SEROLOGICAL TESTS

FOR CANCER
DIAGNOSIS
By:
Prachi (BTB17/114)
Shweta Shukla (BTB/17/121)
Sneha Guha Thakurta
(BTB/17/126)
Shreeya Sharma (BTB/17/156)
 GENERAL
OVERVIEW
o Even with modern methods and with considerable public education with regard to cancer, the

disease is often not diagnosed until it is beyond the stage at which cure might be effected.

o The need for a sero diagnostic test for general screening purposes for cancer detection is

tremendous.

o The major objective of cancer serodiagnostic test methods is to discover a general test that will

detect cancer in a high percentage of cases while it is in an early stage; that can be done in any

laboratory; and that is simple and inexpensive.

o Serological biomarkers play an important role in these serodiagnostic tests.

 GENERAL
OVERVIEW
 Serological markers identify a variety of stages in the process of carcinogenesis such as

inherited or acquired susceptibility to cancer, environmental exposures to carcinogens,

biological effects of exposures, and the presence of pre invasive or invasive cancer.

 Serological markers may be used in clinical trials to select high risk but disease-free

individuals for participation in clinical trials based on susceptibility factors or carcinogenic

exposures.

 Serological markers of carcinogenesis have widespread applications in clinical research and

potentially for clinical practice. Currently, the only limitation to their widespread use is the

availability of validated serological markers .

 CHARACTERISTICS OF
SEROLOGICAL BIOMARKERS
 Accessibility and Acceptability- The accessibility and acceptability of obtaining blood samples
are key practical advantages to using serological markers in clinical research. Because of the
ease and acceptability of blood sampling, serological markers may be used in large-scale
prevention trials with minimum risk to participants.

 Quality of Serological Markers.-Serological markers of carcinogenesis should be valid and

reliable. Validation studies should be carefully conducted to ensure that the marker is
accurately measuring the specific stages in the disease process. The sensitivity and specificity
of the tests defines its validity.
 CHARACTERISTICS OF
SEROLOGICAL
BIOMARKERS
 Sensitivity measures the ability of a positive test for a marker to correctly identify the disease
process, whereas specificity measures the ability of a negative test for a marker to correctly identify
those who do not have the disease process.

 Reliability- This type of application depends on assay reliability on repeated measures to ensure that
the rate of change is due to biological processes and not laboratory variability. Serological tests
should be applied only after establishing the validity and reliability of an assay.

 Rapid & inexpensive - Serological tests for markers should be rapid and inexpensive and should
require small amounts of blood in order to be practical for population-based applications.
 ADVANTAGES AND
DISADVANTAGES
OF SERODIAGNOSTIC TEST FOR
CANCER DETECTION
 It helps in detecting cancer
early before the patients shows  False positive test results are
ADVANTAGE symptoms and when it is easier possible. DISADVANTAGE
S to treat.  False negative results can also
S
 Early detection leads to early occur leading to higher stage of
treatments and less time spent cancer
in recovering and chances of
spread also becomes less.
 Serological Test for
cancer detection

Enzyme based tests Antibody based tests

There are many different

types of serodiagnostic
tests being conducted for
cancer detection. Used for detection of
certain enzymes present
Used for the detection of
specific antibodies
In this presentation we are going to discuss two of them. in the sera of patients present in the sera due to
due to the presence of cancerous cells in the
cancer cells in the body body.
 AUTOANTIBODIES
AN EFFECTIVE SEROLOGICAL BIOMARKER FOR
CANCER
• Cancer cells are capable of inducing an immunological response in the body resulting
in the production of autoantibodies , which target the TAAs of cancer cells .
• As these serum immuno-biomarkers are found in the patient’s body before the
development of any clinical symptoms of cancer, these are now being used for the early
diagnosis for a range of cancers. 

