A streamlined workflow for liquid biopsy sample extraction and

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A streamlined workow for liquid biopsy sample extraction

and highplex Crystal Digital PCR™ analysis using the

Maxwell

RSC system and the 6-color naica

system

INTRODUCTION

Liquid biopsies, such as blood samples, can harbor a wealth of

genetic information from both healthy and unhealthy cells to

inform disease diagnosis and treatment. Rigorously qualied

pre-analytical protocols are vital to ensure the performance of

downstream liquid biopsy workows and thus high-quality re-

sults. Circulating cell free DNA (cfDNA), extracted from liquid

biopsy samples, are an established sample type for character-

izing oncology targets. Nevertheless, cfDNA measurements

require a highly sensitive and reliable detection technology to

quantify, often low-level, genetic aberrations within a complex

background of wild-type sequences.

This technical note details a exible method for automated

plasma sample extraction that seamlessly integrates into a

straightforward and sensitive digital PCR genetic analysis

workow. Combining the Maxwell

RSC 48 instrument for au-

tomated cfDNA extraction with Crystal Digital PCR™ on the nai-

system allows ultrasensitive high-plex detection from liquid

biopsy samples. By bridging the two technologies, 32 common

and rare somatic EGFR mutations in exons 18, 19, 20, and 21,

representing more than 90% of EGFR mutations described in

non-small-cell lung carcinoma (NSCLC) are sensitively and pre-

cisely detected. The combination of the two technologies also

allowed for the detection of a set of PIK3CA mutations from cfD-

NA samples.

The combined workow of automated plasma sample ex-

traction using the Maxwell

RSC 48 and the high-plex and sensi-

tive target detection with Crystal Digital PCR™ thus represents a

streamlined full solution from sample-to-answer that can bene-

t cancer researchers across the biomarker testing landscape.

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 ccfDNA LV Plasma Kit (Promega,

ref AS1840). Before extraction, all samples were spiked with a

known quantity of an exogenous extraction control DNA (EC),

and after extraction, aliquots of the human plasma samples

were spiked with known amounts of synthetic mutant DNA. For

comparison to the automated sample extraction methodology,

plasma samples were also manually extracted with the QIAamp

circulating nucleic acid kit (QIAGEN, ref 55114) according to

supplier recommendations.

The extracted cfDNA samples were then analyzed by Crystal

Digital PCR™ on the 6-color naica

system with two indepen-

dent multiplexed cancer detection assays, a 6-plex and a 33-

plex.

Stilla Technologies denes plex as the number of targets de-

tected in a Crystal Digital PCR™ assay.

A custom 6-plex Rectal Cancer assay detecting six targets:

PIK3CA wild-type, 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), an off-the-shelf 33-plex digital PCR kit detecting

EGFR wild-type and the 32 most common non-small cell lung

cancer EGFR mutations

APPLICATION NOTE

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The Maxwell

RSC 48 Instrument (Figure 1A) is a compact,

automated nucleic acid purication platform that processes

up to 48 samples simultaneously. Using Maxwell

cartridges,

prelled with purication reagents and paramagnetic particles

(Figure 1B), the Maxwell

RSC 48 Instrument enables consis-

tent high-throughput purication of DNA or RNA from a variety

of sample types. The Maxwell

RSC 48 Instrument’s 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 place-

ment. This informs the user of any issues before the run starts.

An integrated bar code reader makes it easy to track sample

information along the extraction workow.

Because the Maxwell

RSC instruments are magnetic particle

movers, not liquid handlers, they offer advantages over other

automated nucleic acid extraction systems. There is minimal

risk of cross-contamination because no liquid handling or

splashing occurs during sample processing. With no clogs and

fewer breakdowns, there are fewer disruptions to the nucleic

acid extraction workow. High-quality nucleic acid purication,

with minimal steps and hands-on time, can be obtained with

a wide range of available extraction kits, including the Large

Volume (LV) ccfDNA plasma extraction kit used for this study

(Figure 1C and 1D).

Figure 1 | Maxwell

system workow. A) Maxwell

RSC 48 (top) and Maxwell

RSC 16 (bottom) instruments, B) the Maxwell

extraction methods start

with prelled cartridges ready for the samples. C) Sample preparation for the use of Maxwell

RSC ccfDNA Plasma Large volume Kit extraction D) After

sample addition, the instrument moves the particles and associated nucleic acids through a series of automated steps, ultimately yielding highly pure

nucleic acids.

