Squamous-cell carcinoma - new biomarkers

1. Squamous-cell carcinoma- newbiomarkers RafalDziadziuszko MedicalUniversity of Gdańsk, Poland

2. Molecularaberrationsin lungadenocarcinoma • Drivingmolecularevents: • EGFR mutations • HER2 mutations • KRAS mutations • BRAF mutations • MEK mutations • ALK rearrangement • RET rearrangement • ROS1 rearrangement • MET amplification • Others… • Markersrelated to progression:- EGFR, MET high copynumber- High plasma ligand levels (EGF, HGF, VEGF etc.) • IL-6 paracrinecircuit • Markers of aquiredresistance • (T790M, MET for EGFR inhib.) • Markers of DNA repaircapacity • Markers of tumor angiogenesis • Others…

3. Whois the driver? • Molecularaberrationfulfillingthe criteria of „oncogeneaddiction” • Usuallymutuallyexclusive with otherdrivers (oralmostmutuallyexclusive) and associated with particularhistology • Cell lineinhibition with nanomolarconcentrations of the target inhibitor; high activity in animalmodels

4. Markers of progression • Associated with tumor progression, not initiation; usuallyquantitativeratherthanqualitative • Usuallynot mutuallyexclusivewith othermolecularevents • Inhibitionrelated to sometheraputic benefit in cell lines and in vivo models; oftentransient and bypassed by othermechanisms

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

6. Lung Cancer Molecular Consortium Analysis in clinicalsamples of lungadenocarcinomas • Mutations found in 54% (280/516) of tumors completely tested (95% CI: 50% to 59%) No Mutation Detected KRAS22% AKT1 EGFR17% NRAS EML4-AKL7% MEK1 MET AMP HER2 PIK3CA 2% BRAF 2% DoubleMutants 3% Kris MG, et al. ASCO 2011. CRA7506. Johnson BE, et al. IASLC WCLC 2011. Abstract O16.01

7. Squamous-cell carcinoma of the lung~25% – 60% of alllungcancerdiagnoses(geographicalvariation)

8. Squamous-cell carcinoma of the lung:The „new” drivers?b- EGFR vIII (del 2-7 EGFR) mutations- FGFR1 amplification- DDR2 mutations- SOX2amplification- PIK3CAamplification

9. EGFR vIII (del 2-7 EGFR) mutations • In-framedeletion of exons 2 – 7 identifiedin a significantproportion of glioblastomas • Demonstrated in 3/56 (5%, Ji et al.) and 7/87 (8%, Sasaki et al.) of SCCsatgenomiclevel • ImmunohistochemicalstainingagainstEGFRvIII protein ispossible but goodcorrelativestudiesarelacking 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 Tet-op-EGFR vIII Bi-Transgenicanimalsdevelop AAH and invasiveadenocarcinomasafter 8 and 16 weeks of doxycyclineadministration, respectively Ji H et al., PNAS 103:7817 – 22, 2006

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

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

13. EGFR vIII (del 2-7 EGFR) mutationsConclusions • Detectiondifficultdue to need for relativelycomplexassays on genomiclevel • Clinical significance and trueprevalenceunknown • Morepreclinical and clinical data needed

14. EGFR vIII (del 2-7 EGFR) mutationsMLPA assay Assaydeveloped by Prof. Piotr Kozlowski, PolishAcademy 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 animalmodels Weiss J et al., SciTransl Med. 2: 62ra93, 2010

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

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

18. FGFR1 amplificationPolish NSCLC cohortanalyzed by SISH, N=59 Squamous FGFR1 2.08 CH 8 2.16 Ratio 0.96 Squamous FGFR1 4.78 CH 8 4.32 Ratio 1.11 16% of SCCsamplified Wynes MW et al., poster presentation; Chicago 2011

19. FGFR1 amplificationConclusions • Observedin ~ 20% of SCCs • Optimalcut-off point to defineamplification? • Attractive target currentlyevaluated in clinicaltrials

20. Discoidindomain receptor 2 (DDR2) mutations ObservedacrossentireDDR2gene, in ~ 4% of SCCs Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

21. DDR2 mutations DDR2mutatedcell lines aresensitive to src inhibitor dasatinib Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

22. DDR2 mutations DDR2mutatedcell lines aresensitive to src inhibitor dasatinib Hammerman PS et al., Cancer Discovery. 1:78-89, 2011

23. SOX2 • Transcriptionfactorinvolved in foregut development and squamousdifferentiationof epithelialesophagaland respiratory cells • Locatedat 3q26 ampliconobservedin ~ 20 – 30% of SCCs • Major regulator of stem-cellfunction and the cellcycle; suppressionleads to antiproliferativeeffect in cell lines Hussenetet al. PLoS ONE 2010Bass et al. Nat Genet 2009

24. PI3KCA • Catalyticsubunitalpha of phosphatidylinositol 3-kinase • Mutationsobserved in ~2-3% of SCCs, amplification in ~ 30% of SCCs (3q26 ampliconcontainingSOX2) • Severaltherapeuticsarecurrentlyin phase I – phase II clinicaltrials

25. Squamous-cellcarcinoma:molecularsubtypes Perez-Moreno P et al. CCR 2012 in press

26. Squamous-cell carcinoma: getting hot and spicy