Going further with 3-color Crystal Digital PCR: characterizing CNV and indels. 4th qpcr & Digital PCR Congress 09/13/2018

Transcription

1 Going further with 3-color Crystal Digital PCR: characterizing CNV and indels 4th qpcr & Digital PCR Congress 09/13/2018

2 Overview of presentation I. Introduction to digital PCR and Naica System II. Reliable HER2 CNV assessment for breast cancer III. Drop-off assay for Del19 detection IV. Applications for drop-off assays Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 2

3 Overview of presentation I. Introduction to digital PCR and Naica System II. Reliable HER2 CNV assessment for breast cancer III. Drop-off assay for Del19 detection IV. Applications for drop-off assays Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 3

4 Introducing the Naica System Sapphire Chip (consumable) Naica Geode Naica Prism3 Crystal Miner (software) An easy-to-use and integrated solution for digital PCR Fast time-to-result (2h30) Reliable multiplex assays with 3-color detection We are extremely satisfied with the Naica System, which fully answers our needs in terms of precision and reproducibility for liquid biopsy testing. Dr. Ludovic LACROIX Dir. Translational Research Institut Gustave Roussy Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 4

5 At The Heart of Our Innovation : The Sapphire Chip A unique and patented partitioning technology: droplet crystals. Sapphire Chip : pre-filled with oil Input volume 25 µl Droplets per sample Droplet volume Number of samples 0.59 nl 4 / chip Droplet crystal: Self-assembled 2D array of droplets Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 5

6 Step 1 Prepare the Sapphire Chip 5 min Unpack Sapphire Chip Pipette 25 µl of PCR mix Seal inlet port with cap Compatible mixes and chemistries : Use with Quanta BioSciences PCR and RT-PCR Mix with no ROX With Taqman Probes, add Fluorescein as reference dye With EvaGreen chemistry, add Alexa Fluor 647 as reference dye Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 6

7 Step 2 Partition & Amplify 2h10 Step 2.1 Partition Transfer chips to the Geode 1-3 chips and 1-12 samples / run ~30,000 partitions / sample Contactless fluid injection The droplet crystals spontaneously forms inside the chip Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 7

8 Step 2 Partition & Amplify 2h10 Step 2.2 Amplify Standard cycling conditions: 95 C 60 C 25 C Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 8

9 Step 3 Detect 10 min (50 s / sample) Transfer chips to the Prism3 1-3 chips and 1-12 samples / run 3 colors fluorescence imaging Blue Ex: nm Em: nm Green Ex: nm Em: nm Red Ex: nm Em: nm FAM, EvaGreen VIC, HEX, Cy3 Cy5, Cy5,5 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 9

10 Step 4 Analyze your data with The Crystal Miner Software User Friendly software with intuitive visuals Simple image analysis and data exploration A trustworthy quality control Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 10

11 Overview of presentation I. Introduction to digital PCR and Naica System II. Reliable HER2 CNV assessment for breast cancer III. Drop-off assay for Del19 detection IV. Applications for drop-off assays Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 11

12 Detection of HER2 copy number amplification with Crystal Digital PCR Motivation: Investigating HER2 amplification status is crucial in breast cancer to determine the indication of anti-her2 targeted therapy. Problem: Assessing HER2 amplification from DNA is challenging when tumor DNA is diluted in DNA from healthy cells: Tumor sample heterogeneity Liquid biopsy samples Chr2 Assay : REF: Chr2 TSN Target 1 : Chr17 HER2 Target 2 : Chr17 MRM1 Result classification: (FAM) (Cy3) (Cy5) Chr17 HER2/TSN <= 1 MRM1/TSN <=1 HER2/TSN > 1 MRM1/TSN <=1 HER2/TSN >1 MRM1/TSN >1 HER 2 Negative HER2 positive Polysomy 17 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 12

13 Reliable CNV measurement in low abundance samples Assay characterisation Genomic DNA extracted from SKBR3 cell line (HER2/TSN ratio of 10:1) was spiked into normal genomic DNA with mutant fractions from 1% to 12%, modeling a theoretical range of HER2/TSN ratios from 1 to SKBR3 CNV SKBR3 % Expected CNV 10 : 1 100% 10 : 1 10 : 1 10% 1.9 : 1 10 : 1 1% 1.09 : 1 10 : 1 0.1% : 1 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 13

14 Assessment of HER2 status from tumor samples with Crystal Digital PCR Result classification: HER2/TSN <= 1 MRM1/TSN <=1 HER2/TSN > 1 MRM1/TSN <=1 HER2/TSN >1 MRM1/TSN >1 HER 2 Negative HER2 positive Polysomy 17 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 14

