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study of EGFR protein expression and mutation

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This document discusses oral squamous cell carcinoma (OSCC), which represents 90% of oral cancers and is characterized by a poor prognosis and lower survival rate. It notes that 0.7 million new cases and 0.3 million deaths occur annually from OSCC globally. In India, approximately 30-40% of all cancer cases are oral cancers, which is much higher than in Western countries. Risk factors for oral cancer include smoking, tobacco use, HPV infection, oral submucous fibrosis, and certain other conditions. The document discusses using immunohistochemistry to examine EGFR expression levels in oral cancer tissue samples and using PCR and restriction enzyme digestion to analyze for EGFR mutations, which can indicate prognosis and treatment options.

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study of EGFR protein expression and mutation
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 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%.
4 of 55 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.
5 of 55 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.
6 of 55 Over-activation of this pathway is considered an etiological factor in human cancer, which contributes to Cancer development, metastasis and resistance to chemotherapy
7 of 55 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 of 55 Extracellular Domain Transmembrae Domain T K Intracellular Domain
9 of 55 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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13 of 55 Total 50 formalin fixed parafin embedded tissue block of buccal mucosa and tongue will be collected from oral carcinoma patients.
14 of 55 Immunohistochemistry (IHC)
15 of 55 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.
16 of 55 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 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.
18 of 55 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 of 55 QIAamp kit FFPE by from extraction method DNA
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
21 of 55 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.
22 of 55 DNA Quantitation by Spectrophotometer
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]
24 of 55 Polymerase chain reaction (PCR)
25 of 55 . . .
26 of 55 Restriction Endonuclease Digestion
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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30 of 55 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.
31 of 55 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 of 55 Ethical Issues : None Concent Forms : Yes
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
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study of EGFR protein expression and mutation

  • 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%.
  • 4. 4 of 55 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.
  • 5. 5 of 55 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.
  • 6. 6 of 55 Over-activation of this pathway is considered an etiological factor in human cancer, which contributes to Cancer development, metastasis and resistance to chemotherapy
  • 7. 7 of 55 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
  • 9. 9 of 55 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
  • 13. 13 of 55 Total 50 formalin fixed parafin embedded tissue block of buccal mucosa and tongue will be collected from oral carcinoma patients.
  • 14. 14 of 55 Immunohistochemistry (IHC)
  • 15. 15 of 55 3-4 μm sections of paraffin embedded tumor tissue sections will be taken on APES coated slides. Incubate overnight at 60Ëšc 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.
  • 16. 16 of 55 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.
  • 18. 18 of 55 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
  • 21. 21 of 55 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.
  • 22. 22 of 55 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]
  • 24. 24 of 55 Polymerase chain reaction (PCR)
  • 25. 25 of 55 . . .
  • 26. 26 of 55 Restriction Endonuclease Digestion
  • 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
  • 30. 30 of 55 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.
  • 31. 31 of 55 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