EPITHELIAL TUMOR MARKERS
Dr. VARUN SURYA
• P.G student
• Dept. of oral & maxillofacial
• Government dental college
• HOD & professor
• Dept. of oral & maxillofacial
• Government dental college
• Earlier the most common determinant or marker
of carcinomatous transformation in a tissue was
the histopathologic presence or absence of
• However the expanding field of oncology has
revealed new and more specific markers that
would help to determine the degree of cell
alteration and enable a better understanding of
the degree of malignant transformation of these
• Several abnormal cellular products are produced
by the neoplastic cells and also by the body in the
presence of such an abnormal situation.
• Such cellular products can be detected in the
various body fluids and on the surface of the
cancer cells either by biochemical methods or by
• These products that are detected and measured
are known as ‘tumor markers’.
• A tumor marker is 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 normal
tissue or to determine the presence of a
tumor based on measurement in the blood or
• Tumor markers can be found in cells, tissues
or body fluids.
• They can be measured quantitatively or
qualitatively by chemical, immunological or
molecular biological methods to determine
the presence of neoplasia
• The distinguishing biological characteristics of
tumor cells, such as their capacity for invasion,
metastasis, unlimited proliferation, evasion of
apoptosis and angiogenesis, are all mediated
by complex molecular pathways, any of the
components of which are potential tumor
• Few markers are specific for a single
individual tumor (tumor specific markers).
• Most are found with different tumors of the
same tissue type (tumor associated markers)
IDEAL TUMOR MARKER
• It should be highly sensitive and should have low false
• It should be highly specific and should have low false
• It should have high positive and negative predictive value
• 100% accuracy in differentiating between healthy
individuals and tumor patients
• It should be able to differentiate between neoplastic and
non-neoplastic disease and show positive correlation with
tumor volume and extent
• It should predict early recurrence and have prognostic
• It should be clinically sensitive i.e., measurable at
early stage of tumor
• Its levels should be preceding the neoplastic
process, so that it should be useful for screening
• It should be either a universal marker for all types
of malignancies or specific to one type of
• It should be easily assayable and be able to
indicate all changes in cancer patients receiving
1.Screening in General Population
• The concept of screening an apparently
healthy population is to find out the presence
of occult disease at an early stage, wherein
effective therapeutic intervention is likely to
• However, inadequate test sensitivity and
specificity in discrimination of a low
prevalence situation, limits the role of these
markers to smaller confines
• The identification of primary disease is an important
function of any tumor marker.
• For definitive results it is essential that the marker be
100% specific and 100% sensitive.
• For example, the presence of Bence Jones proteins in
the urine remains one of the strongest diagnostic
indicators of multiple myeloma.
• In the vast majority of diagnostic situations, circulating
tumor markers are used. If appropriate, in conjunction
with diagnostic imaging and tissue biopsy, perhaps
coupled with immuno-histochemical staining of tumorspecific antigens.
• Most of the tumor markers are present in normal,
benign and cancerous tissue and are usually
• However, they can still be used in differential
diagnosis of suspicious lesions.
• The tumor markers are commonly employed to
decide the histogenetic origin of oral cavity
neoplasms and therefore help ruling out atleast
some of the entities on the differential diagnosis
4.Clinical Staging of Cancer
• Clinical staging of the cancer is aided by
quantitation of the marker, that is, the serum
level of the marker reflects the tumor burden
• Markers can also detect microscopic
metastasis with radioimmune detection
5.Nuclear Scanning of Injected
• Radioactive labeled antibody specific tumor
marker is injected into patients suspected of
having undetected tumor metastasis.
• Accumulation of specific antibody in tumor cells
may be visualized by isotope scanning with
appropriate subtraction techniques to overcome
background activity or by emission tomographic
• The labeled antibody can be injected
intravenously or into the lymphatic circulation for
more efficient binding to metastatic site
6.Prognostic Indicators for Disease
• Tumor markers can provide prognostic
information, which may include indications for
choice of therapy and likelihood of response.
