Mutations detection in plasma-like and plasma samples
We analyzed extracted cfDNA from both plasma-like and plasma samples with each of the two 6-color Crystal Digital
PCR
™
detection assays, the Rectal Cancer 6-color assay and the EGFR 6-color Crystal Digital PCR™ kit. Quantification
of the EC allowed to calculate the extraction yield for each sample. The mean yield obtained for eight samples (3
plasma-like and 5 plasma) was 74,5%. All mutations known to be present in the samples were detected (Table 1).
Example 1D-dot plots generated by the naica
®
system Crystal Miner analysis software resulting from the detection by
the Rectal Cancer 6-color assay (Figure 8), and 2D-dot plots resulting from the detection by the EGFR 6-color Crystal
Digital PCR™ kit (Figure 9) show clear separability between the positive and negative clusters, with minimum droplet
rain.
Comparison of automated Maxwell RSC LV
extraction to a manual reference method
We compared the concentrations of WT and mutant DNA
measured using the EGFR 6-color Crystal Digital PCR™ kit
on cfDNA extracted from plasma-like samples with the
Maxwell® RSC LV kit versus the manual QIAamp
circulating nucleic acid kit. Similar WT and mutant
concentrations were obtained from samples extracted by
the two methods (Figure 6 and Figure 7).
Introduction
Liquid biopsies, such as blood samples, can harbor a wealth of genetic information from both healthy and unhealthy cells (Figure 1) to inform disease diagnosis
and treatment. cfDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high
background of wild-type sequences. Rigorously qualified pre-analytical protocols are vital to ensure the performance of downstream liquid biopsy workflows to
ensure high-quality sample to results (Figure 2). This proof-of-concept study evaluates the performance of the cfDNA extraction protocols of the Promega Maxwell
®
®
system of PIK3CA and EGF R mutations and external extraction controls.
Abstracts
Circulating cell free (cf)DNAs extracted from liquid biopsy samples have become established sample types for characterizing oncology targets. Currently, there are several extraction protocols and genomic platforms for researchers to select when interrogating genetic information. The focus of this work was to identify a flexible method for sample extraction that
seamlessly integrates into a straightforward and sensitive genetic analysis workflow from plasma samples. Here, we present a workflow combining the Maxwell
®
CSC 48 instrument for automated cfDNA extraction and Crystal Digital PCR™ on the naica
®
system for ultrasensitive high-plex detection from liquid biopsy samples of EGFR mutations in non-small cell
lung cancer (NSCLC) and PIK3CA mutations in breast and rectal cancers. By bridging sample extraction with the Maxwell
®
system and high-plex digital PCR with the 6-color naica
®
system, we enabled the detection with high sensitivity and precision of 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, representing more than 90% of
EGFR mutations described in NSCLC. Furthermore, following the same extraction and sample testing workflow, we enabled the detection of a set of PIK3 CA mutations from cfDNA samples. This proof-of-concept workflow creates the foundation for the further development of streamlined sample-to-answer protocols that will better assist cancer researchers
across the biomarker testing landscape.
Maxwell® CSC 48 Instrument
.
Cecile JOVELET, Stéphanie ROY
1
, Myrtille REMY
1
, Mylene MENANTEAU
1
, Doug WHITE
2
, Douglas HOREJSH
2
, Allison MALLORY
1
1
Stilla Technologies, Villejuif France and Beverly, MA USA;
2
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI USA
© Copyright Stilla Technologies 2022
THE MULTIPLEX DIGITAL PCR COMPANY
#527
A streamlined workflow for liquid biopsy sample extraction and highplex digital PCR analysis
®
CSC system and 6-color Crystal Digital PCR™
Figure 2. Complete blood sample to results analysis workflow.
A) This workflow combines cfDNA automated extraction using the Maxwell
®
RSC system and Crystal Digital PCR™ on
the naica
®
system for ultrasensitive high-plex detection. B) cfDNA analysis by the 6-color Crystal Digital PCR™ allows
timely and sensitive monitoring of cancer disease (treatment response, MRD, cancer relapse (resistance mutations).
A
B
Crystal Digital PCR™ on the naica
®
6-color system
The 6-color Crystal Digital PCR™ workflow (Figure 4) enables the highest multiplexing capacity in a single reaction saving both time and precious sample and
providing ultra sensitivity and increased low-level detection of multiple reactions in parallel. By partitioning sample reactions into a large 2D array of droplets,
Crystal Digital PCR™ technology can be used for absolute nucleic acid quantification in a wide range of assays including, but not limited to, oncology (copy number
variation, mutation detection, rare event detection, therapeutic monitoring). Crystal Miner software measures the concentrations of targeted nucleic acids,
providing automatic identification of positive and negative droplet crystals for all fluorescence channels. Through intuitive visuals for image analysis, users can
explore their data in multiplex way, including the direct inspection of droplet crystals for quality control.
