A study of EGFR protein expression and mutation in oral cancer patients.

Guide:Dr.Hemangini H.VoraImmunohistochemisty and Flow cytometry Division

Rakesh S.ParmarRoll No. :CB-0613

M.Sc. Cancer BiologyGCRIGCRI

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Oral cancer is a type of head and neck cancer

It is the sixth most common cancer worldwide.

It forms in the tissue of oral cavity or the oropharynx:Buccal mucosa and alveolar mucosa, lower lip, toungue,hard and soft palate, floor of the mouth, gingiva etc.

Oral squamous cell carcinoma (OSCC), represents 90% of oral cancers which is characterized by poor prognosis and a lower survival rate

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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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These cellular activities carried by activating multiple downstream cell signaling pathways including the RAS/RAF/MAPk , PI3/AKT pathway etc.

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 TKTK

erbB1HER1EGFR

erbB2HER2neu

erbB3HER3

erbB4HER4

No specific ligands Heregulins

NRG2NRG3

Heregulins

EGF,TGFa,

b Cellulin

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T

K Intracellular Domain

Transmembrae Domain

Extracellular 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-Carcinomamay fulfill multiple roles in cancer diagnostics, not only forearly detection but also for prognostic evaluation and treatment selection.

EGFR overexpression or mutation can be treated with anti-EGFR treatment

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To evaluate EGFR protein expression in formalin fixed parafin embedded tissue block by Immunohistochemistry (buccal mocosa and tongue)

To evaluate EGFR (exon 19) mutation in formalin fixed parafin embedded tissue block by PCR

Intercorelation between EGFR protein and

. mutation status

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EGFR protein and mutation incidence will be corelated with

Clinical parameters:Age, Gender,Tobacco habit and Pathological parameters :Tumor size, nodal status,disease stage, histologic grade

Detailed clinical history of patients will be

noted

Statistical analysis will be carried out using SPSS software

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Total 50 formalin fixed parafin embedded tissue block of buccal mucosa and tongue will be collected from oral carcinoma patients.

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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 fixationThe 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.

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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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• 0 : 0% cells

• 1+ : <10% cells

• 2+ : 10-50% cells

• 3+ : >50% cells

Staining positivity • 1+ : weak staining

intensity

• 2+ : moderate staining intensity

• 3+ : strong staining intensity

Stainingintensity

Scoring is done by semi-quantitativemethod.

Score = Staining positivity x Staining intensity [Score range : 0 -12]If, Score range will be 0 – 6 : weak staining If, Score range will be 7 – 12 : strong staining

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(1)Remove paraffin : paraffin is dissolved in xyleneand removed

(2) Lyse : sample is lysed under denaturing conditions with proteinase K

(3) Heat : incubation at 90°C reverses formalincrosslinking

(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 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]

.

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.

.

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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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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

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)

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Ethical Issues : None

Concent Forms : Yes

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(1) Sarkis et al. Head & Neck Oncology 2010, 2:13http://www.headandneckoncology.org/content/2/1/13

(2) Hindawi Publishing Corporation Journal of Dental Surgery Volume 2014, Article ID 158709, 7 pageshttp://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

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(5)Okamoto I: Epidermal growth factor receptor in relation to tumordevelopment: EGFR-targeted anticancer therapy. FEBS J 277:309-315, 2010.

(6)Mitsudomi T and Yatabe Y: Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. FEBS J 277: 301-308, 2010.

(7)http://www.oralcancer-screening.org/events/pdf/OCF-Facts-2014.pdf

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(8)Ramos-Vara, JA; Miller MA (2014). Veterinary Pathology 51 (1): 4287.doi:10.1177/0300985813505879.PMID 24129895. Retrieved 13 February 2014.

(9)BMC Research Notes 6: 513. doi:10.1186/1756-0500-6-513 2013. PMID 24314313

(10)QIAamp DNA FFPE Tissue Handbook 04/2010

(11)http://dx.doi.org/10.1016/j.ctrv.2008.11.005

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