Liquid biopsies to track clonal evolution and resistance to EGFR inhibition in mcrc

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1 Liquid biopsies to track clonal evolution and resistance to EGFR inhibition in mcrc Alberto Bardelli Candiolo Cancer Center IRCCs University of Torino - Medical School

2 Disclosures Horizon discovery Biocartis Trovagene Relationship SAB SAB SAB

3 Tumor evolution in the blood of patients

4 Colorectal cancer as a model system for liquid biopsies Adenomatous Polyps Severe Dysplasia Adenocarcinoma Hepatic Metastasis Early Adenoma Hyperproliferation

5 Actionable targets in CRC

6 Liquid biopsies of actionable targets in CRC crizotinib MET ALK Lapatinib trastuzumab HER2 cetuximab panitumumab EGFR TRKA entrectinib Wnt 1 Porcupine inhibitor Anti RSPO Ab RSPO LRP5/6 Frizzled LGR4-6

7 Publication First clinical response (PR) Validation in animal models First patient treated (Niguarda Hospital) Clinical trial HERACLES

8 HER2+ mcrc xenopatients are sensitive to dual HER2 blockade with lapatinib and trastuzumab vehicle trastuzumab lapatinib HER2+ mcrc PDX or xenopatients lapatinib and trastuzumab Bertotti A. et al, Cancer Discovery 2012

9 HER2 amplification in CRC IHC 0 IHC 1+ IHC 2+ IHC 3+ HER2 IHC 10X 10X 10X 10X 10X 10X 10X 10X 20X 20X 20X 20X 10X 10X 10X 10X 40X 40X 40X 40X FISH 100X 100X 100X 100X Positive tumor cells > 50% Eligible for HERACLES Trial

10 Patient # Patient # Representative CE-CT scans of 2 responders baseline Week 8 - PR Week 54 - PR + baseline Week 8 - PR Week 24 - PR + 10

11 Change in target lesion from baseline (%) Change in target lesion from baseline (%) Response and resistance to HER2 blockade Waterfall plot Spaghetti plot HER2 3+ HER2 2+ Patients on treatment PD NEW LESION 11 *3 patients are not shown: (IHC 2+), not assessed yet; (IHC 3+) and (IHC 3+) early clinical PD. Sartore-Bianchi et al., Lancet Oncology 2016

12 E R B B 2 C N V o r G E * 1 0 ^ -3 / m l p la s m a o r H E R 2 E C D (n g / m l) % P IK 3 C A o r T P 5 3 m u t a t e d a lle le s HER2 blockade secondary resistance G E * 1 0 ^ -3 / m l p la s m a E R B B 2 C N V Patient P O Z Z I 5 0 H E R 2 E C D P IK 3 C A H R 4 0 T P 53 R 17 5H N G S Siravegna et al., Unpublished

13 HER2 CNA or ERBB3 % mutated alleles TP53 % mutated alleles or GE*10^-3/ml plasma HER2 blockade secondary resistance ERBB2 CNV GE*10^-3/ml plasma ERBB3 L652W ERBB3 E928G TP53 G105V Patient PD 42-PD weeks of Trastuzumab+lapatinib treatment 0 Siravegna et al., Unpublished

14 Actionable targets in CRC crizotinib MET ALK Lapatinib trastuzumab HER2 cetuximab panitumumab EGFR TRKA entrectinib Wnt 1 Porcupine inhibitor Anti RSPO Ab RSPO LRP5/6 Frizzled LGR4-6

15 CRC patient: ALK translocated 4X 20X 60X CRC patient: NTRK1 translocated 4X 20X 60X Medico et al., Nature Commun 2015

16 Targeting translocated NTRK1 in CRC B LMNA-NTRK1 Sartore-Bianchi et al., JNCI 2016

17 Monitoring NTRK1 blockade in cfdna Russo et al., Cancer Discovery 2016

18 TRKA resistance mutations

19 Cetuximab Panitumumab Anti EGFR therapy in colorectal cancer Ligand EGFR EGFR/ErbB2/ ErbB3/ErbB4 P P Cytoplasm RAS PI3K RAF AKT MEK ERK Cell Proliferation Survival Migration Nucleus

20 % mutated alleles Monitoring EGFR blockade in cfdna Misale et al., Nature 2012 Siravegna et al., Nat Med 2015

21 Mechanism (1) Downstream signaling reactivation EGFR p.v600e p.g12d p.g13d p.g12r p.g12c p.k117n p.a146t BRAF KRAS NRAS p.g12d p.g13d p.g12c p.q61r MEK p. K57N Approx 70% of patients ERK

22 Mechanism (2) Mutations of the target: EGFR ECD EGFR S492R EGFR R451C EGFR S464L EGFR G465R EGFR K467T EGFR I491M S492K467 I491 G465 S464 R451 EGFR Approx 30% of patients

23 Resistance to EGFR blockade drives lesion-specific responses in colorectal cancer

24 MAP2K1 K57T emerges during to EGFR blockade TP53 p.e171* MAP2K1 p.k57t TISSUE SAMPLES TP53 RAS/RAF MAP2K1 Primary Tumor, 2011 Liver metastasis (pre-cetux), 2012 Post-cetux progression liver biopsy, 2014 p.e171* WT WT p.e171* WT WT p.e171* WT p.k57t