• The estimate of survival time based on
evidence of metastatic disease can also be
7. Evaluating the Success of Treatment /
Monitoring the Response to Therapy
• After successful initial treatment, such as surgery, the marker value should
• The rate of the alteration can be predicted by using the half-life of the
• For e.g. If the marker is supposed to be decreased after t/t, but the
observed half-life of the marker after treatment is longer than the
expected half-life, then the treatment has not been successful in
completely eliminating the tumorous tissue.
• The magnitude of marker alteration may, however, reflect the degree of
success of the treatment or the extent of disease involvement.
• Most tumor marker values correlate with the effectiveness of treatment
and responses to therapy.
• Sometimes, however, the values may show an initial delay before
demonstrating the expected pattern of change.
8.Detecting the Recurrence of Cancer
• It is of utmost importance to precede and to predict
recurrence so that relapse can be picked up as soon as
possible and appropriate measures may be
• A clinically sensitive tumor marker will reflect the
amount of viable tumor burden, assuring homogeneity
of production in tumor cell population.
• This quantitative relationship is of great importance
not only to monitor response to therapy, but also to
alert the clinician to the onset of drug resistance.
• Cytokeratins are also known as keratins and
they are classified as intermediate filament
proteins and are found with in the cytoplasm
of all epithelial cells
• Cytokeratins are a family of 20 members
which are present in normal epithelial cells
and their tumors.
• They are divided in to two broad groups
according to their isoelectric point
Basic group (type 1)
Acidic group (type 2)
MW: 52-67 kD
MW: 40-56 kD
• The keratins are broadly divided into
• 1. Primary keratins are those keratins which are always
synthesized by the epithelial cells on a regular basis, e.g.,
K8/18 in simple epithelia, K5/14 in stratified epithelia.
• 2. Secondary keratins are those types of keratins which are
produced by the epithelial cells in addition to or instead of
primary keratins, e.g., K7/19 in simple epithelia, K15, and
K6/16 in stratified epithelia.
• During epidermal differentiation, low molecular weight
keratins 5/14 of the basal layer are replaced by high
molecular weight keratins 1/10,11 which can be used as a
marker of keratinization
Based on molecular weight
• molecular weight of 40kDa.
• mainly distributed in glandular and simple
• molecular weight between 40kDa and
• found in stratified epithelia.
• molecular weight of 57kDa
• seen in keratinized stratified epithelia
Functions of Keratins
• Keratins fundamentally influence the architecture and mitotic
activity of the epithelial cells.
• Keratins and associated filaments provide a scaffold for
epithelial cells and tissues to sustain mechanical stress,
maintain their structural integrity, ensure mechanical
resilience, to protect against variations in hydrostatic pressure
and establish cell polarity.
• Keratins and its filaments are involved in cell signalling , cell
transport, cell compartmentalization and cell differentiation.
• Keratin filaments also influence cell metabolic processes by
regulating protein synthesis and cell growth.
• Keratins may also be involved in the transport of membrane
bound vesicles in the cytoplasm of the epithelial cells.
• Keratins are usually found in pairs, with one
type I co-existing with one type II , each
encoded by its own gene.
• In the absence of pairing , they are unstable &
vulnerable to degradation by proteases.
• Cytokeratin profile reflects both cell type &
differentiation status in different types &
different layers of epithelia.
CK in basal layer
CK in supra-basal layer
Cytokeratin 4 in human
Simple epithelial keratins
• K8 & k18
• Not normally found in stratified squamous
epithelium, but rather in single cells, such as
glandular tissue (e.g., salivary glands).
Cytokeratin expression in pathology
appear early in
seen in more
from cuboidal or
weight keratins appear
Associated with the
formation of more
seen in more complex
epithelia and their
(i.e., squamous cell
• In epithelial tumors, some keratin
polypeptides are either not expressed or are
• After their release from proliferating or
apoptotic cells, cytokeratins act as useful
markers for epithelial malignancies.
• Therefore, keratins have gained importance as
marker proteins useful in diagnosis of tumors
of epithelial origin.
• It should be remembered that cytokeratin
expression is not strictly restricted to epithelia
and their tumors.
• Some non-epithelial derived tumors, such as
melanoma, schwannoma, glial tumors, large cell
immunoreactivity for cytokeratins.
