The document summarizes a presentation on using gene profiling and biomarkers to better classify and treat non-small cell lung cancer (NSCLC). It discusses current and emerging markers like EGFR mutations, ALK translocations, and FGFR1 amplifications that define NSCLC subtypes and can guide targeted therapies. Integrating multiple genomic analyses may help characterize unknown abnormalities in a third of NSCLC tumors and identify new treatment opportunities.

1. Gene Profiling in Clinical Oncology 8-9 April 2011 Viareggio, Italy New markers to define NSCLC and the potential of genomics L.Lacroix PharmD PhD Institut Gustave Roussy, Villejuif, FR Friday, 8 April 2011

2. Lung cancer Males Females Current smokers in France IARC 2008; INSERM 2007 Sun S et al, Nature Can Rev 2007 32 % 21% 15 Millions people CANCER France Europe World Incidence Mortality Prevalence 320 000 144 000 800 000 2 600 000 1 600 000 8 000 000 12 000 000 7 600 000 28 000 000

3. What is lung cancer ? The old perception Clinical parameters Anatomo Histological parameters Histological microscopic parameters

4. Patient background heterogeneity Tumor molecular heterogeneity Cancer is a gene disease

5. What we do currently… Identify patients in need of treatment Identify more effective therapeutic opportunities tailored to the individual patient The Challenge…… Who to treat? How to treat?

6. Goals of tailoring therapy according to predictive markers Other treatments Gandara R, et al. J Clin Oncol, 2007: Abst 7500 Responders with standard therapy Non responders Toxicity Patients with same diagnosis Improve therapy

7. Deciphering Hanahan, Weineberg Cell 2011

8. Deciphering Hanahan, Weineberg Cell 2011

9. Several possibility to analyze Gene abnormalities simplex gene analysis (PCR/sanger sequencing/IHC), multiplex gene analysis (GE microarray/CGHa/MassiveSequencing). Proteomic Mass Spec. RPPA, Antibody µA, TMA Structural Genomic (Copies Nb or Structure) CGH µA; methylation µA, Sequencing approach Functional Genomic GE µA ; Q-PCR

10. Significantly mutated pathways in adenocarcinomas of the lung Ding et al. Nature 455, 1069, 2008

11. NSCLC key mutations f. treatment

12. EGFR Herbst NJEM 2008 gefitinib erlotinib

13. EGFR – TKI : History Description of EGFR mutation 2000 2002 2004 2006 2008 2010 Trial results EGFR Mutations Gefitinib approval restricted to EGFR mutation 1 st large phase III trial with selected population - IPASS Phase II erlotinib / gefitinib 1st phase III trial - BR21 Erlotinib approval 2/3 rd line

14. EGFR mutations Activating mutation 10% of caucasians Exon 19 in-frame deletions (45%) L858R missence mutation (45%) Less frequent mutation (10%) in exons 18 to 21 Herbst NJEM 2008

15. Lynch et al. NEJM Pao et al. PNAS In vitro “EGFR mutation=TKI sensitivity” Weinberg, book 2006

16. Mutations FISH Hirsch, J Clin Oncol 2006 Response biomarkers

17. Erlotinib & gefitinib inhibit EGFR Mitsudomi et al, IASLC 2007, Abstract Mutations EGFR and response (N=1335)

18. IPASS study (Mok TS et al. NEJM 2009)

19. McDermott, U. et al. J Clin Oncol; 27:5650-5659 2009 Mechanisms of EGFR Inhibitor Resistance Mut

20. Mechanisms of EGFR Inhibitor Resistance T790M mutation (exon20)

21. Engelmann et al. 2006 Bean et al. PNAS 2007 In resistant tumors 9/43 (21%) Met amplification 20/43 (47%) T790M (4 with Both ) (In Naïve tumor 2/62 (3%) Met Amp and 0/62 T790M )

22. Not all mutations are equivalent sensitivity / primary resistance / secondary resistance Sharma et al. Nat Rev Cancer 2007 http:// www.somaticmutations-egfr.info /

23. EGFR TKI and KRAS mutation Sun S et al, Nature Can Rev 2007 Ding et al, Nature 2008 MUT WT KRAS mutated patient = EGFR WT = worth pronostic

