Highly Sensitive, Multiplexed ctdna Mutation Detection on MassARRAY :

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1 Highly Sensitive, Multiplexed ctdna Mutation Detection on MassARRAY : Lung & Colon Cancer Panel Pre-Launch & Data Sharing 栾晓辉, Ph.D. Senior Scientist, Application Development Agena Bioscience, Greater China alex.luan@agenabio.com 2016 Agena Bioscience, Inc. All Rights Reserved. For Research Use Only. Not for use in diagnostic procedures. 1

2 An Ideal Platform for Clinical Liquid Biopsy Sensitivity As much information as possible from limited & compromised samples Cost (test cost, labor cost, validation cost) Time to report Actionable, guideline compliance 2

3 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% As much information as possible from limited & compromised samples Cost (test cost, labor cost, validation cost) Time to report Actionable, guideline compliance 3

4 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% 20ng ctdna, one reaction covers ~100 mutations from multiple genes Cost (test cost, labor cost, validation cost) Time to report Actionable, guideline compliance 4

5 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% 20ng ctdna, one reaction covers ~100 mutations from multiple genes Dollars/mutation, no bioinformatics expert needed, in-house validated Time to report Actionable, guideline compliance 5

6 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% 20ng ctdna, one reaction covers ~100 mutations from multiple genes Dollars/mutation, no bioinformatics expert needed, in-house validated 2 days from ctdna to result. Or 3 days with sample QC step Actionable, guideline compliance 6

7 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% 20ng ctdna, one reaction covers ~100 mutations from multiple genes Dollars/mutation, no bioinformatics expert needed, in-house validated 2 days from ctdna to result. Or 3 days with sample QC step All mutations selected according to ASCO/AMP latest guideline 7

8 An Ideal Platform for Clinical Liquid Biopsy LOD ~0.1% 20ng ctdna, one reaction covers ~100 mutations from multiple genes Dollars/mutation, no bioinformatics expert needed, in-house validated 2 days from ctdna to result. Or 3 days with sample QC step All mutations selected according to ASCO/AMP latest guideline 8

9 Sensitive and Multiplexed Panels for Lung & Colon Cancer Mutation Screening Available from April Gene BRAF EGFR Coverage Codon 469 (exon 11) and codons 594, 600 (exon 15) Exon 19 indels, exon 20 insertions, and substitutions across exons 18, 19, 20, and 21 Single multiplexed PCR reaction 1% mutation frequency detection iplex HS Lung Panel # of Mutations UltraSEEK Lung Panel ERBB2 Exon 20 insertions 2 2 KRAS Codons 12, 13 of exon 2; codon 61 of exon 3 PIK3CA Codons 542, 545 (exon 9), codon 1047 of (exon 20) Single multiplexed PCR reaction iplex HS Panel enables detection as low as 1% allele frequency from solid tumor samples UltraSEEK Panel enables detection as low as <0.1% allele frequency from blood 9

10 Sensitive and Multiplexed Panels for Lung & Colon Cancer Mutation Screening Available from April Gene Coverage BRAF Codon 469 (exon 11) and codons 594, 600 (exon 15) iplex HS Colon Panel # of Mutations UltraSEEK Colon Panel 3 5 EGFR Extracellular domain mutations across exon KRAS NRAS PIK3CA Codons 12, 13 (exon 2), codons 59, 61 (exon 3) and codons 117, 146 (exon 4) Codons 12, 13 (exon 2), codons 59, 61 (exon 3), and codons 117, 146 (exon 4) Codons 542, 545 (exon 9), codon 1047 of (exon 20) Single multiplexed PCR reaction iplex(r) HS Panel enables detection as low as 1% allele frequency from solid tumor samples UltraSEEK Panel enables detection as low as <0.1% allele frequency from blood

11 Detecting Somatic Mutations for More than One Decade Here we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1000 human tumor samples. 11

12 Traditional Reagents Struggle around 5-10% Spectra for a PIK3CA somatic mutation MassARRAY used to screen primary and metastatic colorectal cancer samples Created a subpanel of assays specific to colorectal cancer mutations Achieved 5-10% sensitivity (1% with latest iplex HS) of mutant detection within normal background in FFPE tissue Data example shown: 4.8% PIK3CA mutation A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes Fumagalli et al., 2010, BMC Cancer; 10(101) 12

13 Back to the Principles PCR-based Direct Mass Analysis 13

14 iplex HS Improved Sensitivity by Limiting WT Terminator Supply EGFR_S492R 5% EGFR_S492R 2.5% EGFR_S492R 1.25% EGFR_S492R 0% 14

15 How Does UltraSeek Work? Multiplex PCR Mutant Specific Extension Capture of Mutant Allele Analysis on the MassARRAY A T Mutant Template C G Wild Type Template C G C G C G C G A CT G C G C G C G 15

16 How Does UltraSeek Work? Multiplex PCR Mutant Specific Extension Capture of Mutant Allele Analysis on the MassARRAY U A B C CC C C CCC 16

17 How Does UltraSeek Work? Multiplex PCR Mutant Specific Extension Capture of Mutant Allele Analysis on the MassARRAY Mag Bead U B 17

18 How Does UltraSeek Work? Multiplex PCR Mutant Specific Extension Capture of Mutant Allele Analysis on the MassARRAY 18

19 Relatively Quantitative 1% 0.1% Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry. M. Mosko, etc. JMD, Volume 18, Issue 1, January

20 AZ KRAS Ring Study Data published as 2016 ESMO poster a r r r r r Analysis successful, mutation detected Analysis successful, no mutation detected Analysis unsuccessful, no mutation detected Analysis successful, wild type sample. Kit does not assay codon, no mutation detected Incorrect mutation detected Sherwood et al., KRAS mutation detection technologies. Modern Pathology. Under Review. 20

21 Feasibility Study with China Dx Company 44 samples, first round by NGS, left over for MassARRAY UltraSEEK Colon Panel used Exome QC panel was performed with 1µl of each template to determine the copy number and sample input volume Mutations reported by UltraSEEK cross-checked with NGS reports and raw data 21

22 Mutation Result Example 22

23 Result Summary 23

24 Brief Summary 5 samples excluded due to insufficient quality/quantity of the archived aliquot 27/39 samples had concordant results between UltraSEEK and NGS 14 new mutations detected in 12/39 samples (31%) 9/14 new mutations confirmed by NGS raw data inspection (not called by data analysis pipeline)

25 Fulfilling NGS Workflow 25

26 Voice from Cancer Program Leader 26

27 Sample QC/ID before NGS 27

28 We Have More to Tell 28

29 Thank you for your attention! 29