ADVANTAGES :
• Being present in the serum of the patient , these could be easily
screened using an non-invasive approach .
• These are cost effective .
• Usually these are also found in high concentrations in the serum,
and are generally stable and bind with high specificity to their
specific antigenic proteins, thereby being easily detectable.
 TECHNIQUES USED FOR
AUTOANTIBODY DETECTION IN SERUM

• There was the need that the autoantibodies , which are to be used in diagnosis
of cancer should be
• Highly Specific
• Highly sensitive .
• Panels of multiple tumor- associated autoantibodies with high specificity and
sensitivity are sought therefore for translation into simple biomarker panel
tests for routine clinical diagnosis, since a single biomarker is not available
with all the desired characteristics
 MECHANISM OF PRODUCTION OF AUTOANTIBODIES

Post-translational modifications (PTMs) :

PTMs such as phosphorylation, proteolytic cleavage, Immunogenic modifications :
glycosylation make these self-proteins more • Intracellular proteins that are re-
detectable as foreign bodies , thereby inducing a
immune response.- autoantibodies . localized to the tumor cell surface
Effect – • Structural mimicry - TAAs that have
• These generate neo-epitopes
• Or enhance the self-epitope presentation as well
structural similarity to cross-reacting
as increased affinity to the MHC molecule foreign antigens.
TAAs and their role in
autoantibody production :
The autologous proteins of tumor cells,
commonly referred to as TAAs, are thought
to be altered in a ways like
• Overexpression
• Mutation
• Aberrant degradation
• Misfolded. that renders these proteins
immunogenic.

Aberrant Cell Death – release of TAAs: Over expression and mutation in

• Aberrant tumor cell death, causes the genes:
release of these modified intracellular • The p53 gene by overexpression or point
TAAs, which when presented to the mutation becomes immunogenic in nature.
immune system produces and elicited • CTAs ( cancer testis antigens ) and other
response of autoantibodies . overexpressed proteins increase the
• Tumor cell death also releases proteases antigenic loads on the primary immune
that would generate cryptic self-epitopes to response, thereby eliciting the production
trigger an autoimmune response. of autoantibodies in the body.
TECHNOLOGIES FOR AUTOANTIBODIES DETECTION
 ELISA
•
TEST
Cancer detection and treatment has to be made more broad and new cancer biomarkers and screening technique

strategies need to be employed at a high level.

• One of the most frequent technique used is the ELISA (Enzyme-linked immunosorbent assay) technique for serological

detection of cancer. This technique helps to get new serum markers such as Nicotinamide N-Methyltransferase (NNMT)

and extracellular Protein Kinase A (ecPKA)

• In this type of assay, the cancer antigens are detected and quantified indirectly using the auto-antibodies against the

tumor proteins which are present in the human serum.

• The result of the optimisation and validation process was in the case of ecPKA a reproducible and stable assay. In case

of NNMT the assay was probably not sensitive enough.

 MECHANISM OF
DETECTION USING ELISA
• The tumor protein 53- induced nuclear protein 1(TP53INP1) plays an important role during the cell response in place of
the tumor suppressor gene p53 in many cancers.

• This nuclear protein is expressed at a very high level in some pathological situations such as inflammation and prostate
cancer.

• This successful development of the enzyme-linked immunosorbent assay(ELISA), can detect TP53INP1, which takes
advantage of the molecular tools which include the monoclonal antibodies(mAb) and recombinant proteins.

• The ELISA principle is based upon the sandwich immunoenzymatic system

• The TP53INP1 protein is trapped by the first specific mAb coated on microplate then recognised by the second specific
mAb. This method allows the detection of TP53INP1 in prostate cancer.
 MECHANISM OF
DETECTION USING ELISA
• The TP53INP1 gene encodes two protein isoforms, TP53INP1-alpha and TP53INP1-beta and they are identical in
sequence, except the additional C-terminal part TP53INP1-beta.

• The mAbs are raised against the TP53INP1-alpha in order to recognise both the isoforms.

• The mAb E12 which is efficient in immunohistochemistry on paraffin-embedded organ sections is widely used.

• Interestingly, different studies showed that TP53INP1 is either lost or overexpressed in tumoral part of different organs.