24. KRAS mutation = less responses to TKI CR + PR SD + PD Sun S et al, Nature reviews cancer 2007 R NR

25. KRAS mutations KRAS = predictor of lack of EGFR TKI benefit KRAS should not be used in CT decision algorithm Therapeutics targeting KRAS Farnesyltransferase inhibitor (tipifarnib/lonafarnib) RAF inhibitors (sorafenib/plexicon4032…) MEK inhibitors (AZD6244/RO5126766/ GSK 1120212 …) Loriot S et al, Nature Rev Clin Onco 2009

26. ALK Pathway – ALK Translocation 1. Inamura K et al. J Thorac Oncol 2008;3:13–17 2. Soda M et al. Proc Natl Acad Sci U S A 2008;105:19893–19897 Figure based on: Chiarle R et al. Nat Rev Cancer 2008;8(1):11–23 Mossé YP et al. Clin Cancer Res 2009;15(18):5609–5614; and Data on file. Pfizer Inc. *Subcellular localization of the ALK fusion gene, while likely to occur in the cytoplasm, is not confirmed. 1,2 Or ALK ALK fusion protein* Cell survival Tumor cell proliferation Anaplastic Lymphoma kinase (Chr2q) Translocation in 3-7% of ADC Main partner gene = EML4 ADC (TTF1+), (young) Man Non smoking advance tumor. Exclusive from EGFR/KRAS Inversion Translocation

27. FISH Assay for ALK Rearrangement AT Shaw et al. JCO 20093

28. Crizotinib Selectivity Profile Selectivity findings Crizotinib – ALK and c-MET inhibition at clinically relevant dose levels Crizotinib – low probability of pharmacologically relevant inhibition of any other kinase at clinically relevant dose levels Cellular selectivity on 10 of 13 relevant hits Upstate 102 kinase 13 kinase “hits” <100X selective for c-MET *The cellular kinase activities were measured using ELISA capture method Pfizer Inc. Data on file Crizotinib (PF-02341066) Kinase IC 50 (nM) mean* Selectivity ratio c-MET 8 – ALK 20 2X RON 298 34X 189 22X Axl 294 34X 322 37X Tie-2 448 52X Trk A 580 67X Trk B 399 46X Abl 1,159 166X IRK 2,887 334X Lck 2,741 283X Sky >10,000 >1,000X VEGFR2 >10,000 >1,000X PDGFR  >10,000 >1,000X

29. Crizotinib: First-in-human/Patient Trial Part 2: Molecularly enriched cohorts (ALK and c-MET) Enrolling patients with ALK -positive NSCLC after preliminary observation of impressive activity in a few patients Data from database April 7, 2010 Data presented for 82 patients, study ongoing Part 1: Dose escalation 1 DLT: grade 3 ALT elevation 2 DLTs: grade 3 fatigue ALT = alanine aminotransferase Cohort 1 (n=3) 50 mg QD Cohort 2 (n=4) 100 mg QD Cohort 3 (n=8) 200 mg QD Cohort 4 (n=7) 200 mg BID Cohort 5 (n=6) 300 mg BID Cohort 6 (n=9) 250 mg BID MTD/RP2D

30. Molecular analysis of EML4–ALK breakpoints Kwak et al. NEJM 2010 1500 screened=>82 pos. & treated and 77%continued after data cuttoff

31. NSCLC Patient with ALK amplification/polysomy about 5 % of amplification in our experience Few data about this point and link with therapy. Besse in progress

32. ALK Secondary resistance Choi et al. NEJM 2010

33. FGFR1 : amplification in 22% of SCC Weiss et al. Science Transla Med 2010

34. FGFR1 inhibitor are effective in amplified model FGFR1 the next Target for therapy in NSCLC ?? Dose dependent Growth inhibition in vitro & in vivo Apoptosis induction Weiss et al. Science Transla Med 2010

35. Other mutations suitable for therapies Overexpression (IHC) in 17 to 42% of ADC Amplification and Mutation in 5%-10%of ADC Inframe insersion in exon 20 Associated to Asian/ Women/ Non smoking/ADC. HER2 mutation associated to resistance to EGFR TKI but sensitive to HER2 therapies. HER2 Mutation have been reported in 2-3% of ADC HER4 Wang et al. Cancer Cell 2006

