Coordinación científica:

Dr. Rafael López López

Complejo Hospitalario Universitario de Santiago de

Compostela

Organizado por: Sede:

San Francisco Hotel Monumento

Campillo de San Francisco, 3 - Santiago de

Compostela

The OncoBEAM RAS liquid biopsy experience in

real-world clinical practice

Frederick S. Jones, Ph.D.,

Global Director, Medical Scientific Affairs,Sysmex

OncoBEAM liquid biopsy:

circulating tumor DNA (ctDNA)

• 2

Concept of Liquid Biopsy:

OncoBEAM: BEAMing Digital PCR

• 3

Flow Cytometry

Wild-type

Mutant

Pre-Amplification Emulsion PCR Flow

Cytometry Hybridization

» BEAMing (Beads, Emulsions, Amplification, Magnetics).

4 Vogelstein et al. PNAS 1999; mod. Diehl et al Curr Opin Oncol. 2007

Digital PCR Conventional PCR

Wild-type DNA

Mutant DNA Partitioning of DNA

into many individual

reactions

Digital signal readout Relative signal readout

Conventional vs Digital PCR: Digital PCR method can achieve higher analytical sensitivity of mutation detection

Sysmex Inostics OncoBEAM™ RAS Kit

(Joint Collaboration: Sysmex | Merck)

Bringing Service based Assays

to Hospitals Globally LDT to IVD

• 5

NRAS

• Codon 12

• Codon 13

• Codon 59

• Codon 61

• Codon 117

• Codon 146

KRAS

• Codon 12

• Codon 13

• Codon 59

• Codon 61

• Codon 117

• Codon 146

» 34 mutation expanded RAS panel

RAS mutation

status

Anti-EGFR

therapy

Tissue

Blood

WT

Chemotherapy

(e.g. FOLFIRI)

MUT

Monitoring of RAS mutation status

T0 T1 T2

MUT

No tissue

Therapy Selection

Anti-EGFR Re-challenge?

Blood-based RAS Testing mCRC: Therapy

Selection and Detection of Resistance

RAS Resistance Detection

» Concordance of RAS status in blood vs tissue

OncoBEAM RAS in Spain - Published 2017

7

• 8

OncoBEAM RAS Concordance and Outcomes Studies in Spain:

Study Reference Pts. OPA Comments/Conclusions

Grasselli et al.

2017 146 89.7%

Retrospective concordance;

mPFS & mOS of RAS+ and WT patients 2nd and 3rd line

selected by tissue vs. plasma were comparable.

Vidal, Muinelo et

al. 2017 115 93.0%

Retrospective/Prospective concordance;

mPFS of WT patients 1st line selected by tissue vs. plasma

were comparable.

Overview: OncoBEAM RAS liquid biopsy studies in

Spain - performance vs SOC tissue RAS testing

MAFs in CONCORDANCE study: required sensitivity?

29%

21%

11%

39%

MAFs

0.01-0.1%

0.1-1%

>1%

>5%

Concordance study, VHIO, retrospective, patients anti-EGFR naïve, t esting plasma BEAMing + tissue SOC & BEAMing (RAS panel).

MAF distribution of RAS mutant samples:

-- 48% of patients were < 1% MAF -- Techniques must show robustness in detection in the 0.01-1% MAFs

1% MAF>

Grasselli et al Annals of Oncology 28: 1294–1301, 2017

doi:10.1093/annonc/mdx112 Published online 20 March 2017

• 10

Studies of OncoBEAM RAS Concordance in Spain:

Study Reference Pts. OPA Comments/Conclusions

Grasselli et al. 2017 146 89.7% Retrospective concordance.

Vidal, Muinelo et al.

2017 115 93.0% Retrospective/Prospective concordance

Garcia-Foncillas

et al. 2017 232 90.5%

Real world experience: Prospective concordance study

in clinical practice in 10 hospital labs where OncoBEAM

is installed.

Overview: OncoBEAM RAS liquid biopsy studies in

Spain - performance vs SOC tissue RAS testing

Garcia-Foncillas et al – ESMO 2017

• 11

OncoBEAM RAS concordance is highest in

CRC patients with metastases in the liver

12

Liver Only Liver and

Other Other P-value

Concordance Plasma vs. Tissue

N 91 75 66

0.0007 Discordance 6 (6.6%) 2 (2.7%) 14 (21.2%)

Concordance 85 (93.4%) 73 (97.3%) 52 (78.8%)

OncoBEAM RAS concordance is lowest in

CRC patients with metastases in the lung

13

Lung

Only

Lung and

Other Other P-value

Concordance Plasma vs. Tissue

N 16 53 163

0.0003 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. 2017

14377 KRAS12 KRAS12 (51.815%) 11/15, 12/15, No Liver, lung, peritoneal

14343 KRAS146 KRAS146 (35.23%) 3/16, 4/16, No Liver, lung

14408 KRAS12 KRAS12 (9.88%) 7/16, 8/16, No Liver, lung

14323 KRAS13 KRAS13 (6.477%) 12/15, 1/16, No Liver

14494 KRAS12 KRAS12 (0.804%) 1/16, 2/16, No Liver

14571 KRAS12 KRAS12 (0.465%) 1/16, 2/16, No Liver and peritoneal

14314 KRAS12 KRAS12 (0.0503%) 1/16, 3/16, No Liver

14468 KRAS61 KRAS61 (0.052%) 2/16, 4/16, Yes Liver

Expert Taskforce Recommendation for

OncoBEAM in Clinical Practice

15

“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:10.1093/annonc/mdx501(2017).