25 Clonal evolution and lesion-specific responses Panitumumab + trametinib (5 months) MEK1 p.k57t? Russo et al., Cancer Discovery 2016

26 Clonal evolution and lesion specific response in cfdna

27 Tracing cancer s evolutionary trees with liquid biopsies Russo and Bardelli submitted

28 What are we up against?

29 First cell: 4 billion years Mass extinction events Bottlenecks

30 Tumor evolution and targeted therapies Response to therapy Resistance to therapy

31 Liquid biopsies to monitor cancer evolution 1. Exploiting clonal evolution 2. Therapies that adapt to tumor evolution

32 -1- Exploiting clonal evolution

33 % mutated alleles What happens to resistant clones upon progression? Baseline Par al Response Progressive disease

34 KRAS clones decline upon withdrawal of EGFR antibodies Siravegna et al., Nature Medicine 2015

35 When KRAS clone decline in blood, re-challenging with anti-egfr antibodies can be clinically effective CHRONOS

36 % mut RAS clones CHRONOS 1 st line including anti-egfr RAS Baseline 2 nd line rechallenge Mutational Load (BML) RAS Intermediate Mutational Load (IML) RAS Rechallenge Mutational Load (RML) time Tumor sensitivity to anti-egfr sensitive resistant Andrea Sartore Bianchi, Salvatore Siena, Silvia Marsoni

37 -2- Therapies that adapt to tumor evolution

38 EGFR ECD resistant mutations EGFR S492R EGFR R451C EGFR S464L EGFR G465R EGFR K467T EGFR I491M S492K467 I491 G465 S464 R451 EGFR Sabrina Arena and Clara Montagut Approx 30% of patients

39 Emergence of RAS or EGFR extracellular domain mutations and duration of response to EGFR blockade All (N=27) EGFR only (N=7) RAS only (N=13) EGFR + RAS (N=7) Male Sex (%) 13 (48%) 5 (71%) 6 (46%) 2 (29%) Age (median, range) 60 (31-81) 64 (44-78) 59 (42-81) 55 (31-78) Anti-EGFR drug Cetuximab 23 (85%) 6 (86%) 11 (85%) 6 (86%) Panitumumab 4 (15%) 1 (14%) 2 (15%) 1 (14%) Irinotecan -based 20 (74%) 4 (57%) 10 (77%) 6 (86%) Chemotherapy Oxaliplatin -based 4 (15%) 2 (29%) 1 (8%) 1 (14%) None 3 (11%) 1 (14%) 2 (15%) 0 1st 5 (19%) 2 (29%) 1 (8%) 2 (29%) Line of treatment 2nd 9 (33%) 2 (29%) 6 (46%) 1 (14%) > 3rd 13 (48%) 3 (42%) 6 (46%) 4 (57%) Best Response Stable Disease >16w 12 (44%) 1 (14%) 8 (62%) 3 (43%) Response 15 (56%) 6 (86%) 5 (38%) 4 (57%) Progression Free Survival Median (weeks; 95% CI) 39.1 (33-46) 45 (42-48) 25.6 (24-27) 38.7 (21-56) Clara Montagut

40 Emergence of RAS or EGFR extracellular domain mutations and duration of response to EGFR blockade Van Emburgh et al submitted

41 EGFR ECD mutations appear after K-RAS clones during EGFR blockade Van Emburgh et al submitted

42 Dynamics of RAS and EGFR-ECD clonal evolution in cancer cells populations Baseline 1 Month 2 Months 3 Months 4 Months 6 Months NRAS G12C NRAS G12C+EGFR S492R 0.5-5% <0.5%

43 MM-151 and EGFR ECD mutations Arena et al., Science Transl Med 2016

44 MM-151 on patient s avatar

45

46 MM-151 and EGFR ECD mutations EGFR ECD TRIAL

47 In blood veritas DiAGNOSIS: genotyping cfdna in the blood to determine the tumor profile SURGERY: tumor cfdna is not present, the patient is disease free RESISTANCE: emergence of genetic alterations associated with drug resistance MINIMAL RESIDUAL DISEASE: tumor cfdna is still present in the circulation FOLLOW UP: patient monitoring throughout the treatment course to assess response and resistance TREATMENT: analysis tumor cfdna for real time monitoring of response to treatment

48 Monitoring tumor evolution Russo and Bardelli

49 Josep Tabernero Ryan Corcoran Silvia Marsoni Cosimo Martino Salvatore Siena Andrea Sartore Bianchi Mauro Truini Emanuele Valtorta Federica Di Nicolantonio Livio Trusolino Andrea Bertotti Enzo Medico Clara Montagut Beatriz Bellosillo Gary Li Sabrina Arena Giulia Siravegna Benedetta Mussolin Carlotta Cancelliere Simona Lamba Luca Lazzari Giovanni Germano Beth Van Emburgh Giorgio Corti Ciovanni Crisafulli Michela Buscarino Alice Bartolini Mariangela Russo Sandra Misale

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