What is the impact of assay failure in your laboratory and how do you monitor for it?
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
The clinical application development and validation of cell free dna assays -platforms - horizon diagnostics
1. The Clinical Application, Development and Validation of
Cell-free DNA Assays/Platforms
Dr Jonathan Frampton
Product Manager, Diagnostics
Twitter: @HorizonDX_news #cfDNA
2. 2 #cfDNA
Presenters
Dr. Jonathan Frampton, PhD
Product Manager, Diagnostics
In his role, Jonathan works closely with a broad range of European, North American
and EMEA oncology-focussed Quality Assurance Schemes with the goal of driving
the standardization and normalization of molecular assays across the
globe.Jonathan holds a PhD from University of Sussex in Genomic DNA Damage and
Stability and has extensive product development experience through previous roles
including Cambridge-based antibody company Abcam.
3. #cfDNA
What is the impact of assay failure
in your laboratory and how do you
monitor for it?
3
4. Cancer Patient DNA Extraction Diagnosis
4 #cfDNA
Application of Liquid Biopsies
Blood/Plasma
Sample
Driving better treatment
for cancer patients
5. 5 #cfDNA
Sample Handling and Processing
Blood/Plasma
Sample
DNA
Quantification
DNA Extraction Storage
DNA Quantity & Quality
6. 6 #cfDNA
Analytical Processing and Reporting
Sample
preparation
DNA Sample Analysis
Actionable
Decision
Quality of Diagnostic Result
7. #cfDNA
What is the impact of assay failure
in your laboratory and how do you
monitor for it?
7
8. Challenges Faced by cfDNA Assay Implementation or Development
Sample collection
Handling
DNA quality
DNA quantity
Cancer Patient DNA Extraction Diagnosis
8 #cfDNA
Blood/Plasma
Sample
DNA Fragmentation
Contamination
DNA Recovery
DNA quantification
DNA/Biomarker loss
DNA Fragmentation
False negatives
False positives
Specificity
Sensitivity
Variable
Impact
9. 9 #cfDNA
Presenters
Dr. Hadas Amit
Senior Scientist, Diagnostics
Hadas is a senior scientist with a strong interest in the future of personalised
medicine with focus on advances in cell free tumor DNA (cfDNA). Hadas is leading
the research and development of cfDNA Reference Standards at Horizon
Diagnostics.
10. #cfDNA
How are Reference Standards Manufactured and Validated?
“Wild type cell line”
Single Cell
Dilution
Clonal mutant cell line
Pre-Engineering
Cell Line Validation
SNP 6.0
Sanger Sequencing
Digital PCR
RT-PCR
Post-Engineering
Cell Line Validation
Gene Editing
Platform
10
11. #cfDNA
Analyzed Allelic Frequency Down to 0.05%
Mutant Wild type
Genomic DNA
Stoichiometric Dilutions
Dilutions are accurate down to 0.05%
11
12. Improving Cancer Treatment Through Translational Research & Clinical Intervention
#cfDNA
We are now working with over 700 hospitals, kit developers,
proficiency schemes and pathology labs worldwide…
12
13. Cell-free DNA Reference Standard Case Studies
13 #cfDNA
Case Study 1 – EGFR
Multiplex standard down to
0.05%
Validated by ddPCR
Case Study 2 – Multiplex
cancer panel
Multiplex standard containing
>40 validated mutations
Highlights sensitivity and
specificity of NGS platforms
14. Case Study 1: Cell-free DNA EGFR Multiplex Reference Standard
L861Q Wild type
14 #cfDNA
T790M
G719S
L858R ΔE746-A750
15. DNA Sample Analysis
15 #cfDNA
Analytical Processing and Reporting
How good is digital PCR for detecting
mutations at 0.05%?
Actionable
Decision
Sample
preparation
16. #cfDNA
Case Study 1: Cell-free DNA 0.05% EGFR Multiplex Reference Standard
EGFR Multiplex Sample #6 Wild Type
Effective mutant
0.05% 0%
allele frequency
Expected Observed Expected Observed
Total
Copies
EGFR
Mutant
ΔE746-
A750
25 23 0 2.0
G719S 25 20 0 12.0
L858R 25 31 0 1.0
L861Q 25 32 0 1.0
T790M 25 44 0 16.0
Total Copies EGFR 50000 50380 50000 49700
Reference Standards demonstrate the sensitivity and accuracy of a “cfDNA workflow”
16
18. Case Study 2: Tru-Q HDx™ Reference Standards for Next Generation Sequencing
Quantification by Droplet Digital PCR
Quantification by Droplet Digital PCR
Quantification by Droplet Digital PCR
A Blend
40 Mutations
@ 1.3%
18 #cfDNA
B Blend 1
20 Mutations
at 2.5%
B Blend 2
20 Mutations
at 2.5%
C Blend 1
10 mutations
at 5%
C Blend 2
10 mutations
at 5%
C Blend 3
10 mutations
at 5%
C Blend 4
10 mutations
at 5%
EGFR
mutants
K-Ras
mutants
B-Raf
mutants
N-Ras
mutants
PIKCA
mutants
14 Additonal
Biomarkers
1.3%
20 copies per μl
Quantification by Droplet Digital PCR
Dilution
Series with
wild type
1 copy per μl
19. DNA Sample Analysis
19 #cfDNA
Analytical Processing and Reporting
How good is NGS for detecting
mutations at 1%?
