fibers and an elastin fiber in the lower right corner. The collagen fibers are seen as crosswise strands
traversing the field and along the long axis, at a right angle, as dots. B. A chick embryo dermal fibroblast
with cell surface-associated collagen fibril bundles (B); some bundles are enveloped by fibroblast
membrane (arrows). The fibrils are visualized on the long axis as dots.
Myofibroblast viewed by electron microscopy. Myofibroblasts have an essential role in the repair reaction.
These cells, with features intermediate between those of smooth muscle cells and fibroblasts, are
characterized by the presence of discrete bundles of myofilaments in the cytoplasm (arrows).
Overview of repair. This figure provides an overview that interrelates the early dynamic events in repair.
The time scale in this figure is not linear; initial tensile strength, the first phase, develops almost
immediately. Remodeling is ill-defined, extending from its early beginning in repair for weeks or months.
The methods of detection of biomarkers are classified as:
• Serology: Enzyme assays
• I m mu no lo gi cal: I m mu no his to c h em is try, radioimmunoassay, enzyme-linked immunosorbent
assay
• Flow cytometry cytogenetic analysis: Fluorescent in situ hybridization, spectral karyotyping,
comparative genomic hybridization
• Genetic analysis sequencing (automated): Reverse transcription gel electrophoresis and DNA
microarray
analysis
• Proteomics: Surface-enhanced laser desorption/ionization
REF: Negi M, Bansal S, Puri A, Nangia R. Tumor markers in oral squamous cell carcinoma as an adjunct to
diagnosis: An insight. Indian J Dent Sci 2018;10:190-5. DOI: 10.4103/IJDS.IJDS_53_18 1085
General Points About Tumor Marker Tests According To association of Biochemist in Ireland scientific
Committee Guidelines
 No serum marker in current use is specific for malignancy
 In general, serum marker levels are rarely elevated in patients with early malignancy. With a few
exceptions, high levels are usually found only when patients have advanced disease
 No cancer marker has absolute organ specificity. PSA, however, appears to be relatively specific for
prostate tissue but not for prostate cancer
 Apart from possibly hCG in choriocarcinoma, no marker is elevated in 100% of patients with a particular
malignancy
 Requesting of multiple markers (such as CEA and the CA series of antigens) in an attempt to identify
metastases of unknown primary origin is rarely of use
 Tumor marker assays should not be carried out on biological fluids such as peritoneal fluids, pancreatic
juice, and ovarian cystic fluids as reliable reference ranges are currently unavailable for these types of
specimen
 Reference ranges for cancer markers are not well defined and are used only for guidance. Please note
that a level below the reference range does not exclude malignancy, while concentrations above the
reference ranged onto necessarily mean the presence of cancer. Changes in levels over time are likely
to be more clinically useful than absolute levels at one point in time
 As many tumor markers lack agreed international reference preparations (e.g., CA125, CA15-3, and
CA19-9), different assay kits may give different results for the same sera
 Laboratories carrying out tumor marker tests should state the assay used on their report form.
No tumor marker is ideal and the fact that the substances that are being applied as tumor markers are not
synthesized exclusively as a consequence of malignancy. Some of the most common factors (in addition to
malignant disease) that affect serum concentrations of tumor markers are as follows :1085
False-positive results
Presence of inflammatory processes
Benign liver diseases and consequential disturbances in metabolism and excretion (AFP, TPA, CEA, CA19-
9, CA15-3)
Disturbances of renal function (beta-2-microglobulin, calcitonin, PSA, CEA, CA19-9, CA15-3)
Extensive tumor necrosis
As a consequence of diagnostic and therapeutic procedures (digitorectal examination, mammography,
surgery, and radio and chemotherapy)
As a consequence of different physiological conditions (pregnancy – HCG, CA125, CA15-3, MCA, AFP, and
menstrual cycle – CA125).
False-negative results
Complete absence of production (e.g., CA19-9 in Le (a-b-) persons)
Insufficient expression of a certain antigenic determinant (or production in only some of the tumor cells)
Insufficient blood circulation in the tumor
Production of immune complexes with autoantibodies
Rapid degradation and clearance of antigens
Recommendations for ordering a tumor marker test
A single value or test is unreliable in itself. It is noteworthy that in most situations, elevations of markers
in nonmalignant diseases are often transient, whereas elevations associated with cancer either remain
constant or continuously rise. Ordering serial testing can help detect falsely elevated levels due to
transient elevation
It is imperative to be certain that the marker in question was, in fact, elevated before relying on it for
monitoring disease activity, the reason being that none of the tumor markers are 100% sensitive (may
not be elevated in some cases)
In tumors with multiple raised markers measured before definitive therapy, the marker showing the
highest elevation should be used for follow-up
If in a given case, tumor markers were not evaluated in the pretreatment setting, it is advisable to use
multiple markers for monitoring in the posttherapy setup
As a general guideline, the time interval between serial determinations should be 3 months; but in case
of an abnormal value, a repeat estimate can be ordered within 2–4 weeks irrespective of the initial
reading
Use of multiple markers based on the combination pattern for the selected malignancy will improve
sensitivity and specificity of the detection
An important interfering factor to be considered is the presence of a Hook effect.
On the other hand, if the marker level goes up, then the cancer is not responding and the treatment may
need to be changed. (and exception to such cases is is if the cancer is very sensitive to certain
chemotherapy treatment, in that case, the chemo can cause many cancer cells to die and release large
amounts of marker into the blood, which in turn causes the level of the tumor marker to rise for a short
time.) 1085
To Know How Well Treatment is Working: 1085
When there is:

No change – Tumor marker does not fall to <50% of pretreatment concentration
Improvement – Tumor marker falls to <50% of pretreatment concentration
Response – Tumor marker falls to <10% of pretreatment concentration
Complete response – Tumor marker falls to nonmalignancy reference values.
Difficulty in identifying minute quantities of particular substances in serum
Existence of proliferation-related rather than tumor-associated antigen
Cross-reactive antigens, for instance, a common domain in different proteins
Cross-reaction with degradation products of normal proteins taken up by tumor cells
Malignant tumors with extensive necrosis have increased hydrolytic enzymes. Antigenic degradation
products may then form which would normally be absent from nonnecrotic control tissue
The financial and psychological cost to the society of routine screening for early cancers using currently
available tumor marker would be prohibited.
REF: 1107] Mohammad Sayedur Rahman Khan, Fatima Siddika, Sun Xu, Xiao-Lin Liu, Mei Shuang, and Hao
Fu Liang.Diagnosing oral squamous cell carcinoma using salivary biomarkers. Bang Bandhu Sheikh Mujib
Med Univ. Journal 2018; 11: 1-10. DOI: 10.3329/bsmmuj.v.11i.1 35399.
Products such as 3-(methylnitrosamino)-proprionitrile, nitrosamines, and nicotine initiate the production of

reactive oxygen species in smokeless tobacco, eventually leading to fibroblast, DNA, and RNA damage with
carcinogenic effects in the mouth of tobacco consumers. The metabolic activation of nitrosamine in tobacco
by cytochrome P450 enzymes may lead to the formation of N-nitrosonornicotine, a major carcinogen, and
micronuclei, which are an indicator of genotoxicity. These effects lead to further DNA damage and,
eventually, oral cancer. 892
REF: 892] Kamal Niaz, Faheem Maqbool, Fazlullah Khan, Haji Bahadar, Fatima Ismail Hassan, and
Mohammad Abdollahi. Smokeless tobacco (paan and gutkha) consumption, prevalence, and contribution
to oral cancer. Epidemiol Health 2017;39:e2017009. https://doi.org/10.4178/epih.e2017009 892
SALIVARY BIOMARKERS:
The discovery that saliva contains molecular profiles that reflect systemic diseases has opened the doors to
a new noninvasive diagnostic methodology- ‘salivary diagnostics.’ Using saliva in diagnosis is not only
practical and non invasive but at times is also proving to be more accurate than available alternatives.
Saliva has the advantages that it contains a low background of a standard material and inhibitory
substances as well as fewer complexes than blood. It is an informative body fluid containing an array of
analytes (Protein, mRNA, and DNA) that can be used as biomarkers for translation and clinical applications
1664
The review carried out by Barman I et al. 1664 was to look for the potential oral cancer tumor markers in
saliva detected with the modern, sophisticated technology which makes saliva an attractive alternative to
serum testing for screening and molecular pathology analysis for high-risk patients of oral cancer. The
potential salivary biomarkers for OSCC are listed in the following table.
POTENTIAL OSCC SALIVARY BIOMARKERS
BIOMARKERS REFERENCES
1] Defensin-1 Mizukawa et al. 1665
2] Proteins P53 autoantibody Warnakulasuriya et al. 1666