• TP53INP1 is over-expressed in prostate cancer and thus ELISA method can help detect this gene.
The ELISA principle is based on a sandwich immunoenzymatic system. The first step is a coating of microtitration plates with
TP53INP1-specific antibodies. Then the sample containing TP53INP1 is deposited in the wells and allowed to bind to its
specific mAb (second step). All proteins not specifically bound are eliminated by washing. Then anti-TP53INP1 antibodies
coupled to biotin are allowed to attach to the bound TP53INP1 (third step). The fourth and last step is the detection of
antigen-antibody by a streptavidin-peroxidase (HRP) complex which is visualized by the addition of a chromogenic substrate
(TMB on the scheme). The intensity of the color reaction is proportional to the quantity of TP53INP1 bound in the second
step
HAPTOGLOBIN
A WIDELY USED CANCER BIOMARKER

• Acute phase plasma glycoprotein

• Secreted by the liver into the blood
• Attaches itself to any ‘free’ hemoglobin, ie- hemoglobin not
attached to a red blood cell
• This binding is irreversible and the complex thus formed is
removed by the reticuloendothelial system
• Haptoglobin prevents loss of hemoglobin to urine and
conserves iron
• a tetrameric protein with two α/β dimers
• β-Chains are identical in all haptoglobin types
• polymorphisms of haptoglobin arise from differences in α-
chains
 Increased levels of haptoglobin are found in
infection, inflammation, and various cancers:

1. Lung cancer

2. Bladder cancer

3. Malignant lymphoma

4. Breast Cancer

5. Esophageal squamous cell carcinoma

6. Epithelial ovarian cancer

 HAPTOGLOBIN IN LUNG CANCER
According to the 2016 research by Chang YK et. al
- Lung adenocarcinoma
- A proteomic approach with two-dimensional chromatography
- Serum proteins were separated on ProteomeLab PF2D platform
- The first dimension uses chromatofocusing column and the second uses HPLC
column
- Changes in serum protein expression in lung adenocarcinoma patients and healthy
controls were observed by mass spectrometry
- Eight proteins were identified including HP
- HP levels in lung adenocarcinoma patients were significantly higher than in healthy
controls
- Expression of HP was detected in 40% of lung adenocarcinoma by
immunohistochemistry and further confirmed by Western blot

According to the 2007 research by Hoagland LFM et. al.

- Non small cell lung cancer
- 2‐dimensional difference gel electrophoresis (2D ‐DIGE)
- gel spots were identified as the β chain of Hp
- haptoglobin in serum was increased with stage
 HAPTOGLOBIN IN EPITHELIAL OVARIAN CANCER

According to the 2007 research by Changqing Zhao et. al.

- sandwich enzyme-linked immunosorbent assay
- immunohistochemical analysis indicated intense staining of haptoglobin in the
cytoplasm of ovarian cancer cells obtained from late-stage ovarian cancers when
compared with those from benign ovarian tumors
- malignant ovarian tumor tissues possess the capacity to synthesize this protein
- significantly elevated haptoglobin concentrations are associated with poor
outcome for survival in women with epithelial ovarian cancer
- two-dimensional gel electrophoresis, - decreased intensity of staining for this
protein was observed in samples from patients undergoing chemotherapy
 CONCLUSION

By avoiding the progression of a cancer to an often incurable metastatic stage, early detection of all cancers
may help us to increase the survival rates and better quality of life. The golden standard diagnostic
techniques used today, such as mammography for breast cancer detection, are highly successful, however,
they are often subject to detection bias and may result in false-negative diagnosis of a patient whose tumor
has been overlooked because of the limitations of current diagnostic techniques. To aid the early detection of
all cancers and to ensure that all oncology patients are correctly diagnosed, the focus now lies in finding
biomarkers, indicating a positive diagnosis at an earlier stage. Therefore in this case serological biomarkers
can play critical role in improving such treatment technologies .The above mentioned methodologies as well
as the enzyme based tests have enabled the simultaneous identification of several autoantibodies for different
cancers and these are currently being tested for their potential to serve as diagnostic biomarkers for specific
cancers. So far, the clinical application of most identified autoantibodies has been hindered by their low
sensitivity, specificity, and predictive value percentages as well as poor reproducibility within different
experimental designs and applications of the methodology. Presently these serological markers have been
effectively used to detect lung, breast , prostate , stomach cancers.
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 By:
Prachi (BTB17/114)
Shweta Shukla (BTB/17/121)
Sneha Guha Thakurta
(BTB/17/126)
Shreeya Sharma (BTB/17/156)