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
620
616
608
607
604
916
912
910
905
904
903
899
898
897
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
761
759
758
756
755
753
752
750
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.