Three most applied cytokeratin markers
1. tissue polypeptide antigen (TPA)- broad
spectrum test, and it measures cytokeratins
8, 18, and 19.
2. tissue polypeptide specific antigen (TPS)
3. CYFRA 21-1
• TPS and CYFRA 21-1 assays are more specific
and they measure cytokeratin 18 and
• best IHC marker for Merkel-cell carcinomas
• markers for poorly differentiated squamous cell
Ck 14 & 19
• a marker for odontogenic epithelial origin
Ck in odontogenic tumors
• Odontogenic tumors with epithelial component
frequently express CK 14 and 19.
• Numerous studies have shown that AOT and
ameloblastomas express CKs 5, 14 and 19.
• IHC studies by Martínez- Mata et al. proved that
most of tumors of mesenchymal origin like
odontogenic myxoma (OM) do not express CK 14
• Thus, CK 14 and 19 can be used as markers for
tumors of odontogenic epithelial origin.
• Keratin expression patterns are characteristic for distinct
stages during cellular epithelial differentiation from
embryonal to adult and of the internal maturation program
• Epithelial tumors including metastasis most widely retain their
keratin patterns of their normal origin; thus the determination
of the keratin patterns of tumors is widely exploited for cell
and tumor typing.
• Therefore keratins have evolved to be one of the most potent
epithelial differentiation and tumor markers in cell biology,
embryology, and surgical pathology.
• Specific antibodies against several keratins are routinely used
for immunohistochemical typing of carcinoma in tumor
• Keratins can be used as differentiation markers in normal oral
• markers for simple epithelial differentiation
• markers for keratinized epithelium
• markers for non-keratinized epithelium.
• considered as hyperproliferative markers
• expressed in sites of high epidermal keratinocyte turnover and in pathological
hyperproliferative conditions affecting the skin
• strongly stained in most
carcinoma, renal cell carcinoma,
and neuroendocrine carcinoma.
• monitoring of fragments of these
keratins in the serum as
serological tumor markers to
monitor cancer load, cancer
progression, and response to
• The detection of soluble K19
fragments in the serum released
by carcinoma cells by the CYFRA
21-1 has found broad clinical
application as a marker to
monitor treatment and evaluate
response to therapy.
• K20 is a potent
immunohistochemical marker in
tumor pathology since its
peculiar expression spectrum is
essentially maintained in the
corresponding primary and
• K20 positivity is seen in majority
of gastric adenocarcinoma,
transitional cell carcinoma, and
Merkel cell carcinoma.
• K20 is considered to be a
consistent marker for Merkel cell
• Cyfra 21-1 is a soluble fragments of Ck 19
generated by necrosis of tumor cells.
• This marker is recognized by two monoclonal
antibodies against fragments of CK 19 in the
• The assumption is that Cyfra 21-1 is released into
the bloodstream during cell death, and therefore
its level correlates very well with the tumour
mass, or more specifically with the necrosis in the
tumour, which is a function of the tumour mass.
• Ilana Doweck et al (1995) measured Cyfra 21-1
(cytokeratin fraction 21-1) in OSCC patients
and found Cyfra 21-1 levels were in good
correlation with the tumor stage .
K6 and 16 : typically and strongly expressed in squamous cell carcinoma
of different sites.
K6, 16 and 17 : inducible keratin upon stress, injury and inflammation and
therefore increased expression is seen in squamous cell carcinoma.
The increased expression of K17 which is usually absent in normal
epithelia could be attributed to neo-expression during tumorigenesis.
A unique feature of K17 is its inducibility after skin injury.
After K6/16, K17 is switched on in regenerating and migrating epidermal
keratinocytes upon wound healing
• A study of 18 ﬁne needle aspiration biopsies
obtained by traversing the cyst cavity and
contacting the opposite cyst wall, compared the
CK10 expression of sampled epithelial cells with
CK10 expression of the postoperative biopsies .
• The authors reported that this technique
accurately distinguished OKCs from nonkeratinised dentigerous and radicular cysts in the
• Another paper reported strong expression of
cytokeratin 17 in all layers of the epithelium in all of six
OKCs from patients with the NBCCS.