3] α-amylase Chen et al. 1667
4] Inter Leukin-8 St. John et al. 296

Rhodus et al.1668
Arellano-Garcia et al.1241
Brinkmann et al.1669
Elashoff et al 1670
5] Tumor Necrosis Factor-α Rhodus et al 1668
6] Inter Leukin-1 Rhodus et al 1668
7] Inter Leukin-6 Cheng et al.1243

Katakura et al.854
Saheb-Jamee et al.297
Sato et al.1671
8] Basic fibroblast growth factor Vucicevic et al.1672

Gorugantula et al. 1673
9] Statherin Contucci et al. 1674
10] Cyfra 21.1 Nagler et al.1675
11] Tissue polypeptide antigen (TPA) Nagler et al. 1675
12] Cancer antigen 125 (CA125) Nagler et al.1675

Balan et al.1676
13] Endothelin-1 Pickering et al.1676

Cheng et al.1677
14] Inter Leukin-1β Katakura et al.854

Elashoff et al.1670
15] CD44 Franzmann et al.1678
16] Total salivary protein Shpitzer et al.1679
17] Insulin growth factor 1 (IGF-1) Shpitzer et al.1679
18] Matrix Metallo Proteins-2, 9 Shpitzer et al.1679

19] CD59 Hu et al.1295
20] Catalase Hu et al.1295
21] Profilin Hu et al.1295
22] S100A9/MRP14 Hu et al.1295
23] M2BP Hu et al.1295

24] Carcinoembryonic antigen (CEA) Hu et al.1295
25] Carcinoma associated antigen CA-50 Hu et al.1295
26] Salivary carbonyls Shpitzer et al.489
27] Cyclin D1 Shpitzer et al.489
28] Maspin Shpitzer et al.489
29] Phosphorylated-Src Shpitzer et al.489
30] Ki-67 Shpitzer et al.489
31] Lactate dehydrogenase Shipitzer et al.489

Shety et al.1680
32] Transferrin Jou et al.1681
33] Zinc finger protein 501 peptide Jou et al.1682
34] Hemopexin Jessie et al.1683
35] Haptoglobin Jessie et al.1683
36] Complement C3 Jessie et al.1683
37] Transthyretin Jessie et al.1683
38] α1-antitrypsin Jessie et al.1683

39] P53 gene codon 63 Liao et al.1684
40] Loss of heterozygosity in the combination El-Naggar et al.1685

Of markers D3S1234, D9S156, and D17S799
41] Mitochondrial DNAs (cytochrome c oxidase I Jiang et al.1686

moreover, cytochrome c oxidase II)
42] Hypermethylation of promoters in tumor Carvalho et al.1687

suppressor genes: DAPK, DCC, MINT-31,
TIMP-31, TIMP-3, p16, MGMT, CCNA1
43] Inter Leukin-8 Li et al.1688

Elashoff et al.1670
44] Inter Leukin-1β Li et al.1688
45] DUSP1 (dual specificity phosphatase 1) Li et al.1688

Elashoff et al.1670
Cheng et al.1689
46] H3F3A (H3 histone family 3A) Li et al.1688

Elashoff et al.1670
47] OAZ1 (ornithine decarboxylase antizyme 1) Li et al.1688

Elashoff et al.1670
Cheng et al.1689
48] S100P (S100 calcium binding protein P) Li et al.1688

Elashoff et al.1670
Cheng et al.1689
49] SAT (spermidine/spermine N1-cetyltransferase Li et al.1688

EST) Brinkmann et al.1670
Elashoff et al.1670
50] miR-125a Park et al.768
51] miR-200a Park et al.768
52] miR-31 Liu et al.1690
53] Peroxidase Bahar et al.1691

