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1 Enterprise Interest No

2 SY-05 Pulmonary Pathology: Options for targeted therapy in lung cancer Liquid biopsy in thoracic oncology Where are we now? Paul Hofman Laboratory of Clinical and Experimental Pathology Liquid Biopsy Laboratory & FHU OncoAge University Côte d Azur, Nice, Fr (

3 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

4 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

5 The right drug The right patient The right time Stratified medicine in thoracic oncology The right approach The right cost

6 The right turnaround time The right drug The right patient The right time Stratified medecine in thoraciccer patients Liquid and/or tissue biopsies? The right approach The right cost

7 The different compartments of a liquid biopsy Circulating Tumor Cells Platelets Healthy tissue cfdna ctdna(tumoral) Tumor tissue Red blood cells Immune cells Exosomes Apoptotic cells

8 Free tumor DNA Exosomes Free DNA Free germinal DNA Free RNA Free microrna ***** *** *** ** **** *** ** Hofman et al, Curr Mol Med, 2014

9 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

10 Many opportunities in lung cancer patients Screening and early detection of cancer Risk of metastatic relapse (prognosis) Stratified medicine Development of therapies Monitoring relapse and resistance Tumor heterogeneity

11 Ilie M et al, Ann Oncol 2012; Hofman P, Cancers 2017a; Hofman P, Cancers 2017b

12 Morphology IHC (D5F3 clone) FISH Genotyping Tumor tissue (FFPE) EGFR, KRAS, BRAF pyrosequencing (Qiagen) MGG ICC CTCs (ISET) Abbott Molecular Ilie et al, Ann Oncol 2012 Hofman, Cancers 2017a Hofman, Cancers 2017b

13

14 % Overall Survival % Disease-free survival Detection of CTCs by cytomorphology analysis Prognosis biomarker in NSCLC patients undergoing surgery (stages I-II) < 50 CNHC n = 27 (56%) < 50 CNHC n = 45 (57%) 50 CNHC n = 21 (44%) P = Log-rank = 8.97 P = Log-rank = Time (months) Time (months) Stage IA Stage IB Hofman et al, Clin Cancer Res, 2011 Hofman et al, Int J Cancer, 2011 Hofman et al Am J Clin Pathol,2011 Hofman et al, Am J Clin Pathol, 2011 Hofman et al, Cytopathology, 2011 Hofman et al, Cancer Cytopathol, 2016

15 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

16 Detection of EGFR mutations (both activating and resistant) from cfdna extracted from plasma sample The only test to be used clinically in a daily routine practice to date

17 Clinical utility of fluid/liquid biopsy / cfdna Sensitivity about 65%

18 Clinical indications for liquid biopsy for EGFR mutation detection in lung cancer at baseline WHEN? - No tissue sample available - Low percentage of cancer cells in FFPE sample - DNA of low quality and/or no DNA amplification - Inappropriate fixation - Long turnaround time to get the results Hofman & Popper, Virchows Archiv 2016

19 Stage IIIb/IV lung ADC with EGFR activating mutation treated with EGFR TKI Clinical indications for liquid biopsy for EGFR mutation detection in lung cancer at progression under EGFR TKI Disease progression T790M + 3 rd generation EGFR TKI Liquid biopsy T790M + 3 rd generation EGFR TKI T790M - Tissue biopsy T790M - Chemotherapy

20 ARMS ddpcr NGS Sensitivity Coverage Quantification IVD kits Costs (equip) Costs (reag) TAT

21 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

22 Hofman et al, Curr Mol Med, 2014

23 Advantages and limitations of the detection of circulating tumor cells Advantages Limitations Ilié & Hofman, Ann Transl Med, 2014 Hofman & Popper, Virch Archiv 2016

24 Advantages and limitations of the detection of circulating free DNA Advantages Limitations Ilié & Hofman, Ann Transl Med, 2014 Hofman & Popper, Virch Archiv 2016

25

26 Oncotarget 2017

27 Intensity scoring in tissue and CTCs with the SP44 clone (Ventana) Oncotarget 2017

28 Current development Liquid biopsy in the field of immunotherapy AACR 2016 manuscript in revision, 2017

29 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

30 Improvement of our knowledge in lung cancer pathophysiology Hofman et al, Curr Mol Med, 2014 ;;;;;;;;;

31 Toward the concept of a «liquid microenvironment» Interaction between circulating tumour cells and blood cells (platelets, myeloid cells and lymphocytes) A new world for new perspective in research investigation for lung cancer patients Neutrophils CTCs

32 Neutrophil

33 Platelets CTCs From Best et al, Cancer Cell 2017 ISET filter; LPCE, Nice, Fr TEP RNA profiles allow for blood-based early lung cancer diagnostic

34 Screening test for lung cancer?

35 Blood fitration method COPD Patients rrrrrrrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrryyyy yyyyyyyyyyr rrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrr rrrrrrrrrrryyyr Detection of CTCs using blood filtered through a filter.. Working with a kind of piston coffeemaker! rrrrrrrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrrrrrrr rrrrrrrrrrrrrrrrrrrrrrr! rrrrrrrrrrrrrrrrryyyy yyyyyyyyyyr

36

37 Patient LH/ P/Y Severe COPD November 2009 Ilie & Hofman, PLoS One 2014 Hofman et al, Curr Opin Oncol, 2017

38 ISET Technology Patient LH/ P/Y Severe COPD November mm Ilie & Hofman, PLoS One 2014 Hofman et al, Curr Opin Oncol, 2017

39 Patient LH/298 December 2011 ct1n0m0 Ilie & Hofman, PLoS One 2014 Hofman et al, Curr Opin Oncol, 2017

40 «Sentinel» Circulating Tumor Cells Allow Early Diagnosis of Lung cancer in Patients with Chronic Obstructive Pulmonary Disease Ilie & Hofman PLoS One 2014 Hofman, Curr Opin Oncol, 2017

41 Agenda Brief background Potential usefulness of a liquid biopsy in lung cancer The only current approach in a daily routine practice What s the future for CTCs detection? A couple of perspectives (among others) Conclusion

42 Liquide biopsy for lung cancer in 2017 In routine now, assessment of EGFR status in cf-dna Coming soon, assessment of various genomic alteration by NGS Perspectives: Early diagnosis of lung cancer (RNA, microrna, CTCs, DNA methylation) Predictive biomarker (s) for immunotherapy response Screening of lung cancer onset in a high risk population

43 Compagny X We still need to be aware! Can tissue biopsy be replaced by liquid biopsy? Ilié M and Hofman P, Transl Lung Cancer Res, 2016 Aug;5(4):420-3

44 Team LPCE 2017

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