Figure 4. The naica
®
system: Get absolute
quantification of multiple genetic targets in a single
run. The 6-color naica® system is an easy-to-use digital
PCR platform that harnesses cutting-edge microfluidic
technology to integrate the digital PCR workflow onto a
single consumable chip. The Crystal Digital PCR™
technology partitions samples into a large array of
thousands of individual droplet crystals – each its own
reaction compartment – before amplifying nucleic acid
molecules in each droplet crystal. These reactions are
tagged with fluorophores to be read using up to six
different fluorescence light channels, maximizing
multiplexing capacity. The naica® system makes for a fast
and simple workflow that can be completed with less than
10 minutes of hands-on time.
Material and Methods
Plasma-like samples (SensID reference material ref SID-000002, SID-000016, SID-000089) and human K2EDTA plasma samples collected from healthy donors were extracted with the
Promega Maxwell® RSC LV cfDNA plasma extraction kit (Promega, ref AS1840), according to supplier recommendations. Before extraction, all samples were spiked with a known quantity
of an exogenous Extraction Control DNA (EC), and a portion of the human plasma samples were spiked with known amounts of synthetic mutant DNA. For comparison, plasmas were also
extracted with QIAamp circulating nucleic acid kit (QIAGEN, ref 55114) according to supplier recommendations.
®
6-color Crystal Digital PCR™ system with two independent 6-color cancer detection assays:
- A custom Rectal Cancer 6-color assay detecting PIK3CA wild-type (WT), four PIK3CA mutations (p.E542K, p. E545K, p.H1047L, p. H1047R) and the EC
- The EGFR 6-color Crystal Digital PCR™ kit (Stilla Technologies
®
, Ref R30006) detecting EGFR WT and 32 EGF R mutations (for more information, see poster 75) .
Results
The Maxwell
®
RSC LV kit and the naica
®
6-color
system are compatible workflows
We first evaluated the compatibility of the samples extracted
with the Maxwell
®
RSC LV ccfDNA Plasma extraction kit by
determining the stability of the droplet crystal using Sapphire
chips. Our Crystal Miner software allows a transparent check
of the droplet crystal to visualize the individual droplets. All
samples extracted with the Promega Maxwell
®
CSC system
were highly compatible with the naica
®
Crystal Digital PCR™
droplet chemistry (Figure 5).
.
Figure 6. Total DNA concentrations (copies/µL) of ccfDNA samples
extracted from 2mL of plasma-like samples by Maxwell® RSC LV kit (Left) and
QIAamp circulating nucleic Acid kit (Right)
Figure 7. Mutant DNA concentrations (copies/µL) of ccfDNA samples
extracted from 2mL of plasma-like samples by the Maxwell® RSC LV kit (Blue)
and the QIAamp circulating nucleic Acid kit (Grey)
0
50
100
150
200
250
300
350
400
450
500
Maxwell RSC LV QIAamp Circulating NA
Total DNA concentration
(copies/µl)
0
2
4
6
8
10
12
14
16
DEL19 L858R L861 T790
Mutant DNA concentration
(copies/µl)
Maxwell RSC
LV
QIAamp
Circulating NA
Extraction control DNA Measured concentrations (copies /µL)
Rectal Cancer 6-color assay Rectal Cancer 6-color assay EGFR 6-color Crystal Digital PCR™
Mutants
Expected
Concentrations
(copies/µL)
Measured
Concentrations
(copies/µL)
Extraction
yield (%)
WT DNA
PIK3CA
E545K
PIK3CA
H1047R
PIK3CA
H1047L
WT DNA
Deletion
exon 19
L858R
L861Q,
G719S
T790M
Plasma
-like WT 60,00 47,25 78,75 735,60 0 0 0 0 0 0 0 0
Plasma
-like PIK3 CA PIK3CA E545K, H1047R 60 38,00 63,3 641,95 2,69 6,00 0,00 0 0 0 0 0
Plasma
-like EGFR
EGFR Deletion exon19, L858R,
L861Q, G719S, T790M
60 47,00 78,3 0 0 0 0 411,30 5,30 5,00 14 4,8
Plasma WT
60 45,98 76,6 0 0 0 0 0 0 0 0 0
Plasma
PIK3CA E545K PIK3CA E545K 60 38,17 63,6 0 12,9 0 0 0 0 0 0 0
Plasma
PIK3CA H1047L PIK3CA H1047L 60 46,7 77,8 116,6 0 0 33,05 0 0 0 0 0
Plasma
PIK3CA H1047R PIK3CA H1047R 60 44,55 74,3 109 0 46,5 0 0 0 0 0 0
Plasma
EGFR EGFR Deletion exon19 60 50 83,3 122 0 0 0 105 4,8 0 0 0
Mean Yield (%) 74,51
Table 1. naica® 6-color Crystal Digital PCR™ results: Two assays (Rectal Cancer 6-color assay and EGFR 6-color Crystal Digital PCR™)
were used with the naica
®
6-color Crystal Digital PCR™ to quantify WT and mutant DNA from cfDNA extracted from plasma-like or plasma
with the Maxwell
®
RSC LV kit. Concentrations are in copies/µL. Extraction yield was calculated by dividing the measured EC concentrations
Figure 5. Crystal Miner software Quality Control view of a Sapphire chip
chamber. Droplets containing amplicons amplified from cfDNA extracted with the
Maxwell® RSC system form a stable 2D droplet crystal characteristic of high-quality
Crystal Digital PCR™ data. Inset: a zoom of the droplet crystal structure.