16

Summary: OncoBEAM liquid biopsy

Evaluation of the Sensitivity of KRAS Mutation Detection of the Idylla Platform in Comparison to

the OncoBEAM RAS CRC Assay

Ana Vivancos1, Enrique Aranda2, Manuel Benavides3, Elena Elez4, M. Auxiliadora Gómez-España2, Marta Toledano5,

Martina Alvarez6, M. Rosario Chica Parrado6, Vanesa García-Barberán7, and Eduardo Diaz-Rubio7

1Cancer Genomics Group, Vall d’Hebron Institute of Oncology, Barcelona; 2Department of Medical Oncology, Reina Sofía University Hospital, CIBERONC, Córdoba;

3Department of Medical Oncology, Hospital Universitario Regional y Virgen de la Victoria Málaga; 4Department of Medical Oncology Vall d’Hebron Institute of Oncology Barcelona;

5IMIBIC Instituto Maimonides Investigación Biomédica de Córdoba; 6Laboratorio de Biología Molecular del Cáncer-Centro de Investigaciones Médico Sanitarias Málaga;

7Laboratorio de Investigación Traslacional, IdISSC, Hospital Clínico San Carlos, CIBERONC, Madrid

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 studies1,2,3,4

The Idylla™ ctKRAS Mutation Test (qPCR) • analytical sensitivity of ≤ 1% for KRAS mutations in exons

2 and 3 and ≤ 5% for mutations in exon 4 5

• 78.9% concordance of Idylla™ and SOC RAS tissue test results for mCRC5,6

• 88.3% concordance in subgroup with liver metastases5,6,7

• 90.4% concordance in newly-diagnosed patients with synchronous liver metastases5,6,7

1.. Schmiegel, W. et al. Mol. Oncol. 11, 208–219 (2017); 2.. Vidal, J., Muinelo, L., et al. Ann. Oncol. 28: 1325–1332, (2017); 3. Grasselli, J. et al. Ann. Oncol. 28: 1294–1301, (2017); 4. Garcıa-Foncillas, J. et al. Ann. Oncol. 0: 1–7, doi:10.1093/annonc/mdx501(2017); 5. Biocartis Idylla_Tech_Sheet-ctKRAS-IVD-A4_web.pdf; https://biocartis.com/news; 6. Bachet J. B., et al. J. Clin. Oncol. 2017 35:15 suppl.; 7 . Normanno N., Ann. Oncol. 28, Issue suppl5, 1 2017. mdx393.066.

Scale of Reported Mutation Detection Analytical Sensitivity (%MAF)

19

1 - 5%

1.0%

0.1% - 0.5%

< 0.1%

2- 5%

Idylla™

= Detection of 1 mutant count in 1,000 total counts

> 0.01% = Detection of 1 mutant count in 10,000 total counts

Ana Vivancos et al. - Poster 592 ASCO GI Congress

January 20, 2018 - San Francisco, California Presenters: Drs. Enrique Aranda and Manuel Benavides

• 20

• 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

• 22

Study Schema

• 23

Results - Primary Objective

• 24

02/04/2015

Results - Secondary Objective

Overall, the testing with Idylla ctKRAS resulted in a 16.9% reduction in clinical sensitivity (PPA) for KRAS mutation detection vs SOC FFPE and OncoBEAM plasma testing. Since 37.4% of the 559 mCRC patients in this study were KRAS-MUT+ ≤ 1% MAF using OncoBEAM, it is estimated that a 16.9% drop in clinical sensitivity by Idylla might result in false negative calls for 45 out of 559 patients in this study.

Idylla ctKRAS testing showed WT results in 14 out of 31 patients that were called KRAS-

MUT+ by both OncoBEAM and SOC (PPA, 54.8% ; 17/31). This PPA was similar to that found in objective 1 of this study (61.2% for n= 80 patients; Table 1).

Notably, 8 out of 14 these discordants were seen in patients with liver metastases (see

Table 2).

The overall agreement (concordance) of OncoBEAM vs SOC FFPE KRAS testing results for 43 patients ≤ 1% MAF was 31/43 or 72.1%, whereas concordance of Idylla with SOC FFPE KRAS results was 20/43 or 46.5%.

31 out of 43 patients that were KRAS-MUT+ by OncoBEAM at ≤ 1% MAF were KRAS-

MUT+ by SOC FFPE tissue testing (Table 2). 12 out of 43 patients that were KRAS-MUT+ by OncoBEAM at ≤ 1% MAF were WT by SOC

FFPE tissue testing (Table 2). Interestingly, both Idylla and OncoBEAM calls were RAS-MUT+ in 9 out of 12 patients - - it is therefore likely that some of these patients may in fact be RAS-MUT+.

Only 3 patients called WT by Idylla were in agreement with a WT call made by SOC FFPE

testing.

Concordance for ≤1% MAF: OncoBEAM with SOC : 72.1% (31/43) Idylla with SOC : 46.5% (20/43)

• 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).

WT Calls by Idylla in 14 OncoBEAM and SOC KRAS-MUT+ Patients

RAS-MUT+ Calls by OncoBEAM and Idylla in SOC WT Patients

WT Calls by Idylla and SOC in OncoBEAM KRAS-MUT+ 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)

• 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%

• 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.