Maxwell

RSC 48 Instrument

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Crystal Digital PCR™ on the 6-color naica

system

The 6-color naica

system Crystal Digital PCR™ workow (Fig-

ure 2) enables high multiplexing capacity in a single reaction,

saving both time and precious samples, and provides ultra-sen-

sitivity and increased low-level detection of multiple reactions

in parallel. By automatically partitioning sample reactions into a

2D array of tens of thousands of droplets, Crystal Digital PCR™

technology can be used for absolute nucleic acid quantication

in a wide range of applications. For example, Crystal Digital

PCR™ can be used for oncological analysis applications, includ-

ing copy number variation, mutation detection,rare event de-

tection, and therapeutic monitoring. The accompanying Crystal

Miner software measures the concentrations of targeted nucle-

ic acids, providing automatic identication of positive and neg-

ative Droplet Crystals for the selected uorescence channels.

With intuitive visuals for image analysis, users can easily ex-

plore their multiplex data and directly inspect the Droplet Crys-

tals in different uorophore channels. This visual inspection

allows unparalleled peace of mind for quality control.

RESULTS

Integration of the Maxwell

RSC LV kit and

the 6-color naica

system into a compatible

sample-to-answer workow

To evaluate the Droplet Crystal stability and compare the sam-

ple concentrations of cfDNA using two different extraction pro-

cedures, cfDNA from plasma-like samples were extracted auto-

matically with the Maxwell

RSC LV kit on the Maxwell

RSC 48

system (Promega) and manually with the QIAamp circulating

nucleic acid kit (Qiagen). The samples were processed by Crys-

tal Digital PCR™ on the 6-color naica

system using the EGFR

6-color Crystal Digital PCR™ kit. All samples showed full com-

patibility for Droplet Crystal stability (Figure 3A). Furthermore,

a comparison of the total DNA concentration (Figure 3B) and

mutant DNA concentrations (Figure 3C) revealed highly similar

results across the two extraction procedures.

Figure 2 | The 6-color naica

system: absolute quantication of multiple genetic targets in a single run. The 6-color naica

system is an easy-to-use digital

PCR platform whose cutting-edge microuidic technology automatically integrates the digital PCR workow into a ready-to-use single consumable chip.

With no moving parts, Crystal Digital PCR™ partitions the sample into a 2D array of thousands of individual droplet reaction compartments that individually

amplify nucleic acid molecules. These reactions are tagged with uorophores to be read using up to six different uorescence light channels that can be

combined for multiplex detection of dozens of individual target mutations. The 6-color naica

system makes for a fast and simple workow that can be

completed with less than 10 minutes of hands-on time.

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Figure 3| A. Comparing automated Maxwell RSC LV extraction with another manual extraction method. A. Direct visualization of droplets containing

amplicons amplied from cfDNA extracted with the Maxwell

RSC system. Droplets show a stable 2D Droplet Crystal characteristic of high-quality Crystal

Digital PCR™ data. Inset: a zoom of the Droplet Crystal structure with positive and negative droplets clearly distinguishable. B. Total DNA concentrations

(copies/µL) of circulating cfDNA samples extracted from 2mL of plasma-like samples with the Maxwell

RSC LV kit and the QIAamp circulating nucleic

acid kit. C. EGFR mutant DNA concentrations (copies/µL) of cfDNA samples extracted from 2mL of plasma-like samples by the Maxwell

RSC LV kit (dark

blue) and the QIAamp circulating nucleic acid kit (light blue).

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Example 1D-dot plots generated by the Crystal Miner software resulting from the detection by the 6-plex Rectal Cancer assay are

shown in Figure 4.

Table 1 | Crystal Digital PCR™ 6-color naica

system results: Two assays, 6-plex Rectal Cancer assay and EGFR 6-color Crystal Digital PCR™ kit, were used

with the 6-color naica

system to quantify wild-type and mutant DNA from cfDNA extracted from plasma or plasma-like samples with the Maxwell

RSC

LV kit. Concentrations are in copies/µL. Extraction yield was calculated by dividing the measured EC DNA concentrations by the expected concentrations.

Mutation detection in plasma and plasma-like samples

Extracted cfDNA from both plasma and plasma-like samples were analyzed by two multiplexed detection assays customized for Crys-

tal Digital PCR

™

workow: a custom designed 6-plex Rectal Cancer assay and the commercially available EGFR 6-color Crystal Digital

PCR™ kit. Quantication of the EC allowed to calculate the extraction yield for each sample. The mean yield obtained for eight samples

(three plasma-like and ve plasma) was 74.5%. All mutations known to be present in the samples were detected (Table 1).

Figure 4 | 6-plex Rectal Cancer assay analysis with Crystal Miner software. 1D dotplots generated by Crystal Miner software after Crystal Digital PCR™

partitioning, amplication and scanning using the 6-plex Rectal Cancer assay of DNA extracted from 2mL of clinical plasma with the Maxwell RSC LV kit.