Actionable
Decision
Sample
preparation
20. Case Study 2: Analysing PGM Workflow down to 1%
5% blend 2.5% blend 1.3% blend
20 #cfDNA
Source:
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
Platform: N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
Gene Mutation
BRAF V600M 4.0 4.4 3.5
EGFR T790M 4.2 3.9 4.3
EGFR L858R 4.2 4.2 3.5
EGFR L861Q 4.2 4.1 3.6
KIT D816V 5.0 5.4 6.4
KRAS G12A 5.0 5.7 4.9
KRAS G12R 5.0 5.2 4.6
NRAS Q61K 5.0 4.9 3.3
Specific and Sensitive down
to 5% allelic frequency
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
2.0 2.2 2.1
2.1 2.0 2.1
2.1 2.0 2.3
2.1 2.1 1.8
2.5 2.6 3.2
2.5 3.0 2.5
2.5 2.9 2.6
2.5 2.5 2.5
Horizon
Diagnostics
Predicted %
Horizon
Diagnostics
Observed %
Partner
N/A
QX100™
Droplet
Digital™ PCR
System
Ion Torrent
1.0 1.0 1.9
1.0 1.1 missing
1.0 1.1 missing
1.0 1.0 missing
1.3 1.3 1.5
1.3 1.4 missing
1.3 1.3 missing
1.3 1.2 missing
Specific and Sensitive down
to 2.5% allelic frequency
Not sensitive to detect down
to 1% for all variants
21. 21 #cfDNA
What’s Coming Next?
Blood/plasma
sample
HDx™ Reference Urine sample
Standard
HDx™ Reference
Standard
HDx™ Reference Standards will confirm your specificity and sensitivity
22. What are the Outstanding Questions?
What is the impact of assay failure
in your laboratory and how do you
monitor for it?
22 #cfDNA
What sensitivity
do you want to
achieve?
What mutations
are you looking
at?
How can we
support your
workflow
23. Last Slide #cfDNA
What to do Now?
Molecular Lab Next Generation
Sequencing Lab
Kit, Assay, Platform
Developer?
HDx™ Reference Standards
Every Assay, Every Run, Every Confidence
Contact me at j.frampton@horizondiscovery.com
24. Your Horizon Contact:
Jonathan Frampton
Product Manager
j.Frampton@horizondiscovery.com
+44(0) 1223 655 580
Horizon Discovery Group plc, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 655 581 Email: info@horizondx.com Web: www.horizondx.com
Notas del editor
Before I begin, I would like to ask you one question – what is the impact of assay failure in your laboratory and how do you monitor for it?
And that is what we are going touch on and expand together in this webinar:
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
How do we manufacture our reference standards?
At Horizon, we have developed a library of over 550 genetically defined cells harbouring mutation found in cancer patients as well as their control cell lines from the same genetic background.
As part of the cell line establishment process it is critical that we single-cell dilute the original “wild type” cell line to ensure clonality. These cells are extensively validated before undergoing cell line engineering to produce the mutant cell line of interest. For pre- and post-validation we run SNP 6.0 analysis to confirm cell line identity, sanger sequencing to confirm engineering as been accurately targeted to the endogenous gene, digital PCR to confirm copy number and allelic frequency and RT-PCR to analyse gene expression.
Using our genetically defined cells lines we manufacture a range of reference standards – DNA, RNA, FFPE blocks and sections, and cells slides for IHC and FISH
DNA Reference Standards
These are ideal for the routine validation of your molecular assay workflow and can be used initially to determine the limit of detection of your platform. We can provide pre-diluted standards ready to go; for example a 5% EGFR T790M or alternatively the mutant and matched wild type separately and allow you to generate the specific dilution curve most useful for you. Dilution guidelines can be found on our website and we are always happy to discuss specific projects as needed. We typically provide our DNA standards at 100ng @ 5ng/ul.
