LUNG CANCER AND EGFR

1. LUNG CANCER AND EGFR Prof.Dr.Gülay ÖZBİLİM Akdeniz University Faculty of Medicine,Pathology Department ANTALYA

3. *. Lung cancer, is one of the most common and most lethal malignancies.* Every year, approximately 1.2 million new lung cancer is diagnosed.* 480 new cases worldwide each day (one case/ 3 minutes )are diagnosed with lung cancer

4. RISK FACTORSCIGARETTE-The most important risk factor.-More than 80% of lung carcinoma are due to smoking -Passive smoking increases the risk by 1.6%-Exposed in childhood, the risk is 2.6 times greater than that.

5. RECURRENT INFECTION • Tuberculosis and pneumonia increases the risk of developing adenocarcinoma in scar tissue.Berilliosis and Silicosis increases lung carcinomas riskTALC • Although the use of talc as Cosmetics doesn’t increase, Talc miners have an increased risk.FAMILY STORY • Having lung carcinoma increases the risk of developing another lung carcinoma. • Family story of lung cancer increases the risk .

6. VITAMINSVitamins C and E have preventative effects.Antioxidant intake is helpful.* ALCOHOL30gr/day increases lung cancer risk • * AIR POLLUTIONIncreases the risk in 11% of cases.* PHYSICAL ACTIVITYModerate and heavy exercise reduce the risk by 13-30%

7. Genetic studies relieved Ch 15 relation with lung cancer . 30% increased risk with one copy, 70-80% with 2 copies. • 3 genes space coding nicotinic acetyl choline receptor,cell surface receptor, increases mutation risk. • Real effect is to increase nicotine dependence

8. Precancerous lesions • Squamaus dysplasia/carcinoma insitu • Atypical adenomatous hyperplasia • Diffuse idiopathic NE cell hyperplasia

9. HISTOPATHOLOGIC DIAGNOSTIC METHODS *Sputum, lavage cytology *Transthoracic or transbronchial FNAB *Bronchoscopic biopsy or needle biopsy *Thoracoscope *Wedge biopsy *Lobectomy or pneumonectomy

10. Histopathologic classification of the lung carcinoma • 1981,1994,1999 classificationsIn 2004 (WHO / IASLC)   World Health Organization and the international working group for lung cancer with lung and pleural tumors by the revised histopathological classification is used.

12. Small Cell • Non small cell --Squamaus carcinoma --Non squamous carcinoma • Approximately 80% of these patients constitute non small cell lung cancer (NSCLC) .

13. Average 5-year survival in lung cancer is 15% . • Stage is the most important prognostic factor in lung cancer. • CT and PET in preoperative staging is carried out. • Transbronchial FNAB or mediastinoscopy is performed to evaluate the mediastinum.

14. The most curative therapy is surgery, • The basic approach in early stages is surgery, adjuvant KT and RT. • In locally advanced disease, RT and CT, real-time application is made in appropriate cases.

15. 75% of cases are in advanced staged when diagnosed. • Basic treatment in patients with advanced NSCLC is chemotherapy, and palliative RT is activated in selected cases.

16. Standard first choice treatment with platinum based drugs response rate is approximately 20%,median survival 9 months. • Second choice Pemetrexet (antimetabolic) or Docetaxel (microtubule inhibitor) have high toxicity and response rate is below 10%. • The third choice chemotherapy is to be effective under 2% of patients.

17. Toxicities with existing cytotoxic agents in this group of patients have advanced age, researchers have directed less toxic treatments. • In recent years the use of targeted agents in NSCLC, treatment modalities have changed from conventional treatment towards targeted therapies.

18. Normal cell growth and proliferation, are regulated by many signaling pathways. • Signaling pathway is controlled by extracellular growth factors , neurotropins, cytokines and hemautopoetins. • These growth factors are also secreted by cancer cells and interact with receptors on cell surface by autocrine ways to stimulate tumor growth.

19. If a cell has growth factor and its receptors then it has a growth circle to stimulate its proliferation.This is called as "Ring of autocrine growth’’ . • Autocrine growth rings found in normal cells, but only respond to physiologic stimuli. This balance is disturbed in cancer cells.

20. In the development of lung cancer, not a single growth factor but several growth factors and receptors are affected. • As a result of interaction of these growth factors and receptors metastasis and invasion are assumed to occur.

21. Today,Tyrosine kinase (TK)reseptor,epidermal growth factor reseptor,is one at the molecular targeted agent that inhibits specific pathologic pathways and key molecules in tumor growth and progression. • In NSCLC, EGFR and its ligands are activated while neuropeptides and their receptors are activated in small cell carcinomas.

22. Family of ErbB Receptor Tyrosine Kinase ErbB receptors are Type 4: ErbB1 = Her1, EGFR ErbB2 = Her2, neu ErbB3 = Her3 ErbB4 = Her4 These 4 receptors act by forming homo-or hetero-dimers.

23. EGFR EGFR (Epidermal Growth Factor Receptor) is a member of ErbB family of tyrosine kinase and a receptorEGFR , transmembrane glycoprotein with tyrosine kinase activity in weight 170 KD, and transmits extracellular signals into the cell.Located on 7p12 chromosome.

24. EGFR, consists of three areas:1.Cell ligands (EGF, TGF-a, Amfiregülin) connects the N terminal2.Hydrophobic transmembrane region3. located within the C terminal with tyrosine kinase activity. Ligand PI3 Kinase P85 P P SHC P100 ekstracellulerregion Membrane intracelluler region Proliferation, Differentiation, ApoptosisTK receptor ligand binding to EGFR activation and signaling mechanism following a series of proliferation, migration, invasion and suppression of apoptosis improves.