Detected alleles, are the PIK3CA wild-type in the teal channel, the PIK3CA H1047R, E542K, E545K and H1047L mutations in the blue, green, yellow and red

channels respectively. The IR channel quanties the EC. Sample names are shown on the X-axis and uorescence intensities for each color channel on the

Y-axis. Threshold lines are automatically generated to separate the negative and positive clusters of droplets.

Mutants

6-plex Rectal Cancer assay

EGFR 6-color Crystal Digital PCR™ kit

Extraction control DNA Measured concentrations (copies /µL)

Expected

Concentration

(copies/µL)

Measured

Concentration

(copies/µL)

Extraction

yield (%)

DNA

PIK3CA

E545K

PIK3CA

H1047R

PIK3CA

H1047L

DNA

EGFR Del

exon 19

EGFR

L858R

EGFR

L861Q,

G719S

EGFR

T790M

Plasma-like WT No mutant 60.0 47.3

78.8 735.6

0.0 0.0 0.0 N.A N.A N.A N.A N.A

Plasma-like PIK3CA

PIK3CA E545K,

H1047R

60.0 38.0

63.3 642.0 2.7 6.0

0.0

N. A N.A N.A N.A N.A

Plasma-like EGFR

EGFR Deletion exon19,

L858R, L861Q, G719S,

T790M

60.0 47.0

78.3

N.A N.A N.A N.A

411.3 5.3 5.0 14.0 4.8

Plasma WT No mutant 60.0 46.0

76.6 175.3

0.0 0.0 0.0

N.A N.A N.A N.A N.A

Plasma PIK3CA E545K PIK3CA E545K

60.0 38.2

63.6 190.4 12.9

0.0 0.0

N.A N.A N.A N.A N.A

Plasma PIK3CA H1047L PIK3CA H1047L

60.0 46.7

77.8 116.6

0.0 0.0

33.1

N.A N.A N.A N.A N.A

Plasma PIK3CA H1047R PIK3CA H1047R

60.0 44.6

74.3 109.0

0.0

46.5

0.0

N.A N.A N.A N.A N.A

Plasma EGFR EGFR Deletion exon19

60.0 50.0

83.3 122.0

0.0 0.0 0.0

105.0 4.8

0.0 0.0 0.0

Mean Yield (%) 74.5

N.A. Not applicable

STILLA TECHNOLOGIES

info@stillatechnologies.com For Research Use Only. Not for use in diagnostic procedures.

Furthermore, 2D-dot plots resulting from the detection by the EGFR 6-color Crystal Digital PCR™ kit show clear separability between the

positive and negative clusters. (Figure 5).

CONCLUSIONS

This study demonstrates compatibility between the automat-

ed Maxwell

RSC cfDNA extraction system and high-plex de-

tection from liquid biopsy samples by Crystal Digital PCR™ on

the 6-color naica

system. This full solution workow enables

largely automated highly sensitive detection and quantication

of the main somatic EGFR mutations described in NSCLC and a

set of PIK3CA mutations in breast and rectal cancers. Both tech-

nologies produce results quickly with minimal hands-on-time,

enabling a complete sample-to-result workow in less than a

day. This proof-of-concept workow creates the foundation

for the further development of streamlined sample-to-answer

protocols for high multiplex assays that will benet cancer re-

searchers across the biomarker testing landscape.

Stilla Technologies provides a full set of comprehensive

Application Notes and Technical Notes to support the Crystal

Digital PCR™ workow. In case further support is needed,

contact [email protected]

For further information on Maxwell Extraction systems and

technologies, contact Maxwell-dP[email protected]

Reference:

Harrison PT, Vyse S, Huang PH, Rare epidermal growth factor receptor (EGFR)

mutations in non-small cell lung cancer, Seminars in Cancer Biology (2019),

doi: https://doi.org/10.1016/j.semcancer.2019.09.015

Figure 5 | EGFR 6-color Crystal Digital PCR™ kit analysis with Crystal Miner software. Examples of 2D dotplots, generated by Crystal Miner software, of the

uorescence intensities of each indicated color channel after Crystal Digital PCR™ partitioning, amplication and scanning of two cfDNA (A and B), extracted

from 2mL of plasma-like sample with the Maxwell

RSC LV kit. Colored polygon thresholds separate the negative (orange polygon) and positive clusters as

indicated on the axes.

HIGHLIGHTS

y The Maxwell

RSC cfDNA extraction system and

Crystal Digital PCR™ on the 6-color naica

system for a

seamless, largely automated, highly sensitive detection

and quantication end-to-end workow.

y Demonstrated detection and quantication of the main

somatic EGFR mutations described in NSCLC and a set

of PIK3CA mutations described in breast and rectal

cancers.

y Minimal hands-on-time, enabling a complete full

solution sample-to-result workow in less than a day.

A B