Adenocarcinoma of the lung: a molecular perspective

1. Adenocarcinoma of the lung:a molecular perspective Rolf Stahel Zürich, Switzerland Lugano, 8.7.07

2. Morpholgical classification of lung cancer • Small cell lung cancer (15%) • Non-small cell lung cancer (85%) • Squamous cell carcinoma • Adenocarcinoma • Bronchoalveolar carcinoma • Large cell carcinoma • Rarer entities

3. Changing distribution of lung cancer histology (U Texas Galvaston) Mary Wahbah, Ann Dign Pathol, 2007

4. Oncogene mutations lung adenocarcinoma • About 50% of lung adenocarcinoma harbor somatic mutations of six genes that encode proteins in the EGFR signaling pathway: • KRAS mutations • EGFR mutations • Her-2 mutations • Her-4 mutations • BRAF mutations • Phosphatidylinositol 3-kinase (PI3K) mutations

5. EGFR: downstream signaling EGFR, HER2 Cell membrane SOS EGFR-TK RAS ATP ATP PI3K GRB2 RAF P MAPK Akt STAT MEK EGFR-TKpathways Gene transcriptioncell-cycle progression Nucleus Proliferation Resistance to apoptosis Invasion Metastasis Angiogenesis

6. Ras mutations • 1979-1981: Cloning of H-ras and K-ras • 1987: Rodenhuis, NEJM 1990: Slebos, NEJM: K-ras mutation in 30% of lung adenocarcinoma. Associaton with smoking. Poor prognostic factor in resected tumors • 1989-1990:Isoprenylation (farnesyl-tranferase) necessary for biological activity

7. RAS mutation in NSCLC • Induction of lung adenocarcinoma in the mouse model by conditional activation of mutated KRAS (Meuwissen, Oncogene 2001) • Meta-analysis of 23 clinical studies with mutational data: HR 1.4 (CI 1.18-1.65)(Mascaux, Br J Cancer 2005) • Adjuvant cisplatin/vinorelbine subgroup analysis according to ras mutation: No apparent benefit in patients with ras mutated tumors (however not significant in interaction analysis)(Winton, NEJM 2005) • Lack of sensitivity of K-ras mutated tumors to gefitinib or erlotinib(Pao, PLoS 2005)

8. Gefitinib responders Non-responders EGFR mutations 8/9 0/7

9. Case report 75 y/o man retired, held many jobs from working as driver, attendant at gasoline station to sexton. Hobby trumpet • 2/06 follow-up CT one year after TUR-P: solitary lesion L lower lobe. 3-months follow-up CT: Increasing seize • 6/06 thoracoscopic wedge resection abandoned because of non-small cell lung cancer with carcinomatosis of pleura: referral for palliative chemotherapy • Histology: adenocarcinoma • Smoking history: Never smoker • EGFR mutation analysis: deletion in exon 19 (delL747-E749, A750P)

10. Case report • 7/06: erlotinib and bevacizumab (SAKK 19/05) 10.7.06 29.8.06 22.6.07

11. EGFR mutations in lung adenocarcinoma Sharma, Nat Rev Cancer, 2007

12. EGFR mutations and adenocarcinoma histology Yatabe and Mitsudomi, Pathology International, 2007 Mucinous differentiation of adenocarcinoma with broncheoalveolar features correlates with absence of EGFR mutations and presence of KRAS mutations(Finberg, J Mol Diagn 2007)

13. 11 ligands each with a common EGF like structure EGF TGF-a Amphiregulin Betacellulin Epiregulin HB-EGF Epigen Neuregulin 1 (NRG1) Neuregulin 2 (NRG2) Neuregulin 3 (NRG3) Neuregulin 4 (NRG4) EGF The ErbB family 4 types of erbB receptors ErbB1 (HER1, EGFR) ErbB2 (HER2, neu) ErbB3 (HER3) ErbB4 (HER4)

14. HER2 and HER4 mutations lung cancer • HER2 mutation in 10% of lung adenocarcinomas(Stephens, Nature 04) • HER2 mutations mainly in adenocarcinoma with broncheoalveolar features and mutually exclusive with KRAS and EGFR mutations(Buttitta, IJC 2006) • Cells transfected wit mutated HER2 remain sensitive to lapatinib, but become resistant to EGFR TKIs(Wang, Cancer Cell 2006) • HER4 mutations in 2.3% of lung cancers (adeno and squamous cell)(Soung, IJC 2006)

15. EGFR: downstream signaling EGFR, HER2, HER4 Cell membrane SOS EGFR-TK RAS ATP ATP PI3K GRB2 RAF P MAPK Akt STAT MEK EGFR-TKpathways Gene transcriptioncell-cycle progression Nucleus Proliferation Resistance to apoptosis Invasion Metastasis Angiogenesis