54] Reactive nitrogen species (RNS) such as Bahar et al.1691
nitricoxide (NO), nitrites (NO2) and
nitrates (NO3)
55] Glutathione S-transferase (GST) Bahar et al. 1691
56] Superoxide dismutase (SOD) Agha-Hosseini et al.1692
57] 8-hydroxy-2-deoxyguanosine (8-OHdG) Agha-Hosseini et al.1692
58] Glutathione Almadori et al.1693
59] Malondialdehyde (MDA) Agha-Hosseini et al.1692
60] pyrroline hydroxycarboxylic acid Sugimoto et al.1694
61] leucine plus isoleucine Sugimoto et al.1694
62] choline Sugimoto et al.1694
63] tryptophan Sugimoto et al.1694
64] valine Sugimoto et al.1694
65] threonine Sugimoto et al.1694
66] histidine Sugimoto et al.1694
67] pipecolic acid Sugimoto et al.1694
68] glutamic acid Sugimoto et al.1694
69] carnitine, Sugimoto et al.1694
70] alanine Sugimoto et al.1694
71] piperidine Sugimoto et al.1694
72] taurine Sugimoto et al.1694

73] alpha-aminobutyric acid Sugimoto et al.1694
74] phenylalanine Sugimoto et al.1694
75] betaine Sugimoto et al.1694
76] serine Sugimoto et al.1694
77] tyrosine Sugimoto et al.1694
78] glutamine Sugimoto et al.1694
79] beta-alanine Sugimoto et al.1694
80] cadaverine Sugimoto et al.1694
81] Phenylalanine Wei et al. 1695