• In a sample of seven sporadic OKCs
– two cases : strong expression of cytokeratin 17
– other 5 : showed weaker and irregular staining
• Dentigerous cysts showed weak positivity for
cytokeratins 13, 17 and 18.
• It was suggested that cytokeratin 17 might be a useful
– to distinguish OKCs from other jaw cysts
– to distinguish syndrome from sporadic OKCs
Ck in oral squamous cell carcinomas
Well -differentiated oral squamous cell carcinomas.
• increased expression of K1/10, K4/13
Less well-differentiated oral squamous cell carcinoma
• express neither of these keratin pairs to any significant extent but do express K19.
Moderated and poor oral squamous cell carcinoma
• May express K8 and sometimes K7 and 18.
• The expression of K8 and K18 in oral squamous cell carcinoma is considered to be
an independent prognostic marker .
• Also K5 and K6 are considered as useful markers for squamous cell carcinomas in
histologically uncertain, poorly differentiated or metastatic tumor cases.
OSCC vs mucoepidermoid carcinoma
OSCC (grade III)
carcinoma (high grade)
strikingly positive focally positive
areas of squamous
positive for CK10
positive for CK13
• also expresses CK7, 8, and 19 and negative for CK10.
• usually absent in these tumors, reflecting that true keratinization is an uncommon
finding in mucoepidermoid carcinoma.
• found to be useful in differentiating mucoepidermoid carcinoma from other salivary
• Consistent expressions of K13 and K19 may
provide a useful marker of odontogenic
epithelium in general.
• K19 scores are found to reflect histological
differentiation as well as predicting the clinical
• Combining K19 immunostaining with regular H &
E stain may be helpful to facilitate and assure
assigning a more accurate grade for oral epithelial
Desmoplakins are highly conserved proteins
present within the desmosomal plaques of
desmoplakin I and II, immunoreactivity can be
localized to peripheral punctate regions in a
wide variety of epithelial cells, meningeal cells,
• Desmoplakins can also be identified within the
glandular component of synovial sarcomas but
not in other sarcomas.
• Thus desmoplakins represent an additional
marker of epithelial differentiation independent
• Mutations in this gene are the cause of several
cardiomyopathies and keratodermas as well as
Desmoplakin I/II . Immunoperoxidase staining of formalin
fixed, paraffin-embedded human skin tissue showing
cytoplasmic staining of epidermal cells.
• AFP is a very popular and extensively studied
carcinoembryonic glycoprotein / Oncofetal
antigen. Alpha-fetoprotein (AFP) is a 70kD
• AFP is normally produced during fetal
development by the liver and yolk sac. After
birth, the levels drop off rapidly, and by the
second year only trace amounts are
detectable in serum.
• Serum AFP measurement is of valuable clinical
aid in diagnosis, prognosis and monitoring
carcinoma, hepatoblastoma, non-seminomato
us testicular germ cell tumors the embryonal
carcinoma, teratoma, choriocarcinoma and
yolk sac carcinoma etc., germ cell tumors of
ovary and extragonadal germ cell tumors
Human fetal liver tissue stained with
alpha Fetoprotein antibody
Carcinoembryonic antigen (CEA)
• CEA is an oncofetal glycoprotein.
• It is expressed in normal mucosal cells .
• Elevated levels of CEA is seen in malignancies
such as salivary gland tumors i.e. carcinomas with
glandular differentiation and can also be
observed in squamous cell carcinomas.
• Non-neoplastic conditions associated with
elevated CEA levels include cigarette smoking,
peptic ulcer disease, inflammatory bowel disease,
pancreatitis, hypothyroidism, biliary obstruction,
Epithelial membrane antigen (EMA)
• High molecular weight protein (265 to 400)
• Shows apical staining on a wide variety of
normal & neoplastic epithelial cells.
• Expression of CEA and EMA, as with that of
keratin 10/11, appeared to be dependent on
epithelial structure and diﬀerentiation.