Figure 8. cfDNA analysis with naica
®
6-
color Crystal Digital PCR
™
1D dotplots generated by Crystal Miner
software after Crystal Digital PCR™
amplification using the the 6-color Rectal
Cancer assay of DNA extracted from 2ml of
clinical plasma with the Maxwell RSC LV kit.
Fluorescence intensities for each color
channel (Y-axis) and sample names (X-axis)
are indicated. Threshold lines separate the
negative and positive clusters.
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
Figure 9. cfDNA analysis with the EGFR
6-color Crystal Digital PCR
™
A) 2D dotplots generated by Crystal Miner
software of the fluorescence intensities of
each color channel after Crystal Digital
PCR™ amplification of DNA extracted from
2ml of plasma-like with the Maxwell RSC LV
kit. B) 2D dotplots generated after Crystal
Digital PCR™ amplification of DNA extracted
with Maxwell RSC LV kit from 2ml of
plasma spiked with an exon 19 deletion
mutant DNA. Colored polygon thresholds
separate the various negative and positive
clusters.
A B
Conclusions
In this study, we show high compatibility between the automated Maxwell® CSC cfDNA extraction system and
high-plex detection from liquid biopsy samples by Crystal Digital PCR™ on the naica® system. This workflow
enables the highly sensitive detection and quantification of the main somatic EGFR mutations described in
NSCLC and a set of PIK3CA mutations described in breast and rectal cancers. Both technologies have a fast
time-to-result with minimum hands-on-time, enabling a complete sample-to-result workflow in less than a day.
This proof-of-concept workflow creates the foundation for the further development of streamlined sample-to-
answer protocols that will better assist cancer researchers across the biomarker testing landscape.
Figure 1. Schematic of the liquid biopsy composition.
Liquid biopsy obtained from peripheral blood is composed of
different tumoral components such as circulating tumor cells
(CTCs), circulating cell-free DNA (cfDNA), extracellular
vesicles (EVs), and micro-RNA (miRNA). From Biomedicines,
Ayuso-Sacido et al., //doi.org/10.3390/biomedicines9080906
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
Plasma
WT
Plasma
E545K
Plasma
H1047L
Plasma
H1047R
The Maxwell
®
CSC 48 Instrument (Figure 3A) is a compact, automated nucleic acid purification
platform that processes up to 48 samples simultaneously. Using Maxwell® cartridges prefilled with
purification reagents and paramagnetic particles (Figure 3B), the Maxwell
®
CSC 48 Instrument brings
the same consistent, reliable purification of DNA or RNA from a variety of sample types and a higher
throughput. The intuitive graphical interface makes the instrument easy to use. The integrated vision
system with its large LED indicator reduces the potential for user error by detecting proper cartridge
placement and lets you know before a run starts if there is an issue. An integrated Bar Code reader
makes it easy to track samples.
Because the Maxwell® CSC instruments are magnetic particle movers, not liquid handlers, they offer
advantages over other automated systems. There is minimal risk of cross-contamination because no
liquid handling or splashing happens during sample processing. With no clogs and fewer breakdowns,
there are fewer disruptions to your workflow. High-quality nucleic acid purification with minimal steps
and hands-on time can be obtained with a wide-range of available extraction kits, including the Large
Volume (LV) cfDNA plasma extraction kit used for this study (Figure 3C).
Figure 3. Maxwell® system workflow. A) Maxwell®
CSC 48 (top left) and Maxwell® CSC 16 (bottom right)
instruments, B) the Maxwell® extraction methods start
with prefilled cartridges ready for the samples. After
sample addition, the instrument moves the particles and
associated nucleic acids through multiplex steps,
ultimately yielding highly pure nucleic acids. C) Maxwell®
cfDNA plasma kit extraction protocol overview..
A B C
Automated extraction of
nucleic acids with Promega Maxwell®
PROCESS TIME
HANDS-ON TIME
LESS THAN 10 MIN
LESS THAN 3 HOURS
Wild-type DNA
Tumor DNA
GMO DNA
miRNAs
Viral RNA
Etc..
Droplet crystal:
Self-assembled
CRYSTAL DIGITAL
PCR™ STEP
C
1
C
2
C
3
C
4
C
5
C
6
NAICA® SYSTEM
WORKFLOW
Single pipetting step
