1. Squamous-cell carcinoma - new biomarkers Rafal Dziadziuszko Medical University of Gdańsk, Poland

2. Molecular aberrations in lung adenocarcinoma Driving molecular events: -EGFR mutations -HER2 mutations -KRAS mutations -BRAF mutations -MEK mutations -ALK rearrangement -RET rearrangement -ROS1 rearrangement -MET amplification -Others… Markers related to progression: - EGFR, MET high copy number - High plasma ligand levels (EGF, HGF, VEGF etc.) -IL-6 paracrine circuit -Markers of aquired resistance (T790M, MET for EGFR inhib.) -Markers of DNA repair capacity -Markers of tumor angiogenesis -Others…

3. Who is the driver? Molecular aberration fulfilling the criteria of „oncogene addiction” Usually mutually exclusive with other drivers (or almost mutually exclusive) and associated with particular histology Cell line inhibition with nanomolar concentrations of the target inhibitor; high activity in animal models

4. Markers of progression Associated with tumor progression, not initiation; usually quantitative rather than qualitative Usually not mutually exclusive with other molecular events Inhibition related to some theraputic benefit in cell lines and in vivo models; often transient and bypassed by other mechanisms

5. Lung adenocarcinoma: molecular subtypes according to driver mutations PDGFR PIK3CA ALK MET ROS EGFR ERBB2 BRAF MEK1 Mutations in NSCLC cell lines. Sharma, et al. Nat Rev Cancer 2010 K-RAS

6. Kris MG, et al. ASCO 2011. CRA7506. Johnson BE, et al. IASLC WCLC 2011. Abstract O16.01 Lung Cancer Molecular Consortium Analysis in clinical samples of lung adenocarcinomas Mutations found in 54% (280/516) of tumors completely tested (95% CI: 50% to 59%) No Mutation Detected KRAS 22% EGFR 17% EML4-AKL 7% Double Mutants 3% BRAF 2% PIK3CA 2% HER2 MET AMP MEK1 NRAS AKT1

7. Squamous-cell carcinoma of the lung ~25% – 60% of all lung cancer diagnoses (geographical variation)

8. Squamous-cell carcinoma of the lung: The „new” drivers? b - EGFR vIII (del 2-7 EGFR) mutations - FGFR1 amplification - DDR2 mutations - SOX2 amplification - PIK3CA amplification

9. EGFR vIII (del 2-7 EGFR) mutations In-frame deletion of exons 2 – 7 identified in a significant proportion of glioblastomas Demonstrated in 3/56 (5%, Ji et al.) and 7/87 (8%, Sasaki et al.) of SCCs at genomic level Immunohistochemical staining against EGFRvIII protein is possible but good correlative studies are lacking Ji H et al., PNAS 103:7817 – 22, 2006; Sasaki H et al., Oncol Rep 17: 319-23, 2007

10. EGFR vIII (del 2-7 EGFR) mutations Ji H et al., PNAS 103:7817 – 22, 2006 Tet-op-EGFR vIII Bi-Transgenic animals develop AAH and invasive adenocarcinomas after 8 and 16 weeks of doxycycline administration, respectively

11. EGFR vIII (del 2-7 EGFR) mutations Ji H et al., PNAS 103:7817 – 22, 2006 EGFR vIII transformed Ba/F3 cells are sensitive to EGFR inhibition with irreversible HKI-272 and less sensitive to erlotinib

12. EGFR vIII (del 2-7 EGFR) mutations Sasaki H et al., Oncol Rep 17: 319-23, 2007 EGFR vIII tumors had higher EGFR gene copy number as compared to EGFR WT tumors (mean 4.7 vs. 2.2 copies by qPCR)

13. EGFR vIII (del 2-7 EGFR) mutations Conclusions Detection difficult due to need for relatively complex assays on genomic level Clinical significance and true prevalence unknown More preclinical and clinical data needed

14. EGFR vIII (del 2-7 EGFR) mutations MLPA assay Assay developed by Prof. Piotr Kozlowski, Polish Academy of Science, Poznan

15. FGFR1 amplification Detected in 34/153 SCCs (22%) by FISH (defined as mean>4 copies per nucleus) Associated with sensitivity to FGFR1 inhibitor PD173074 in cell lines and animal models Weiss J et al., Sci Transl Med. 2: 62ra93, 2010

16. FGFR1 amplification Dutt A. et al., PLoS ONE. 6: e20351, 2011

17. FGFR1 amplification Weiss J et al., Sci Transl Med. 2: 62ra93, 2010

18. FGFR1 amplification Polish NSCLC cohort analyzed by SISH, N=59 Wynes MW et al., poster presentation; Chicago 2011 Squamous FGFR1 2.08 CH 8 2.16 Ratio 0.96 Squamous FGFR1 4.78 CH 8 4.32 Ratio 1.11 16% of SCCs amplified

19. FGFR1 amplification Conclusions Observed in ~ 20% of SCCs Optimal cut-off point to define amplification? Attractive target currently evaluated in clinical trials

20. Discoidin domain receptor 2 (DDR2) mutations Observed across entire DDR2 gene, in ~ 4% of SCCs Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

21. DDR2 mutations DDR2 mutated cell lines are sensitive to src inhibitor dasatinib Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

22. DDR2 mutations DDR2 mutated cell lines are sensitive to src inhibitor dasatinib Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

23. SOX2 Transcription factor involved in foregut development and squamous differentiation of epithelial esophagal and respiratory cells Located at 3q26 amplicon observed in ~ 20 – 30% of SCCs Major regulator of stem-cell function and the cell cycle; suppression leads to antiproliferative effect in cell lines Hussenet et al. PLoS ONE 2010 Bass et al. Nat Genet 2009

24. PI3KCA Catalytic subunit alpha of phosphatidylinositol 3-kinase Mutations observed in ~2-3% of SCCs, amplification in ~ 30% of SCCs (3q26 amplicon containing SOX2) Several therapeutics are currently in phase I – phase II clinical trials

25. Squamous-cell carcinoma: molecular subtypes Perez-Moreno P et al. CCR 2012 in press

26. Squamous-cell carcinoma: getting hot and spicy