• Their expression by OKC epithelium was
restricted to weak and patchy staining of the
surface layers, although areas with ’disordered
structure’ showed cytoplasmic staining of most
cells particularly for EMA.
• In the dentigerous cysts both antigens, more so
EMA, were found in most epithelial cells and in
the radicular cyst their distribution was variable.
• Diﬀerences in cytokeratin, EMA and CEA between the
parakeratinised OKC and the orthokeratinised variety have
demonstrated that the latter, having a considerably less
aggressive behaviour, is a diﬀerent entity and should bear a
diﬀerent name, ’orthokeratinised odontogenic cyst’ (OOC) .
• Keratins associated with squamous diﬀerentiation or
corniﬁed epithelium showed pronounced staining in all but
the basal cell layer of the OOC whereas in the
parakeratinised OKC staining was found only in the upper
and surface parakeratin layers.
• Both EMA and CEA were consistently present in the surface
parakeratin layer of the OKC but completely absent in the
Leu M1 (CD15)
• Useful in identifying carcinomas
distinguishing them from mesotheliomas.
• It is also granulocytic marker & stains reedsternberg cells of hodgkin’s disease.
• Ber –EP4 is another widely distributed
epithelial cell antigen which react with a pair
of glycoprotein (34 & 49 kD).
• Many carcinomas
are +ve ,whereas
mesothelial cells & mesotheliomas are usually
• TAG-72 (tumor associated glycoprotein)
• Helpful in distinction between carcinoma &
1. Too many markers
• Each year clinicians are faced with the discovery
of a multitude of new molecular markers, often
with claims that they will provide new
information that is important for determining
prognosis or improving cancer treatment.
• The clinician is then faced with the arduous task
of trying to keep with the technology and sorting
through the literature to determine which of
these tumor markers are relevant to the care of
their individual patients
2. Creates un-necessary confusion
• The relevant question that needs to be answered is
whether the tumor marker in question will alter the clinical
decision-making to result in a favorable outcome for the
patient and unfortunately most often it does not!
• The absence of large, prospective, multicenter trials to
assess the utility of any of the tumor markers only adds to
• Despite the specificity of some tumor markers, a negative
marker value does not rule out recurrent disease and other
diagnostic modalities such as imaging still have to be
performed as part of the surveillance program.
3. Technique is not fully developed
• Despite the advent of monoclonal antibody
and immunoassay technologies, which have
dramatically increased our ability with a high
degree of reproducibility to identify minute
quantities of particular substances in serum,
there are currently no tests for tumor markers
of adequate sensitivity and specificity to
permit routine screening or early diagnosis of
a particular type of cancer.
4. Inadequate sensitivity and
• The lack of adequately sensitive and specific test can be attributed to a
• Most tumor markers are substances produced by some types of nonneoplastic cells; although perhaps in much lower quantities than they are
produced by tumor cells.
• Varying levels of these markers may be present at all times in different
tumor free individuals and varying levels of the particular marker may be
present in different individuals with a particular tumor type.
• Most tumor markers show some overlap between the levels seen in
controls and in cancer-affected individuals. Thus it becomes necessary to
choose a threshold at which level particular marker is considered
abnormal and suggestive of the presence of that tumor type.
• Setting the threshold lower increases sensitivity by including a higher
number of patients with a particular tumor, but decreases the specificity
by also including more tumor free individuals, while raising the threshold
will have the reverse effect
• If a tumor marker assay was 95% sensitive or 95%
specific and were to be applied to population
containing 5,00,000 cases in a population of 100
million people, it would yield 4,75,000 true positive
and 5 million false positive results.
• Thus for every cancer case detected there would be 10
cancer free individuals who would needlessly undergo
further work up and psychological stress resulting from
a false positive test. These numbers in reality would
become even worse because tumor marker in current
use have sensitivity and specificity below 95%
particularly for small lesions.
5. Financial and psychological cost
• Knowledge by the patient of a rising tumor
marker may cause significant anxiety,
particularly, if this information does not alter
the treatment plan as patients equate rising
tumor marker levels with worsening disease.
• Thus the financial and psychological cost to
the society , of routine screening for early
cancers using currently available tumor
marker would be unreasonable.
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