Comparison of OncoBEAM and NGS methods to

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T790M mutation during progression in Non Small Cell Lung Cancer. Methodology ... screening of other EGFR TKI resistance mechanisms;. - Monitoring EGFR p ...
Comparison of OncoBEAM and NGS methods to detect plasma EGFR p.T790M mutation during progression in Non Small Cell Lung Cancer Jessica Garcia 1,2,3, Aurélia Delherme1,3, Florence Geiguer1,3, Patrick Merle4, Claire Tissot5, Frederick S. Jones6, Daniel L. Edelstein6, Chassidy Johnson7, Pierre-Jean Souquet8, Claire Rodriguez-Lafrasse1, Zhenuy Xu9, Léa Payen1,2,3, and Sébastien Couraud3,8 1. Laboratoire de Biochimie et Biologie Moléculaire, Groupe Hospitalier Sud, Hospices Civils de Lyon, Lyon, 69003, France / 2. Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Cancer Research Center of Lyon, Equipe Labellisée Ligue contre le Cancer, Lyon, France / 3. Cancer Institute of Hospices Civils de Lyon, CIRculating CANcer (CIRCAN) program, 69495 Pierre Bénite, France / 4. Service de Pneumologie et cancérologie thoracique, CHU de Clermont-Ferrand, France / 5. Service de Pneumologie et cancérologie thoracique, CHU de Saint Etienne, France / 6. Medical Scientific Affairs, Sysmex Inostics, Inc., Mundelein, IL, USA 60060 / 7. Clearbridge Biomedics Inc., Singapore / 8. Service de Pneumologie Aigue Spécialisée et Cancérologie Thoracique, Groupement Hospitalier Sud, Cancer institute of Hospices Civils de Lyon, Pierre Bénite, 69495, France / 9. SOPHiA Genetics, Saint Sulpice, Switzerland

Results

Purposes

Samples from stage 4 NSCLC patients with princeps EGFR mutation and who progress under 1st line TKI were sent to our lab as part of our routine program for detection of EGFR p.T790M mutation (CIRCAN). We used both assays (NGS and BEAMing) to detect this mutation in 183 paired samples. BEAMing Technology – Sysmex Inostics

A

1

Pre-Amplification

Emulsion PCR

Hybridization

Flow Cytometry

Wild-type

Mutant

Figure 1 : Overview of the workflow of OncoBEAM digital PCR used for the research of EGFR p.T790M in cfDNA samples. BEAMing

PCR (Beads, Emulsion, Amplification and Magnetics) is based on Biorad a multiplex PCR targeting EGFR somatic alterations and a PCR B Digital Droplet PCR amplification performed on magnetics Emulsion beads in the followed by an hybridization with fluorescent probes. The wildstepoil emulsion PCR Cytometry cfDNA extraction type and corresponding mutated forms are quantified by flow cytometry.

0.10

n=3 Mean = 0.0078 SD : 0.0019

AF(%) = 0.1% 15 - 20 ng

AF(%) = 0.1% 11 - 5 ng

AF(%) = 0.1% 5 - 2 ng

Library Preparation

Flow Cell Loading

Clustering

AF(%) = 1% 10 - 5 ng

AF(%) = 1% 5 - 2 ng

5B Correlation of p.T790M allelic frequency between OncoBEAMTM-EGFR and NGS-56G § 183 paired samples of cfDNA analyzed with both p.T790M OncoBEAMTM-EGFR and NGS-56G results § Good correlation between OncoBEAMTM-EGFR and NGS56G § BUT: 21 (11%) discordant cases with p.T790M found with BEAMing

Sequencing

Kit « QIAamp circulating acid kit »

Clinical case 1,8 1,6 1,4

1 0,1 0,01

1,2 1 0,8 0,6 0,4

0,001

0,2

0,0001

0

7B

Horizon cfDNA control Figure 4: Allelic frequency (AF) of p.T790M mutation observed in wild-type (WT) control and Horizon cfDNA control mutated at 0.1% and 1% with BEAMing p.T790M assay. N= Number of experiments. Error bars =SD Association of p.T790M with 5A sensitizing EGFR mutations § In 43.4% the p.T790M was BEAMing p.T790M NGS 56G assay associated with a sensitizing L858R/del 19 positive L858R/del 19 negative L858R/del 19 positive L858R/del 19 negative mutation with OncoBEAMTM-EGFR 33 (43.4%) 7 (6.6%) 20 (26.3%) 0 T790M positive (N; %) 1.4261 (3.1315) 0.1146 (0.1102) 6.2 (12.7) Mean allelic frequency in % (SD) and 26,3% with NGS 43 (56.6%) 100 (93.4%) 56 (73.7%) 107 (100%) T790M negative (N; %) § In 7 cases, the p.T790M was no 0.0086 (0.0074) 0.01 (6x10-19) Mean allelic frequency in % (SD) associated with sensiziting Total (N) 76 107 76 107 mutation with OncoBEAMTM-EGFR

