Evaluation of Restriction Enzymes for the Analysis of Circulating Free DNA by Droplet Digital PCR

Transcription

1 Evaluation of Restriction Enzymes for the Analysis of Circulating Free DNA by Droplet Digital PCR Amanda Weaver Cancer Genomics Consortium 2017 Summer Meeting August 7, 2017

2 Acknowledgements Everyone at Biodesix, especially the Lab Ops Team G. Pestano, PhD H. Mellert, PhD T. Jennings T. Pelletier W. Hahn C. Tschida B. Sage A. Audetat Our collaborators at Bio-Rad 2

3 Liquid Biopsy Session Remember Kristin s talk Optimization of blood-based liquid biopsy assays for the rapid identification of 19 actionable RNA fusion variants in NSCLC Kristin Alexander Research Associate 3

4 Biodesix A fully-integrated multi-omic diagnostics company 4

5 Variant GeneStrat DNA Test Process Processing cfdna from plasma of NSCLC patients concludes with detection of EGFR, KRAS, and BRAF variants. Day 1 Day 2 Day 3 Control Whole Blood in Streck BCT from Physician to Biodesix Secure LIMS Accessioning Plasma cfdna Isolation Droplet Digital PCR Data Analysis using Bio- Rad QuantaSoft Test Results to Physician 5

6 Blood Based Circulating Cell-Free DNA Macromolecules in plasma of blood Tumors can release or shed DNA into the blood Highly fragmented DNA bp No invasive tissue biopsy Mellert at all. "Development and Clinical Utility of a Blood- Based Test Service for the Rapid Identification of Actionable Mutations in Non Small Cell Lung Carcinoma." The Journal of Molecular Diagnostics 19.3 (2017):

7 Droplet Digital TM PCR A powerful solution for high-resolution cancer research 1. Make Droplets 2. Cycle Droplets 3. Read Droplets Droplet Generator Bulk PCR Thermal Cycler Droplet Reader 7

8 T790M Mutation EGFR T790M Representative clinical validation samples Mutation Positive Wild Type Positive Mut and WT Positive Negative Variant Negative Variant Positive Wild-Type EGFR Wild-Type EGFR 8

9 Restriction Enzymes Cut DNA at certain sequences Restriction enzymes recommended by Bio-Rad for the ddpcr system Separates tandem repeats for copy number quantitation Cleaves template DNA to allow DNA polymerase to more easily access template 9

10 Restriction Enzymes Cut DNA at certain sequences Restriction enzyme selection and usage is variant specific Ensure enzyme does not cut within the target amplicon sequence for each variant Restriction enzymes used during development of GeneStrat Need to prove removal does not affect patient results EGFR KRAS Variant del19 (ΔE746 -A750) L858R T790M G12C G12D G12V Restriction Enzyme AluI HindIII HindIII MseI MseI MseI BRAF V600E HindIII 10

11 Hypothesis: Removal of restriction enzymes Restriction enzymes necessary for ddpcr with genomic DNA which is very long The GeneStrat test uses highly fragmented cfdna Our hypothesis is that restriction enzymes are not necessary in the GeneStrat test workflow cfdna already small enough (~ bp) so restriction enzymes are not needed to further cleave DNA into smaller pieces 11

12 Hypothesis: Benefits of restriction enzyme removal: Reduces possible source of contamination in PCR Master Mix preparation Exclude a step where human error could occur Make the process more efficient to save time 12

13 Hypothesis: Risks of restriction enzyme removal: Change to current processing can introduce undesired effects on data quality and accuracy of clinical results Example: It is possible that viscosity is imperative for droplet generation and removal of restriction enzymes stored in glycerol will detrimentally impact results 13

14 On-market Improvement Process for a Clinical Test Working within design controls Feasibility Plan and Protocol Drafting and Approvals Execution Report Drafting and Approvals SOP Updates Training Lab Personnel Update Inventory Management 14

15 Study Design Evaluate the GeneStrat test in the presence or absence of restriction enzymes Sample selection key to show equivalency 21 known positive cfdna samples from NSCLC patients run with restriction enzymes 3 samples for each of the 7 DNA variants 1 high, 1 medium, and 1 low mutation copy number Positive control and no template control All run in duplicate wells Replace restriction enzymes with nucleic acid/nuclease free water in PCR master mix 15

16 Metrics for Method Comparison Study Acceptance criteria determined before execution Predefined Acceptance Criteria Positive or negative result concordance All positive control and no template control runs pass defined metrics Absolute copy number for mutation and wild type are similar Clear droplet separation in 2D QuantaSoft Plots to allow for accurate thresholding Average fluorescence intensity in the mutation and wild type channels is the similar or better 16

17 Results Copy number: Mutation Positive Copies Wild type copy numbers also similar average absolute copy difference= 24 copies Average copy number= 418 RE=Restriction Enzyme Mutation Copy Number Difference Between No RE and RE Low Mid High Sample Sample Sample EGFR del EGFR L858R EGFR T790M KRAS G12C KRAS G12D KRAS G12V BRAF V600E Average Copy Number

18 Copy Number High Copy Number Samples Mutation positive copy number Restriction Enzymes No Restriction Enzymes EGFR del19 EGFR L858R EGFR T790M KRAS G12C KRAS G12D KRAS G12V BRAF V600E 18

19 Percent Bias Under 20% for all variants Percent Bias By Variant for WT Copy Numbers Percent Bias By Variant for Mut Copy Numbers RE NoRE % Bias RE NoRE % Bias EGFR del % % EGFR L858R % % EGFR T790M % % KRAS G12C % % KRAS G12D % % KRAS G12V % % BRAF V600E % % 19

20 Fluorescent Intensity Channel 1 Average Fluorescent Intensity Droplet separation Fluorescent Intensity Channel 2 20

21 Average Fluorescent intensity Average Fluorescent Intensity Mutation positive channel N=42 Restriction Enzymes No Restriction Enzymes EGFR del19 EGFR L858R EGFR T790M KRAS G12C KRAS G12D KRAS G12V BRAF 21

22 Data Summary Passed all predefined acceptance criteria Tested 21 positive cfdna samples low, medium, and high copy number sample for each of 7 variants Replaced restriction enzymes with water in the PCR reaction mix Positive or negative result concordance, copy number, and fluorescent intensity All results were 100% concordant There was no significant difference between the resulting copy number Fluorescent intensity slightly better without restriction enzymes in all but one case 22

23 Conclusion Restriction enzymes not necessary for GeneStrat Test Our hypothesis was that restriction enzymes are not necessary in the GeneStrat test workflow Our data supported our hypothesis and showed that removal of restriction enzymes did not affect patient results Saves time and simplifies ddpcr workflow Removes a step where human error could occur Diagnostic labs that utilize cfdna may benefit from testing their workflow to remove restriction enzymes 23

24 Thank YOU for listening to my presentation! Questions? Amanda Weaver Research Associate, Lab Ops P: Biodesix 2970 Wilderness Place, Suite 100 Boulder, CO Biodesix, Inc. All rights reserved 24