36. Other mutations suitable target PIK3CA-Akt-mTor pathway Mutation in 1-5% of NSCLC Interesting target regarding drug in development (melanoma) BRAF Overexpression (IHC) of AKT1 Amplification and Mutation or PIK3CA1 1-17% of NSCLC Capuzzo et al. 2005 ; Daniele et al. 2007 Yamamoto et al. Can Res 2008

37. Other mutations suitable for therapies LKB1 Tumor suppressor Gene Repress mTOR Collaboration with KRAs in oncogenesis 30% of NSCLC with mutation ADC/smokers/caucasians/EGFRWT MET High expression associated to poor survival 3 % with amplification Involved in 10-20% of resistance to EGFR TKI

38. Treatments opportunities Molecular alteration Frequency EGFR mutation 10% EML4-ALK translocation 3-5 % HER 2 alteration 2% PI3K mutation 2% MET amplification 1% RAS and RAF mutation 3-30% Drug Erlotinib, gefitinib New pan-HER Crizotinib Trastuzumab New Pan-HER GDC-0941; XL-147 XL-765 ; PX-866 BEZ-235; BKM120 XL184, ARQ 197 Sorafenib, GSK 1120212 AS703026, AZD 6244

39. Molecular Biomarker in France in 2009 Molecular analysis – Molecular Pathology Sce 26 platforms INCa’s Somatic genetic platform Granted. New Biomarker grant for Prospective evaluation in 2011 10 000 tests expected Cancer Target Lung EGFR KRAS HER2 exon 20 BRAF PI3KCA t EML4-ALK CCR KRAS BRAF MSI Breast amplification HER2 BRCA1/2 PI3KCA / AKT1 Melanoma BRAF cKIT

40. IGR SeqCAN Selection of Candidates Genes screening of >50 cancer driver genes Selection of about 350 sequencing systems targeting most mutated exons Covered over 90k bp. and several thousands of putative mutations.

41. Screening Format 96 amplicons of all Hot spot exons. adapted to tumors board (Lung/melanoma..) Consent of patient for use of tumor for Biomedical research IGR SeqCAN Project applications *autorisation for temporary use delivered by AFSSAPS (French drug agency) MET, AKT1, BRAF, FGFR2, FGFR3, CTNNB1, NRAS, NRAS, ERBB2, PIK3CA, STK11, PTEN, FLT3, FCGR2A, FGFR4, FGFR2PTEN, ERBB4, FGFR4, KDR, ALK, PTEN, PDPK1;PIKR1…

42. Lung cancer evaluation in the XXIst century will have to include Large Sequencing / CGHa approaches… Ding et al. Nature 455, 1069, 2008

43. 33% unknown or unfrequent abnormalities pangenomic analysis Unknown therapeutic impact Xxx? markers (FISH/Mut°) 33% Trials 3 markers EGFR/KRAS/ALK Approval or Trials 33%

44. DNA microarray (Gene Expression/miRNA/CGHa…) Marqueurs tumoraux PanGenomic Expression analysis

45. First Studies in lung cancer N =Normal/ADC=Adenocarcinomas/SQ =Squamous cell Carcinomas/COIID=Carcinoïd/SMLC=small cell lung carcinomas Molecular classification in accordance to histology ADC molecular subtypes Bhattacharjee A et al. PNAS,2001 (17N,139 ADC, 21SQ, 20COIID,6SMLC) AffyU95Av2 12 600 gènes Garber ME et al. PNAS,2001 (5 N, 40ADC, 13SQ, 4LCL, 4SMCL) cDNA 24 000 gènes Beer DJ et al. Nature Medicine,2002 (10 N, 86 ADC,) AffyHuGeneFL 6 800 gènes Molecular subtypes of ADC associated to various prognostics.