16. BRAF Mutations in NSCLC V600E Missense mutations in 4/35 lung adenocarcinoma cell lines and in 2/127 lung adenocarcinoma tissues (Davis, Nature 2002, Noaki, CR 2002 ) Lung-specific expression of mBRAF in mice induced MAPK pathway activation and development of lung adenocarcinomas, which were sensitive to a MEK inhibitor(Ji, Cancer Res 2007) Expression of mBRAF induced lung adenomas in mice. For the development of adenocarcinomas additional TP53 mutation was needed. (Dankort, Genes and Development, 2007)

17. EGFR: downstream signaling EGFR, HER2, HER4 • PI3K mutations: • 5% NSCLC cell lines • 1.2% NSCLC tumors Cell membrane SOS EGFR-TK RAS ATP ATP PI3K GRB2 RAF P MAPK Akt STAT MEK EGFR-TKpathways Gene transcriptioncell-cycle progression Nucleus Proliferation Resistance to apoptosis Invasion Metastasis Angiogenesis

18. Two mutually exclusive pathways to lung adenocarcinoma(pooled data from East Asian and western countries) Tobacco KRAS mutations (14%) Smokers Adenocarcinoma Genetic factors ? ? Carcinogen EGFR mutations (31%) HER2 mutations (4%) BRAF mutations (1%)HER4 mutations (rare) Never Smokers Gazdar, IASLC Workshop 2006

19. Mutational profiling of resected lung adenocarcinomas 235 lung adenocarcinomas from MSKCC tissue bank, 39 kinases: • EGFR mutations: 6% • BRAF mutations: <1% • PI3K mutations: 2% • KRAS mutations: 12% • 1 not previously described mutation of the fibroblast growth factor receptor-4 (FGFR4) Conclusion: The majority of gain of function mutations in lung adencocarcinoma have been identified Marks, PLOS 2007

20. Gene amplification in non-small cell lung cancer • EGFR • Her-2 • Met

21. Correlation between mutation status and TKI sensitivity: HER2 overexpression confers sensitivity 1000 100 T790M 10 PTEN absent TKI IC50 (mM) 1 HER2 amplified 0.1 0.01 mEGFR mKRAS mBRAF WT Mutation status Gazdar, IASLC Workshop 2006

22. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3s signaling Concurrent inhibition of MET by PHA665725 und EGFR suppresses growth of MET amplified, EGFR mutant adenocarcinoma cell line Engelman, Science 2007

23. Findings associated with resistance to EGFR TKI • Lack of activating activating mutations in EGFR exons 18–21 • K-ras mutation: Screening of cell lines and introduction of mutated KRAS into EGFR mutated cell line(Pao, PLoS 2005, Uchida, Cancer Sci 2007) • Presence of T790M mutation: 50% of tissue samples from patients with acquired gefitinib resistance(Kosaka, Clin Cancer Res 2006) • ADAM17 mediated heregulin autocrine loop inducing ERBB2/3 signalling(Zhou, Cancer Cell, 2006) • Amplification of MET: 22% of patients with acquired gefitinib or erlotinib resistance(Engelman, Science 2007)

24. Molecular classification of lung cancer and morphology: Terminal respiratory unit (TRU) derived adenocarcinoma TRU Yatabe, Cancer Chemother Pharmacol 2006

25. Proposed schema of molecular classification of lung cancer Yatabe and Mitsudomi, Pathology International, 2007

26. Expression profiling of adenocarcinoma and underlying genetic changes Takeuchi, JCO 2006

27. Lung mutagene model(Potti, NEJM 2006) Identifying gene expression profiles predictive of recurrence after surgery

28. Adenocarcinoma of the lung:a molecular perspective (1) • 50% of tumors harbor somatic mutations of six genes encoding proteins in the EGFR signaling pathway: KRAS, EGFR, BRAF, PI3K, HER2 and HER4. • With the exception of PI3K mutations, they are mutually exclusive • The carcinogens of tumors with oncogenic mutations other than KRAS remain to be identified • The identification of an activating EGFR mutations has therapeutic implications today(USA 15’000 cases/y, CML and GIST 4500 cases/y each)

29. Adenocarcinoma of the lung:a molecular perspective (2) • The following genetic alterations confer resistance to EGFR TKIs: KRAS mutations, EGFR mutation T790M, MET amplification • The correlations between molecular and histologcial characteristics of lung tumors will lead to new proposals for the classification of lung adenocarcinoma • Gene expression arrays are likely to become an integral part of clinical decision making