25. When connected to EGF receptor subunit can dimerize and intracellular part consists otofosfarilizasyonThis activates multiple signal paths. Ligand-independent activation mutants makes otofosforilation Akt and STAT pathways are activated by MAPK pathway Molecular signals is responsible that cellular proliferation, apoptosis resistance, cellular invasion, metastasis

26. Mechanism of EGFR activation • EGFR overexpression • EGFR mutation • Heterodimerization • Increase of ligand expression • Decrease in phosphatase activity • Decrease in downstream signaling functions

27. The effects of EGFR • Increases mitosis • Inhibits apoptosis • Increases cell motility • Protein secretion increases • Enhances cell adhesion • Enhances cell invasion • Enhances cell survival • Stimulates angiogenesis • Stimulates cytokine release • Extracellular matrix stimulates

28. Mutations in different regions have different effects:Mutations in exons 19 and 21 survival are better and more sensitive to TKI H. Yamamoto ve ark, Lung Cancer 63 (2009) 315–321

29. EGFR is expressed largely in four major tumor types

30. EGFR is located in the lung;* intercellular membranes of basal cells, Clara cells and type II pneumocytes * No expression of apical surface of ciliated cells • EGF is found , bronchial secretory granules of serous acinar cells and the endoplasmic reticulum.

31. Increased EGFR expression found associated with ;Advanced disease,Development of metastasis,Decreased survival Poor prognosis.

33. ErbB receptors inhibitions 1) pharmaceutical extracellular region of ligand (monoclonal antibody)2) intracellular kinase domain - Tyrosine Kinase Inhibitors (TKI)                                                 Gefitinib, Erlotinib3) multiple receptor types

34. The effects of EGFR inhibitors • a. Inhibition of cell cycle in G1 phaseb. Inhibition of tumor cell adhesionc. Induction of apoptosisd. Antiproliferative activitye. Antiangiogenic, and antimetastatic antiinvaziveactivityf. Cytotoxic agents used in conjunction with the additive increased interaction with the antitumoral activity (chemosensitivity and increased radiosensitivity)g. Reduction of treatment-related cytotoxic side effects h. Other:* An increase in survival time* Improvement in symptoms* An increase in quality of life

35. 65 % of NSCLC patients with EGFR mutations is respond to TKI. • EGFR-TKI treatment of adenocarcinoma is particularly sensitive.

36. Patient selection; (most effective) * Women,* Non-smoker,* Asian,* Adenocarcinoma* Skin toxicity

38. Patient View for EGFR 10-15% in Europe and America, 15-25% (35%) in Asia, nonsmoker Female sexAdenocarcinoma type (30-50% in Asia) Cancer Sci 2007; 98: 1817–1824

39. EGFR mutation status in advanced NSCLC 5241 patients included ( AstraZeneca) 2755 patients from studies in Asian countries (1380 M+) 1861 patients from studies in non-Asian countries (473 M+) 625 patients from studies of mixed or unknown location (90 M+)

40. Recommendations for tumour samples for EGFR mutation analysis Tumour biopsy from primary tumour or metastases is the “gold standard” for mutation analysis It is recommended that DNA samples are extracted from formalin-fixed, paraffin-embedded tumour biopsy diagnostic samples Robust well validated DNA extraction methodologies are recommended to avoid assay failures and false negative results Mutation testing in surrogate tissues such as serum/plasma, bronchoalveolar lavage fluid or cytology specimens is not currently recommended Eberhard et al 2008, Kimura et al 2006

41. FREQUENTLY USED METHODS OF EGFR ANALYSIS • Immunohistochemistry (IHC) for EGFR protein expression • Fluorescent In Situ Hybridization (FISH) for EGFR gene copy number determination • Sequence for determination of the EGFR mutations*immunohistochemistry and EGFR mutations were not correlated

42. Determination of Protein Expression of EGFR by IHC a study pictures by using antibodies specific to mutation

43. *EFGR gene copy numbers were determined using Five major FISH patterns, as follows; (1) normal disomy, ≤two genecopies in more than 90% of cells; (2) trisomy, three gene copies in more than 10% of cells; (3) low polysomy ≥ four gene copies in more than 10% but in less than 40% of cells; (4) high polysomy,≥four gene copies in more than 40% cells; (5) gene amplification, the presence of tight gene clusters and a ratio of gene/chromosome of more than 2 or ≥15 gene copies in ≥10% of cells FISHE.-A. Park et al. / Lung Cancer 64 (2009) 179–186

45. Determination of FISH and EGFR Gene Copy Number A: EGFR gene amplification (EGFR gene ratio of> 2 or analysis of the cancer cells,> 10 's in the EGFR gene copy number> 15 in case)B: High polysomy (Analysis of the cancer cells were> 40% 'When the EGFR gene copy number> 4 is the case)

47. Mutation Screening Methods PCR/Sequencing Nested PCR/Sequencing Melt Analysis PCR/HRMAA (High resolution melting amplicon analysis) PCR/dHPLC (Denaturing high performance liquid chromatography) Pyrosequencing Targeted Mutation Detection Methods Real Time PCR ARMS (Amplification Refractory Mutation System) PNA/LNA Clamp (Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection) RFLP (Restriction fragment length polymorphism) EGFR GENE MUTATİONS DETECTİON METHODS

48. PCR/SEQUENCİNG

50. Nowadays, the studies often were combined with bi-directional sequence analysis and real-time ARMS (RT) PCR analysis are used. • Analysis of mutant DNA in the presence of 15-20% was made by clasic sequence analysis , mutant DNA detection of 1% is possible by RT-PCR • Mutations can not be determined with FISH and IHC