15 Assessment of HER2 status from plasma samples with Crystal Digital PCR Tumor Status Plasma Status Patient IHC FISH Status No Amplification No Amplification HER2 Amplification No Amplification No Amplification HER2 Amplification Results Summary Tumor Plasma HER2 Amplification 2 2 Chr17 polysomy 0 0 No Amplification 4 4 Ratio MRM1/TSN 2 Chr17 polysomy 1,8 1,6 1,4 1, ,8 5 0,6 No Amplification HER2 Amplification 0, Ratio HER2/TSN Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 15

16 What ratios do I need to detect in cfdna? % tumor DNA CNV Table 1. Ratios to be measured to discriminate copy number variation of a specific gene, depending on the fraction of tumor DNA present in the sample. Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 16

17 Power Calculations for CNV The two following parameters can be adjusted to quantify ratios close to 1: Number of replicates (= Sapphire chip chambers = droplets) Amount of DNA assayed per reaction Figure 1. Measurable ratio vs number of replicates (ie number of chambers) needed to measure said ratio. The different lines represent different amount of total DNA input into the reaction It is also possible to get more reliable CNV determination by using more than one reference gene, taking advantage of the multiplexing capabilities of the Naica system. Method from Whale AS et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 17

18 Overview of presentation I. Introduction to digital PCR and Naica System II. Reliable HER2 CNV assessment for breast cancer III. Drop-off assay for Del19 detection IV. Applications for drop-off assays Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 18

19 Drop-off assay for EGFR Del19 detection Motivation: Deletions in EGFR exon 19 are activating genomic alterations, which predict response to TKI (tyrosine kinase inhibitors) in NSCLC patients Problem: There are multiple types of deletions reported for EGFR exon 19. We want to detect a maximum of deletions in a simple and reliable assay, that can be used for monitoring ctdna in patients plasma samples Assay : Chr7: EGFR Exon19Ref (FAM) Chr7 EGFR Del19WT (Atto 700) Chr7 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 19

20 Detecting EGFR exon 19 deletions Principle WT probe Ref probe Q Q 5 3 Known deletion hotspot Wild-type DNA WT probe Cy5 Both probes hybridize to the amplicon, yielding double positive droplets Ref probe FAM Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 20

21 Detecting EGFR exon 19 deletions - Principle WT probe Q Ref probe Q 5 3 Deleted exon 19 WT probe Cy5 DNA bearing EGFR exon 19 deletions Only the FAM-labelled probe hybridizes since the Cy5- labelled probe cannot hybridize due the absence of its target Ref probe FAM Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 21

22 Detecting EGFR exon 19 deletions Patient Samples Patient samples contain WT and tumoral DNA bearing exon19 deletions WT probe Cy5 Wild- type DNA DNA bearing EGFR exon19 deletions Ref probe FAM Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 22

23 Detecting EGFR exon 19 deletions Co-encapsulation The target can be coencapsulated with the background population in the same droplet partition WT probe Cy5 Wild- type DNA or co-encapsulation DNA bearing EGFR exon19 deletions Ref probe FAM Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 23

24 Detecting EGFR exon 19 deletions Co-encapsulation The target can be coencapsulated with the background population in the same droplet partition Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 24

25 Monitoring EGFR exon 19 deletions and EGFR T790M mutation in a single assay WT EGFR Del19 Motivation: Monitoring activating mutations (EGFR Del19) and resistance mutations (EGFR T790M) in a single multiplexed assay EGFR T790M Assay : Chr7: EGFR Exon19Ref (FAM) Chr7 EGFR Del19WT (Atto 700) Chr 7 EGFR T790M (Cy3) Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 25

26 Monitoring EGFR exon 19 deletions and EGFR T790M mutation in a single assay Jovelet et al, Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer, PLoS One 2017 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 26

27 Overview of presentation I. Introduction to digital PCR and Naica System II. Reliable HER2 CNV assessment for breast cancer III. Drop-off assay for Del19 detection IV. Applications for drop-off assays Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 27

28 Example : Drop off assay for mutations WT DNA NRAS WT 5 NRAS WT NRAS ref 3 NRAS G13R NRAS G12D NRAS G12-G13Mutant NRAS WT 5 X G12-G13 Mutant NRAS ref 3 NRAS G12S NRAS G12V Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 28

29 Drop off assays Deletions Insertions Mutations Chromosomal rearrangements Fragment size discrimination Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 29

30 Going further with Crystal Digital PCR View PCR assays differently A new world to explore! Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 30

31 Thank you! Stilla Technologies Rémi DANGLA Magali DRONIOU Jordan MADIC Barbara ANDRE Imane DEHRI Mylène MENANTEAU Chloé DUJARDIN Romain GIRARD Alexandra MARTIN Laura CAVE And everybody else at Stilla!! Institut Gustave Roussy Ludovic LACROIX Cécile JOVELET Etienne ROULEAU Aurélie HONORE Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 31

32 Application Notes We are at booth #1 Stilla Technologies 1 Mail du Professeur Georges Mathé, Villejuif 32

33 Questions?