Sugimoto et al.1694
82] Valine Wei et al. 1695

Sugimoto et al.1694
83] Lactic acid Wei et al.1695
84] Sialic acid Vajaria et al.1696
85] α-L-fucosidase Vajaria et al.1696
REF: 1664] Barman I and Talukdar A. Saliva –An imperative medium for Oral Cancer Biomarker Detection - A
Brief Review Int J Cont. Med Res. 2014;1(2):45-55
Molecular markers for oral cancer
REF: 1710] Krishna Prasad, R. B., Sharma, A., & Babu, H. M. (2013). An insight into salivary markers in oral
cancer. Dental research journal, 10(3), 287–295.
No scientific review on inflammation, wound healing, and fibrosis would be complete without providing
examples of how our new-found knowledge will prove meaningful for patient care. We have clearly come a
long way since the four cardinal features of inflammation were defined. Yet we still have a way to go before
we achieve clarity in understanding all the pieces that drive a successful inflammatory and wound healing
response or promote a pathological fibrotic one.1708
As living beings that encounter every kind of traumatic event from paper cut to myocardial infarction, we
must possess ways to heal damaged tissues. While some animals are able to regrow complete body parts
following injury (such as the earthworm who grows a new head following bisection), humans are sadly
incapable of such feats. Our means of recovery following tissue damage consists largely of repair rather
than pure regeneration. Thousands of times in our lives, a meticulously scripted but unseen wound healing
drama plays, with cells serving as actors, extracellular matrix as the setting and growth factors as the
means of communication. This article briefly reviews the cells involved in tissue repair, their signaling and
proliferation mechanisms and the function of the extracellular matrix, then presents the actors and script
for the three acts of the tissue repair drama. 1709
REF: 1709] Krafts K. P. (2010). Tissue repair: The hidden drama. Organogenesis, 6(4), 225–233.
doi:10.4161/org.6.4.12555
The process of oral carcinogenesis is complex and not yet fully understood. Consequently numerous
potential biomarkers have been hypothesised though controversial results across the board hamper their
clinical implementation. Of greatest advantage would be biomarkers signalling early events preceeding OC.
Biomarker targets predominately involve deregulated molecular events that participate in cell signalling,
growth, survival, motility, angiogenesis and cell cycle control but can also use changes in metabolic genes
to discriminate healthy form disease state. Promising potential biomarkers include the growth signalling
oncogenes, Epidermal Growth Factor Receptor and Cyclin D1, the anti-growth signalling components p53
and p21, apoptotic effectors such as Bcl-2 and also components involved in immortalisation, angiogenesis,
invasion and metastasis processes. Translation of these potential biomakers to the patients is closer than
ever though few issues remain to be resolved. Firstly large clinical trials are needed to validate their clinical
applicability but also standardised methods of collection, storage and processing methods are needed to
minimise variability.1716
Normal wound healing response through a series of overlapping phases: inflammation, proliferation, and
remodeling. Several cells at coagulation phase and following phases release growth factors that are
essential for a normal wound healing process to proceed.
REF: 1717] Kuroyanagi, Misato & Kuroyanagi, Yoshimitsu. (2017). Tissue-engineered products capable of
enhancing wound healing. AIMS Materials Science. 4. 561-581. 10.3934/matersci.2017.3.561.
Challanges in Salivary Biomarkers:
1. A lack of standardization of conditions and methods of saliva sample collection, processing, and storage.
2. Variability in the levels of potential salivary biomarkers in both non-cancerous individuals and oral
cancer patients, suggest unknown confounding factors
3. The need for further validation of oral cancer salivary biomarkers. 1300
Much research effort has been dedicated to investigating potential salivary biomarkers for OSCC, and more
than 100 such biomarkers have been reported in the literature. However, some important issues and
challenges have emerged that require solutions and further research in order to find reliable OSCC salivary
biomarkers for clinical use. Cheng et al 1304 have provided an up-to-date list of potential OSCC salivary
biomarkers reported as of the fall of 2013, and discussed the emerging issues.
In the field of medical research and technology, the science of Proteomics and Genomics have made great
strides. The development in these two disciplines has made molecular profiling of healthy and diseased
states possible. The recognization of specific proteins responsible for that particular disease provides
material for drug development which will help to counteract the action of that protein. This will assist in
establishing targeted therapies. Personalized medicine will provide treatment based on the genetic makeup
of an individual and by taking into account the variations noted in a patient. Translation of this research in
diagnostics and treatment planning is essential for the benefit of the patient. 831
REF: 831] Revati D, Priya N D. Proteomics and Genomics: The Future of Personalized Medicine. Biomed J Sci
&Tech Res 2(1)- 2018. Biomed J Sci & Tech Res (BJSTR) MS.ID.000679. DOI :
10.26717/BJSTR.2018.02.000679
REF: 831] Revati D, Priya N D. Proteomics and Genomics: The Future of Personalized Medicine. Biomed J Sci
&Tech Res 2(1)- 2018. Biomed J Sci & Tech Res (BJSTR) MS.ID.000679. DOI :
10.26717/BJSTR.2018.02.000679
Tsui 2051 proposed three hypotheses:
(i) Genetic alterations critical to oral cancer development will be present in oral premalignant lesions
(OPLs), and candidate genes will reside within recurrent regions of alterations in OPLs.
(ii) DNA amplification occurs at the premalignant stage of oral cancer development and harbors genes
involved in key oncogenic pathways.
(iii) The oral cancer field is genetically heterogeneous, and the clonal evolution between biopsies from the
field will be revealed by their genetic signatures.
The author conducted research by employing the following methods. DNA copy number data from OPLs
were generated using tilingpath DNA microarrays. Recurrent regions of alterations on chromosome 3p were
identified and associated with clinical information. Copy number data were associated with public
expression datasets to identify genes and pathways contributing to oral carcinogenesis. Genetic
breakpoints were used to evaluate the clonal expansion of genetically altered cells within a single field.
The author reported the following results:
1] identified the genetic alterations that are recurrently altered in different individuals, supporting
hypothesis 1.
2] Regions of high-level amplification are frequently detected in OPLs, and genes mapped in amplicons are
significantly enriched in the FGF signaling network, supporting hypothesis 2.
3] Furthermore, the genetic relatedness among biopsies from an oral cancer field is revealed by comparing
their genetic signatures, and the field is found to be genetically heterogeneous, supporting hypothesis 3.
Possibly these findings by Tsui 2051 compelled us to study not only biomarkers related to OPMDs, including
OSF, but biomarkers of OSCC as well. With this aim in mind we have elaborated upon each biomarker in
details, both in OSF and OSCC.
REF: Miriam Rosin, Sok Ching Cheong, Anil K Chaturvedi, J. Silvio Gutkind, Scott Lippman, Eva Szabo, Paul
Brennan, The Global Oral Cancer Forum. Henry Schein Cares. 271
REF: Anant Narayan Bhatt, Rohit Mathur, Abdullah Farooque, Amit Verma & B.S. Dwarakanath. Cancer
biomarkers - Current perspectives. Indian J Med Res 132, August 2010, pp 129-149 343
POTENTIAL FIELDS OF APPLICATION OF A CANCER BIOMARKER TEST