Library Preparation by Swift Biosciences and Illumina Technology

2

BEAMing performances with DNA control § Threshold of positivity set up at 50 mutated beads with an AF at 0.02% § Very good specificity determined with WT Horizon control § Sensitivity at 1% with only 2 ng of starting material and at 0.1% with at least 11 ng of starting material

0.01 AF(%) = 0.1% 50 - 20 ng

T4

10

n = 21 Mean = 0.008 SD : 0.005

WT DNA

p.T790M BEAMing

Osimertinib

1

n=6 Mean = 0.00815 SD : 0.0034

n=4 Mean = 0.0403 SD : 0.0173

T2

T1

p.delEX19 NGS

T3

Gefitinib

n=8 Mean = 0.0890 SD : 0.0416

p.T790M NGS

7A

n=5 n=4 Mean = 0.5840 Mean = 0.3264 SD : 0.2529 SD : 0.2991

10

T1

T2

T3

T4

60y male patient diagnosed with adenocarcinoma of the lung. 1. Detection of p.delEX19 in cfDNA and in FFPE samples (T1) è Gefitinib 2. Asymptomatic disease progression on left adrenal gland è cfDNA testing 3. cfDNA show a p.T790M EGFR mutation è switch for osimertinib (T3). 4. Complete response on CT-scan and cfDNA (T4).

Figure 7: (A) Monitoring of the EGFR p.T790M and p.delEX19 allelic frequency under Gefinitib and Osimertinib treatments for each time point available. (B) Abdominal CT scan performed at each time-point.

8A

n=2

n=2

100

n=2

n=2

ClearCell Fx -

Allelic frequency of mutated copies of p.T790M

Methodology

4 Allelic frequency of p. T790M mutated copies %

The aim of this work was to compare EGFR p.T790M mutation detection in circulatingfree DNA (cfDNA) using two assays: OncoBEAMTM-EGFR digital PCR (Sysmex Inostics), and next generation sequencing (NGS, Illumina), with the 56G oncology panel (Swift Biosciences). We also assess detection of this mutation in mimicked circulating tumor cells (mCTC) using the ClearCell FX device (ClearBridge Biomedics).

10

ClearCell Fx + Cell Pellet

n=3 n=3 1

n=2 0.10

n=2

n=2

0.01 0 cells

10 cells (1.3 cells/mL)

50 cells (6.7 cells/mL)

100 cells HCC827 H1915 (13.3 cells/mL) (p.T790M -)(p.T790M +)

NGS 56G oncology panel

8B

732

730

729

728

726

725

724

721

717

716

715

713

712

710

709

701

697

696

691

690

689

684

677

666

660

649

646

642

637

631

622

621

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616

608

607

604

916

912

910

905

904

903

899

898

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895

893

892

890

888

884

883

881

877

875

873

871

869

865

863

861

858

856

854

848

843

837

836

834

833

832

830

Sample ID

829

BEAMing

0 0 0 Princeps 0 0 0,3 40 0 5,3 0 0 1,3 0 0 0 0 0 0 1,3 0 0 0 0 25 1 0 21 9,6 0 0 0 0 0,4 0 0 0,4 32 0,5 0,7 0,3 3,5 0 0 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### Princeps

825

NGS

Sample origin

1005

1004

1003

1002

999

998

989

988

987

981

977

975

974

973

970

969

968

966

965

963

962

961

958

957

956

954

953

952

951

948

944

943

942

941

940

938

937

Sample ID

929

BEAMing

926

NGS

0 0 0 Princeps 0 3,5 3,1 0 0,9 0 0 0 0 0 0 0 0 0 50 0,5 0 0 0,8 0 14 19 0 0 4,6 0 15 0 0 3,5 0,5 0,5 0 8,6 17 0 0 2,5 0 0,5 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### Princeps

921

FFPE

Sample origin

1072

1065

1064

1061

1058

1056

1052

1050

1043

1040

1039

1037

1036

1034

1033

1032

1030

1025

1024

1022

Sample ID

1014

BEAMing

31 0 0 0 0 0 0,7 1 53 0 3 0 0 0 0 0 0 0 5,5 0 33 0,1 0,5 0 0 0,4 40 0 0 0 0 0 2,6 1,4 0,9 0 0 2 29 2,5 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### Princeps

