The OncoBEAM RAS liquid biopsy experience in real-world clinical practice Frederick S. Jones, Ph.D., Global Director, Medical Scientific

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1 The OncoBEAM RAS liquid biopsy experience in real-world clinical practice Frederick S. Jones, Ph.D., Global Director, Medical Scientific Affairs,Sysmex

2 2 OncoBEAM liquid biopsy: circulating tumor DNA (ctdna) Concept of Liquid Biopsy:

3 OncoBEAM: BEAMing Digital PCR Pre-Amplification Emulsion PCR Hybridization Flow Cytometry Flow Cytometry Wild-type Mutant» BEAMing (Beads, Emulsions, Amplification, Magnetics).

4 Conventional vs Digital PCR: Digital PCR method can achieve higher analytical sensitivity of mutation detection Conventional PCR Digital PCR Wild-type DNA Mutant DNA Partitioning of DNA into many individual reactions Relative signal readout Digital signal readout 4 Vogelstein et al. PNAS 1999; mod. Diehl et al Curr Opin Oncol. 2007

5 Sysmex Inostics OncoBEAM RAS Kit (Joint Collaboration: Sysmex Merck) Bringing Service based Assays to Hospitals Globally LDT to IVD KRAS Codon 12 Codon 13 Codon 59 Codon 61 Codon 117 Codon 146» NRAS Codon 12 Codon 13 Codon 59 Codon 61 Codon 117 Codon mutation expanded RAS panel 5

6 Blood-based RAS Testing mcrc: Therapy Selection and Detection of Resistance Tissue Therapy Selection RAS Resistance Detection No tissue RAS mutation status WT Anti-EGFR therapy T0 T1 T2 Monitoring of RAS mutation status Blood» MUT Chemotherapy (e.g. FOLFIRI) Concordance of RAS status in blood vs tissue MUT Anti-EGFR Re-challenge?

7 OncoBEAM RAS in Spain - Published 2017

8 Overview: OncoBEAM RAS liquid biopsy studies in Spain - performance vs SOC tissue RAS testing OncoBEAM RAS Concordance and Outcomes Studies in Spain: Study Reference Pts. OPA Comments/Conclusions Grasselli et al Vidal, Muinelo et al % % Retrospective concordance; mpfs & mos of RAS+ and WT patients 2 nd and 3 rd line selected by tissue vs. plasma were comparable. Retrospective/Prospective concordance; mpfs of WT patients 1 st line selected by tissue vs. plasma were comparable.

9 MAFs in CONCORDANCE study: required sensitivity? MAF distribution of RAS mutant samples: MAFs 39% 29% % 0.1-1% >1% >5% 11% 21% -- 48% of patients were < 1% MAF 1% MAF> -- Techniques must show robustness in detection in the % MAFs Concordance study, VHIO, retrospective, patients anti-egfr naïve, t esting plasma BEAMing + tissue SOC & BEAMing (RAS panel). Grasselli et al Annals of Oncology 28: , 2017 doi: /annonc/mdx112 Published online 20 March 2017

10 Overview: OncoBEAM RAS liquid biopsy studies in Spain - performance vs SOC tissue RAS testing Studies of OncoBEAM RAS Concordance in Spain: Study Reference Pts. OPA Comments/Conclusions Grasselli et al % Retrospective concordance. Vidal, Muinelo et al % Retrospective/Prospective concordance Garcia-Foncillas et al % Real world experience: Prospective concordance study in clinical practice in 10 hospital labs where OncoBEAM is installed.

11 Garcia-Foncillas et al ESMO 2017

12 OncoBEAM RAS concordance is highest in CRC patients with metastases in the liver Liver Only Liver and Other Other P-value Concordance Plasma vs. Tissue N Discordance 6 (6.6%) 2 (2.7%) 14 (21.2%) Concordance 85 (93.4%) 73 (97.3%) 52 (78.8%)

13 OncoBEAM RAS concordance is lowest in CRC patients with metastases in the lung Lung Only Concordance Plasma vs. Tissue Lung and Other Other N P-value Discordance 7 (48.3%) 4 (7.6%) 11 (6.7.%) Concordance 9 (56.3%) 49 (92.4%) 152 (93.3%)

14 Detection of RAS mutations by OncoBEAM covers large dynamic MAF range in patients with liver metastases Patient SOC RAS Result OncoBEAM Result (%MAF) 1) Date tumor 2) Date blood 3) Tumor removed? Location of metastases Concordants Plasma vs. SOC - Garcia-Foncillas et al KRAS12 KRAS12 (51.815%) 11/15, 12/15, No Liver, lung, peritoneal KRAS146 KRAS146 (35.23%) 3/16, 4/16, No Liver, lung KRAS12 KRAS12 (9.88%) 7/16, 8/16, No Liver, lung KRAS13 KRAS13 (6.477%) 12/15, 1/16, No Liver KRAS12 KRAS12 (0.804%) 1/16, 2/16, No Liver KRAS12 KRAS12 (0.465%) 1/16, 2/16, No Liver and peritoneal KRAS12 KRAS12 (0.0503%) 1/16, 3/16, No Liver KRAS61 KRAS61 (0.052%) 2/16, 4/16, Yes Liver

15 Expert Taskforce Recommendation for OncoBEAM in Clinical Practice The clinical utility of the OncoBEAM RAS test allows patients to benefit from international guideline-recommended expanded RAS testing with rapid turnaround. The high degree of concordance of results generated by blood-based OncoBEAM RAS vs. standard tissue testing methods supports the conclusion that detection of RAS mutations in the blood with BEAMing may be a useful replacement to tumor testing. Garcıa-Foncillas, J. et al. Ann. Oncol. 0: 1 7, doi: /annonc/mdx501(2017).