REF: Alvaro Mordente, Elisabetta Meucci, Giuseppe Ettore Martorana, and Andrea Silvestrini. Cancer
Biomarkers Discovery and Validation: State of the Art, Problems and Future Perspectives. R. Scatena (ed.),
Advances in Cancer Biomarkers, Advances in Experimental Medicine and Biology 867, DOI 10.1007/978-94-
017-7215-0_2 1042
REF: Rybinski, B., Franco-Barraza, J., & Cukierman, E. (2014). The wound healing, chronic fibrosis, and
cancer progression triad. Physiological Genomics, 46(7), 223–244.
http://doi.org/10.1152/physiolgenomics.00158.2013 1048
KEY POINTS
■ Current cancer biomarkers suffer from low
diagnostic sensitivity and specificity and have
not yet made a significant impact in reducing cancer
burden
■ The impressive growth of large-scale and high
throughput
biology has resulted in increased
the popularity of the concept that novel biomarkers
can be discovered through various emerging
technologies
■ A better understanding of the mechanisms
behind biomarker elevation in biological fluids
may facilitate the discovery of new tumor
markers
■ Some of the new promising strategies for
biomarker discovery include microarray-based
profiling at the DNA and mRNA level, and mass
spectrometry-
based profiling at the protein or
peptide level
■ Study of tumor markers that include current
biomarkers or examination of fluids and tissues
that are near the tumor might also
assist in the identification of novel tumor markers
■ New tumor markers must undergo rigorously
validation before they are introduced into routine
clinical care
REF: Kulasingam, Vathany, and Eleftherios P Diamandis. n.d. “Strategies for Discovering Novel Cancer
Biomarkers through Utilization of Emerging Technologies.” Accessed September 14, 2018.
https://doi.org/10.1038/ncponc1187 1086
Basic molecular involvement in the transformation of normal cell to cancer cell

REF: 1697] Gudiseva S, Katappagari KK, Kantheti LP, Poosarla C, Gontu SR, Baddam VR. Molecular biology of
head and neck cancer. J NTR Univ Health Sci 2017;6:1-7.
According to Brücher BLDM, and Jamall IS 1698 the role of fibrosis in carcinogenesis can be examined by
analogy to tissues of various cancers. The orchestration of letters in the interplay of manifold components
with signaling and crosstalk is incompletely understood but available evidence suggests a hitherto
underappreciated role for fibrosis in carcinogenesis. Complex signaling and crosstalk by pathogenic stimuli
evoke persistent subclinical inflammation, which in turn, results in a cascade of different cell types,
ubiquitous proteins and their corresponding enzymes, cytokine releases, and multiple signaling pathways
promoting the onset of fibrosis. There is considerable evidence that the body’s attempt to resolve such a
modified extracellular environment leads to further disruption of homeostasis and the genesis of the
precancerous niche as part of the six-step process that describes carcinogenesis. The precancerous niche is
formed and can be understood to develop as a result of (1) pathogenic stimulus, (2) chronic inflammation,
and (3) fibrosis with alterations of the extracellular matrix, stromal rigidity, and mechano-transduction. This
is why carcinogenesis is not just a process of aberrant cell growth with damaged genetic material but the
role of the PCN in its entirety reveals how carcinogenesis can occur without invoking the need for somatic
mutations.
Epistemology of the origin of cancer” consisting of a 6-step sequence proposed by Brücher BLDM, and
Jamall IS. 1698
(1) a pathogenic stimulus
(2) chronic inflammation
(3) fibrosis with associated remodeling of the cellular microenvironment.
(4) precancerous niche (PCN), a product of fibrosis, with remodeling by persistent inflammation
develops
(5) a chronic stress escape strategy failure
(6) normal cell to cancerous cell transition (NCCCT) by PCN-induced cell matrix stress.
Disruption of signaling homeostasis induced crosstalk in the carcinogenesis paradigm “Epistemology of the
origin of cancer”. Simplified scheme of the Disruption of signaling homeostasis induced crosstalk in the
carcinogenesis paradigm “Epistemology of the origin of cancer” consisting of a 6-step sequence:
(1) a pathogenic stimulus followed by
(2) chronic inflammation from which develops
(3) fibrosis with associated remodeling of the cellular microenvironment; and from these changes a
(4) precancerous niche (PCN), a product of fibrosis, with remodeling by persistent inflammation, develops which
triggers the deployment of
(5) a chronic stress escape strategy and when this fails resolve it by
(6) normal cell to cancerous cell transition (NCCCT) by PCN-induced cell matrix stress occurs

Introduction To Biomarkers

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Introduction To Biomarkers

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CHAPTER NO.

