2. 2 of 55
Oral squamous cell carcinoma (OSCC), represents 90% of
oral cancers which is characterized by poor prognosis and
a lower survival rate
3. 3 of 55
0.7 million new cases and 0.3 million deaths are being
noted every year.
In India, approximately 30-40% of all cancer cases are
oral cancers, which are much higher as compared to
Western countries.
In GCRI incidence of oral cancer is 30.03% of total
cancer.
If oral cancer is detected early in stage 1 or 2, the
survival rate may be 80% to 90% ; but
when detected in stage 3 or 4, the chances of survival
may decrease 20% to 30%.
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Smoking and tobacco use are associated with about 75
percent of oral cancer cases.
HPV16 are linked to throat cancer including cancer of
the oropharynx.
Other :
oral submucous fibrosis, erythroplakia, leukoplakia,
candidiasis, endocrine disturbances, syphyllis etc.
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Modern oncology focuses on signal transduction pathways
to derive more knowledge on cancer development.
One of the various molecules studied for this purpose is
the EGFR (170-kDa) , a tyrosine kinase (TK) receptor
located at the cell membrane.
This cell membrane tyrosine kinase is involved in a
variety of cellular activities including proliferation,
differentiation, migration, adhession, survival and death.
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Over-activation of this pathway is considered an
etiological factor in human cancer, which contributes to
Cancer development, metastasis and resistance to
chemotherapy
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TK TK TK
erbB1
erbB2
erbB3
HER1
HER2
HER3
EGFR
neu
erbB4
HER4
No specific
ligands Heregulins
NRG2
NRG3
Heregulins
EGF,TGFa,
b Cellulin
8. 8 of 55
Extracellular
Domain
Transmembrae
Domain
T
K Intracellular
Domain
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EGFR overexpression without gene re-arrangement is
frequently observed in human oral cancers.
structural alterations in the receptor or defective EGFR
pathway contribute to oral carcinogenesis.
EGFR biomarker detection in oral squamous cell-Carcinoma
may fulfill multiple roles in cancer diagnostics, not only for
early detection but also for prognostic evaluation and
treatment selection.
EGFR overexpression or mutation can be treated with
anti-EGFR treatment
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3-4 μm sections of paraffin embedded tumor tissue sections
will be taken on APES coated slides.
Incubate overnight at 60˚c in hot air oven for tissue fixation
The slides will be warmed up to 75°C and incubated for 4
minutes
EZ prep solution will be applied and the slides warmed up to
76°C and incubated for 4 minutes
The slides will be rinsed, warmed upto 90°C and cell
conditioning will be given for 1 hour (extended) for unmasking
of antigen binding sites
Again slides will be rinsed with reaction buffer, warmed up to
37°C and incubated for 4 minutes.
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The slides will be rinsed with reaction buffer, one drop of UV
inhibitor will be added and incubated for 4 minutes.
The slides will be rinsed with reaction buffer, and primary
antibody (EGFR, instrument: cell signaling, dilution: 1:50) will be
added and incubated at 42°C for 1 hour
The slides will be rinsed with reaction buffer, warmed up to
37°C and incubated for 4 minutes
The slides will be rinsed with reaction buffer, one drop of UV
HRP multimer will be added, and incubated for 8 minutes.
17. 17 of 55
The slides will be rinsed with reaction buffer, one drop of UV
DAB and UV DAB H2O2 will be added, and incubated for 8
minutes.
The slides will be rinsed with reaction buffer, one drop of UV
Copper will be added, and incubated for 8 minutes.
The slides will be rinsed with reaction buffer, one drop of
Hematoxylin (counter stain) will be added, and incubated for 8
minutes.
The slides will be rinsed with reaction buffer, one drop of Bluing
reagent (post counterstain) will be added, and incubated for 4
minutes.
The slides will be rinsed with reaction buffer and taken out
from machine, washed in running tap water and dehydrated with
3 washes of acetone and of xylene each and mounted with DPX.
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Scoring is done by semi-quantitative
method.
Score = Staining positivity xx Staining intensity
[Score range : 0 -12]
If, Score range will be 0 – 6 : weak staining
If, Score range will be 7 – 12 : strong staining
19. 19 of 55
QIAamp kit
FFPE by from extraction method
DNA
20. 20 of 55
(1)Remove paraffin : paraffin is dissolved in xylene
and removed
(2) Lyse : sample is lysed under denaturing
conditions with proteinase K
(3) Heat : incubation at 90°C reverses formalin
crosslinking
(4) Bind : DNA binds to the membrane and
contaminants flow through
(5) Wash : residual contaminants are washed away
(6) Elute : pure, concentrated DNA is eluted from
. the membrane
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DNA is eluted in buffer ATE is immediately ready for
use in amplification reaction or for storage at -20ºc.
Purified DNA is free of proteins, nucleases and other
impurities.
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DNA Quantitation by Spectrophotometer
23. 23 of 55
.
DNA will be quantified with 1:1000 dilution of sample and
ratio of A260/A280 will be measured.
A260/A280 ratio will give DNA purity which should be
between 1.65 and 1.85.
A260 is a wavelength of light absorbed by DNA and
proteins have UV absorbance maximum at 280nm.
Absorbance of DNA sample at 280nm. Gives an estimate
of the protein contamination of the sample.
The formulla is used for calculating unknown concentration
of DNA in sample are:
Unknown DNA conc.(μg/ml) = [O.D. A260] Χ Dilution
Factor Χ 50 μg DNA/ml Χ [1 O.D. A260 unit]
27. 27 of 55
PCR product will be digested by Restriction Enzyme
EcoRI to evaluate undigested and digested bands of
EGFR
Undigested bands will be considered as EGFR mutant
gene
Digested bands will be considered as EGFR wild type
gene
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Inclusion:- Patients of oral squamous cell
. carcinoma will be included.
Exclusion:- patient should not have any .
. major illness(diabetes / hypertension).
- patient should not be
. HIV/HbsAg positive.
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The EGFR was found to act as a strong prognostic indicator in
head and neck, ovarian, cervical and bladder cancers.
The EGFR antibodies specifically cetuximab which are directed
against the EGFR, have proven efficacy in the treatment of
metastatic colorectal cancer (mCRC)
Gefitinib, is a drug used for certain breast, lung and other
cancer. Gefitinib is an EGFR inhibitor, like erlotinib, which
interrupts signaling through the epidermal growth factor
receptor (EGFR) in target cells
32. 32 of 55
Ethical Issues : None
Concent Forms : Yes
33. 33 of 55
(1) Sarkis et al. Head & Neck Oncology 2010, 2:13
http://www.headandneckoncology.org/content/2/1/13
(2) Hindawi Publishing Corporation Journal of Dental
Surgery Volume 2014, Article ID 158709, 7 pages
http://dx.doi.org/10.1155/2014/158709
(3) Soler R.P. HER1/ EGFR Targeting :Refining
the . trategy. Oncologist 2004 ; 9 : 58 – 67.
(4) Strausberg R.L, Simpson A.J.G, Old L.J, Riggins
. G.J. Oncogenomics and the development of
new cancer therapies. Nature 2004 ; 429:46949