All of our DNA products undergo extensive validation and we use digital PCR to confirm the allelic frequency of any of our reference standards to an accuracy of 0.01%. If needed for custom work we can provide standards with allelic frequencies as low as 0.05%.
Our controls have already demonstrated to have a profound effect on the labs that have adopted them. We work together with Proficiency Testing Schemes, technology developers and scientists around the world interested in for example comparing assay performance across platforms, validation of lod, maintaining high standard quality control.
Moving on to same examples, I will talk you through two case studies. And in between we will have a pole so make this webinar a bit more interactive and fun.
In the first case study we produced an EGFR validated multiplex standard with mutations at low Afs down to 0.05% and
the second study where we produced a multiplex standard containing over 40 validated mutations which can help to understand the sensitivity and specificity of NGS platforms.
So, for case study number 1 we have generated a five mutant multiplex containing the following EGFR mutations as you can see on the top right and have generated the standards in 6 formats highlighted below ranging from 0.05% mutant allele burden to 2% allele burden with the associated copies of mutant in total EGFR copies.
Prior to blending, our singleplex gDNA samples were analysed by digital PCR to determine total and mutant gene copy number and based on the copy number results we determined the gDNA ratios for blending.
Once the ratio of mutants was confirmed at high frequency, the multiplex was then diluted in wild type to generate lower frequency mixes.
What you’re looking at here is the data for the 0.5% and 0.05% blends. This data was produced using digital PCR. And the slide shows the copies data of each mutant in a total of 50,000 total copies of EGFR.
You can see a very good correlation between the expected and observed values for most mutations for example if you take a look at the EGFR deletion and L858R mutations where you expect to get 250 copies and we observed 240 and 260. This highlights our capability to develop low copy number reference standards.
Within the wild type you expect to see 0 copies of each mutant. However, what you will notice is that for some assays in particular the T790M assay you see that there were some false positive mutant copies. This supports that some assays are more challenging that others and this is consistent with the literature and others are struggling with this region probably due to its complexity.
Finally, In the cell-free DNA space it is very important to have reference standards down to 0.05% because this is in the region of frequencies of physiological relevance.
So for case study number 2 we are focussing on an NGS reference standard where we have taken many of our engineered cell lines covering 40 mutations and many different other genes and generated four “C Blend” Multiplex’s each containing 10 mutations at 5% - PURPLE.
Two of these have been combined to generate two unique “B Blends” each containing 20 mutations at 2.5% allelic frequency – BLUE - and these have been combined to generate the “Megaplex A Blend” containing 40 mutations at 1.3% - GREEN.
This blend can be potentially further diluted and go down to one copy per uL.
We call this blend the Tru-Q (True-Quality) NGS Reference Standards recapitulates the complexity of tumour composition.
In cancer patients, cell-free DNA carries tumour-related genetic alterations that are relevant to cancer development, disease progression and response to therapy.
Cell-free DNA detection allows:
Early detection
Frequent sampling
Monitoring of disease progression
Measure response to therapy
Detection of resistance mutation
Non-invasive diagnostic tool development
Looking at the data, as this is a very large datasets I have chosen to highlight a few mutations across 5 genes and show you some of the ion torrent results provided by one of our partners.
For the 5% blend you can see a good correlation between our data and our partner’s ion torrent data, this is the same for the 2.5% panel. This highlights that the detection method is specific and sensitive down to 2.5% allelic frequency. However as you move lower to the 1.3% blend this dataset highlights that the Ion torrent platform is not sensitive enough to detect down to 1% for all variants, which is actually consistent with the platform specifications.
For anyone setting up their platform and optimising assays these are valuable reference standard to demonstrate how good you are at detecting and calling the different mutations correctly.
Take home message from Hadas:
Critical that cell-free DNA assays are routinely validated to ensure accurate, precise and robust reporting
Highly validated reference standards are required to support assay monitoring and development
We will be launching a new type of reference standards soon.
Finally in conclusion, I would like to say this…
We’ve seen here that cell free DNA reference standards can be precisely defined and manufactured, allowing for accurate analysis of cfDNA platforms and assays. The next step for all of you now is to ask how low do you want to go to with your assay, which mutations do you want to cover and when will you need reference materials to validate your workflow?
What questions do you have?
Ladies and gentleman I would just like to emphasize once again why this is so important – cell free DNA reference standards can be precisely defined and manufactured, allowing for accurate analysis of cfDNA platforms and assays. The next step for all of you now is to ask how low do you want to go to with your assay, which mutations do you want to cover and when will you need reference materials to validate your workflow? Please come and find me during the meeting and I look forward to your feedback.