6 BIOMARKERS IN ORAL SUB-MUCOUS FIBROSIS

Immunohistochemistry (IHC) is a globally available tool that complements histopathological analysis by

(i) show specificity for a particular disease (diagnostic),

(ii) have prognostic value, and

(iii) correlate with disease activity.

(iv) be reasonably stable

(v) present in an easily accessible tissue

(vi) cost-effective to measure reproducibly on a large scale.

This then allows treatment efficacy to be assessed. 1199

HISTORICAL LANDMARKS IN THE DEVELOPMENT OF BIOMARKERS

YEAR FIGURES LANDMARKS

1847 Henry Bence Jones FRS (31 December 1813 – 20 April

REF: Jones HB (1848). "On a new substance occurring in

The The term “biomarker” started to appear in the literature

1967 Compared with other enzyme tests for myocardial

1] Gold P, Goldenberg NA. The carcinoembryonic antigen

1987 , Troponin I as a biomarker for myocardial infarction

1] Katus HA, Looser S, Hallermayer K, Remppis A,

Early Accelerator mass spectrometry used for the analysis of

1995 Applications of proteomics for discovery of biomarkers

1999 The emergence of metabolomics for the study of

2000 Sequencing of the human genome completed opening

Biomarkers are classified in various ways.

1) Biomarkers of Exposure Or Antecedent Biomarkers

[2] Biomarkers can also be categorized as pharmacodynamic, prognostic or predictive 8

2] antibodies (e.g., anti-citrullinated protein antibodies for rheumatoid arthritis),

3]cell types (e.g., white blood cell counts in infection or cancer),

4]metabolites (e.g., phenylalanine in the urine of newborns with phenylketonuria),

5] lipids (e.g., cholesterol and other lipid levels in cardiovascular disease),

6] hormones (e.g., thyroid stimulating a hormone in Hashimoto's Disease),

7] enzyme levels (e.g., various hepatic enzymes for liver cancer),

8] physiological states such as blood pressure or fever,

Malati’s classification: 1080

IMMUNOHISTOLOGICAL MARKERS MARKERS FOR TUMOUR PROGNOSTICATION: 1081

2. Markers to monitor drug resistance: - P-glycoprotein (a transmembrane protein)12,13 - c-erbB-2

4. Tumour angiogenesis: Microvascular density is an independent marker of prognostic relevance.

5. Tumour growth fraction: Ki 67 ANTIBODY

Proliferating Cell Nuclear Antigen (PCNA) & P27 KiP1 Gene

7. Anti-apoptosis genes: bcl-2

Schliephake’s classification: 1082

Classification of Speight and Morgan: 1082

Waxman’s classification: 1084

Manikantan et al’s.Classification: 1079

b. Epithelial membrane antigen

Three main categories (US-NCI):

2. Risk biomarkers (or screening biomarkers)

Correlate with the probability of clinical response to treatment:

They could be:

“Proximal” and “Distal” biomarkers

“The Matrix” (The body fluid or tissue to be tested)

BIOMARKERS AND HUMAN BIOMONITORING

(Human biomonitoring is a scientific technique for assessing human exposures to environmental

Disease Diagnostic Biomarkers to

Predisposition biomarker To identify predisposition to a disease, e.g., genetic

Screening biomarkers To identify those suffering from a disease

Staging biomarker To determine the stage of progression of the disease

Prediction biomarker To predict the course of the disease

Prognostic biomarker To assess disease progression and outcome

Recurrence monitoring biomarkers To identify the recurrence of the disease

The terminology of biomarkers of disease

In oncology, biomarkers have many potential uses. These are

5] Prediction of response to treatment, and