46. Metaanalysis of molecular profiles Meta-analysis of 231 ADC (31 Stanford, 72 Michigan, and 128 Dana-Farber) Identification of molecular subtypes of ADC by comparison with gene profile of histology (BAC,SCC, LCLC). Hayes DN et al. JCO 2006 (1) ADC Bronchoïds (33% to 52% of samples), EGFR mutated15%, 75% with BAC component. Gene expression related to Cell growth, differentiation, cellular survival. (2) ADC Squamoïds (33% to 52% of samples ), EGFR mutated15%,Gene expression related to angiogenesis (HIF1a pathway), TGFb pathway and WNTsignalisation pathway (3) ADC Magnoïds (10% to 26% of samples ), 1/33 EGFR mutation, Gene expression related to inflammation, cytoskeleton, metabolism and proliferation. IHC AU et al. J.Path 2004

47. E.g gene signature to identified High Risq Lau et al. JCO 2009 158genes 3 Stage I Toronto dataset Stage I Duke data set Stage I Havard data set

48. Genomic Prognosis in NSCLC Many more……need prospective Phase III validation Source Gene Signature Beer D, et al Nature Medicine, 2002 56 Gene Signature Potti A, et al NEJM, 2006 (retired 2011) Metagene Profile Lu, Y et al PLOS Medicine, 2007 64 Gene Cox Signature Lau SK, et al JCO, 2007 3 Gene Signature Chen, et al NEJM, 2007 5 Gene Tree Classification Shedden K et al Nat Med, 2008 NCI Director’s Challenge Multiple Gene Sets Tsao M, et al ASCO, 2008* 15 Gene Signature

49. Genomic for pathway ID in NSCLC Bild et al. Nat Med2006

50. Subramanian & Simon JNCI 2010 Chen, et al NEJM, 2007 Lau SK, et al JCO, 2007 Stg IA Stg IB Stg IA Stg IB 3 years survival among groups Validation of 5 genes and 3 genes signature on Shedden et al. large data set

51. Mainly studies of “ biomarker development ” but not “biomarker validation ” studies that will provide adequate level of evidence. - Right selection of patients - Unbiased evaluation of accuracy - Standardized assay Prospective design (e.g CALGB-30506)

52. Chemores Study (KI) (IGR) (INSERM) (CNIO) (EU) (INT) (NKI) (LiU) (KTH) (KUL) (TUM) (CRUK) (UNIMAN) (EGT) (EORTC) (IMM) (APHP) European study 123 NSCLC with paired normal tissu Histology : SCC (n=50) & ADC (n=57) Stage I (n=56) & II (n=81) adjuvant therapy Untreated (n=61) & Cpt treated (n=62) Large Scale Analysis Gene Expression array CGHa miRNA expression array Targeted Sequencing Analysis questions Prognostic Diagnostic Integrated analysis

53. Limitation in real Life… Adequate Biopsies Inadequate Biopsies Squamous Cell Ca Adenocarcinoma Necrosis Fibrosis

54. Limitation in real Life… Quality of nucleic acid FISH and/or frozen

55. Discrepancies between the primary tumor and its metastases even when they are synchronous Italiano Ann Oncol 2006 Gow Ann Oncol 2007 Bozzetti JTO 2008 Kalikaki BJC 2008 Gomez Roca JTO 2009 Which sample is the right one???

56. When must be done the sampling samples from resected NSCLC or from 1st-line metastatic biopsies The molecular portrait on diagnosis material … not predict for molecular portrait of the current disease … . is not appropriate for studying the behavior of 2nd/3rd line meta NSCLC

57. Tumor heterogeneity Analyzed 3 representative area Type of sample Paraffin Bloc > slides Cytology : limitation Minimum FISH 100 cells IHC/mutations 2000 cells Sensitivity of methods : percentage of tumor cells Fixation Fresh frozen>FFPE Type and type of storage Standardization of tests

59. In conclusion Mandatory EGFR mutation (not all are equivalent) Strongly recommended ALK KRAS (BRAF) Innovative PIK3CA/HER2/MET… FGFR1 TS/ERCC1/RRM1 Advanced research Large scale analyses CGH/large sequencing GE array/ prognostic signature

60. Witch Algorithm is the good one for daily practice?

61. tomorrow Harber et al. Cell, 2011

62. Value of markers and points of view Authorities : Benefice/Risk/Cost for population Health Care provider : Benefice/Risk/ for patients Patient : Benefice/Risk for personal value