1013

NGS

Princeps

1006

FFPE

Sample origin FFPE NGS

Acknowledgement & funding Authors would like to thank all patients, families and prescribing physicians. This work was supported by Astra Zeneca, Sysmex Inostics, ClearBridge Biomedics, Merck, Sophia Genetics, and Ligue Nationale contre le Cancer of Saône et Loire. Contact: [email protected] / [email protected] / [email protected]

ClearCell Fx -

10

ClearCell Fx + Cell Pellet

1

n=3

n=3

n=3

0.100

n=2 0.010

n=2

n=2

Sample origin FFPE

Figure 3 : Enrichment of Circulating Tumor Cells (CTS’s) by a Label-Free Inertial microfluidic method : The ClearCell Fx (A). The red blood cells were eliminated with a lysis buffer. The residual white blood cells and CTC’s were loaded in the ClearCell CTChip FR1 (B). The CTC’s enrichment is based on the Dean Flow Fractionation principle (C). The smaller hematological cells [8 – 15 µm] are affected by the Dean Drag and migrate to waste outlet whereas the CTC’s [>15 µm] migrate to the upper outlet.

n=1

821

819

816

813

810

808

807

806

804

802

801

800

799

797

790

788

786

784

778

775

773

772

769

768

766

762

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749

748

745

Sample ID

742

NGS

0 0 Princeps 0 2,3 0,2 0,1 0 7,2 0 2,9 0,2 0 0 12 1,8 1,6 2,4 0 0 0 0 37 0 0 0 0,9 42 3,9 8 8,6 0 0 0,5 0 0 0 0 47 1,1 0 0 0 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### Princeps

739

FFPE

823

3C

603

Sample origin

734

3B

602

Sample ID

BEAMing

3A

Mutation 588

Assay

822

Figure 2 : NGS (Next Generation Sequencing) library preparation workflow (Library preparation, flow cell loading, clustering and paired-end sequencing) is performed with a kit based on multiplex PCR targeting 56 genes including EGFR gene, named 56G provided by Swift Biosciences. The indexed library was sequenced by the Illumina’s NextSeq 500. The Illumina’s technology is based on reversible terminator nucleotide and the utilization of 2 fluorescently-labelled nucleotides detected by 4 cameras. The G is not fluorescent.

6

n= 1 n=1

Allelic frequency of mutated copies of p.T790M

Figure 5: Recapitulative table of the distribution of p.L858R and p.delEX19 positive and negative cases patients detected by NGS correlated with the number of patients harboring the resistance mutation p.T790M detected with OncoBEAMTM-EGFR (first part of the table) and with NGS-56G (second part of the table) (A). Correlation of p.T790M allelic frequency between OncoBEAMTM-EGFR p.T790M and NGS-56G assay. The correlation is excellent (R squared at 0.94) but 21 cases (11%) were found discordants (B).

100

BEAMing

0 Princeps 0 0 0 0 0 0 0 0 0,1 5 0 3,5 0,8 0 0 0 0 0 1 0 0,3 1 12 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### 0,6 p.T790M ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ### ###

L858R

AF >1% Del 19 AF 0.5-1% AF 0.1-0.5%

EGFR

Other sensitizing Mt Wild-type Missing Technic failure

Princeps

AF >1% T790M AF 0.5-1% AF 0.1-0.5% Sample origin

AF >1% AF 0.5-1% AF 0.1-0.5% Lyon University Hosp. External

Figure 6: Heatmap representing FFPE results (at initial diagnosis), as well as cfDNA results (at disease progression) with both BEAMing and NGS assays for the whole cohort.

0.001 0 cell

5 cells (0.7 cells/mL)

10 cells 25 cells 50 cells HCC827 (1.3 cells/mL) (3.3 cells/mL) (6.7 cells/mL) (p.T790M -)

BEAMing p.T790M Figure 8: p.T790M allelic frequency detected by NGS-56G assay (A) and BEAMing (B) regarding the specified number of mCTC (0 / 5 / 10 / 25 / 50) spiked into total blood and enriched by the ClearCell FX (violet bar) or non-enriched (pink bar). Two negative controls were analyzed : one is total blood sample without spiked cells (« 0 cells ») and one is the HCC827 cell line negative for p.T790M. The H1975 cell line is used as positive control. Dotted lines are positivity threshold (0.5% for NGS-56G, and 0.02% for OncoBEAMTM).

Conclusion & Perspectives - OncoBEAMTM-EGFR assay has a higher sensitivity than NGS-56G to detect EGFR p.T790M mutation (+11% of positive cases); - NGS-56G allows larger coverage on longer gene regions for the screening of other EGFR TKI resistance mechanisms; - Monitoring EGFR p.T790M may be clinically useful; - OncoBEAMTM-EGFR assay is suitable for detecting p.T790M on mCTC’s enriched using the ClearCell Fx assay.