16 Summary: OncoBEAM liquid biopsy

17 Evaluation of the Sensitivity of KRAS Mutation Detection of the Idylla Platform in Comparison to the OncoBEAM RAS CRC Assay Ana Vivancos 1, Enrique Aranda 2, Manuel Benavides 3, Elena Elez 4, M. Auxiliadora Gómez-España 2, Marta Toledano 5, Martina Alvarez 6, M. Rosario Chica Parrado 6, Vanesa García-Barberán 7, and Eduardo Diaz-Rubio 7 1 Cancer Genomics Group, Vall d Hebron Institute of Oncology, Barcelona; 2 Department of Medical Oncology, Reina Sofía University Hospital, CIBERONC, Córdoba; 3 Department of Medical Oncology, Hospital Universitario Regional y Virgen de la Victoria Málaga; 4 Department of Medical Oncology Vall d Hebron Institute of Oncology Barcelona; 5 IMIBIC Instituto Maimonides Investigación Biomédica de Córdoba; 6 Laboratorio de Biología Molecular del Cáncer-Centro de Investigaciones Médico Sanitarias Málaga; 7 Laboratorio de Investigación Traslacional, IdISSC, Hospital Clínico San Carlos, CIBERONC, Madrid

18 OncoBEAM vs Idylla Reported differences in analytical and clinical sensitivity OncoBEAM RAS CRC (digital PCR) 16 KRAS and 18 NRAS mutations analytical senstivity < 0.1% mutant allelic fraction (MAF). MAF = calculated by counting mutant versus WT beads and determining proportion or ratio > 90% concordance in several clinical studies 1,2,3,4 The Idylla ctkras Mutation Test (qpcr) analytical sensitivity of 1% for KRAS mutations in exons 2 and 3 and 5% for mutations in exon % concordance of Idylla and SOC RAS tissue test results for mcrc 5,6 88.3% concordance in subgroup with liver metastases 5,6,7 90.4% concordance in newly-diagnosed patients with synchronous liver metastases 5,6,7 1.. Schmiegel, W. et al. Mol. Oncol. 11, (2017); 2.. Vidal, J., Muinelo, L., et al. Ann. Oncol. 28: , (2017); 3. Grasselli, J. et al. Ann. Oncol. 28: , (2017); 4. Garcıa-Foncillas, J. et al. Ann. Oncol. 0: 1 7, doi: /annonc/mdx501(2017); 5. Biocartis Idylla_Tech_Sheet-ctKRAS-IVD-A4_web.pdf; 6. Bachet J. B., et al. J. Clin. Oncol :15 suppl.; 7. Normanno N., Ann. Oncol. 28, Issue suppl5, mdx

19 Scale of Reported Mutation Detection Analytical Sensitivity (%MAF) 2-5% 1-5% 1.0% Idylla 0.1% - 0.5% < 0.1% = Detection of 1 mutant count in 1,000 total counts > 0.01% = Detection of 1 mutant count in 10,000 total counts 19

20 Ana Vivancos et al. - Poster 592 ASCO GI Congress January 20, San Francisco, California Presenters: Drs. Enrique Aranda and Manuel Benavides 2

21 Objectives KRAS detection sensitivity: OncoBEAM <0.1% MAF vs. Idylla 1% MAF Primary Objective: PPA of Idylla vs OncoBEAM across three categories of KRAS MAF: 1) 5%, but > 1% 2) 1%, but > 0.1% 3) 0.1% Secondary Objective: Clinical sensitivity of Idylla and OncoBEAM plasma KRAS mutation detection at 1% MAF vs FFPE tumor RAS testing on primary tumor specimens 2

22 Study Schema 2

23 Results - Primary Objective 2

24 Results - Secondary Objective Concordance for 1% MAF: OncoBEAM with SOC : 72.1% (31/43) Idylla with SOC : 46.5% (20/43) 02/0 4/20 24

25 WT Calls by Idylla in 14 OncoBEAM and SOC KRAS-MUT + Patients 14 of 31 were called WT using Idylla while being mutated using OncoBEAM and SOC PPA of Idylla vs OncoBEAM and SOC: 54.8% (red). 8 of these 14 discordants where Idylla was WT vs SOC and OncoBEAM that were KRAS-MUT+ were observed in patients with liver metastases (yellow).

26 RAS-MUT + Calls by OncoBEAM and Idylla in SOC WT Patients 12 out of 43 patients were WT by SOC FFPE RAS testing. 9 out of these 12 were KRAS-MUT + by both OncoBEAM and Idylla --- a signal that some of these patients may be KRAS-MUT + (beige) WT Calls by Idylla and SOC in OncoBEAM KRAS-MUT + Patients Only 3 out of 43 patients called WT by Idylla were also WT and in agreement with call made by SOC FFPE RAS testing.

27 Summary of Key Findings Idylla shows reduced analytical sensitivity < 1% MAF compared to OncoBEAM ; (PPA, 61.2% vs OncoBEAM, N=115) Clinical sensitivity of Idylla < 1% MAF vs OncoBEAM and SOC FFPE RAS testing : 54.8% Reduction in overall clinical sensitivity by Idylla ctkras vs SOC FFPE and OncoBEAM plasma testing = 16.9% Concordance for 1% MAF: Idylla ctkras vs SOC FFPE RAS is 46.5% 2 7

28 Conclusions Gray zone below 1% MAF: Idylla shows reduced RAS mutation detection accuracy vs both OncoBEAM and SOC FFPE tumor RAS testing. Reminder: Liquid biopsy assays with diminished sensitivity may lack the dynamic range to provide the accurate RAS mutational status to properly guide highly individualized anti-egfr treatment decisions that may benefit patient outcomes. 2