Clinical Applications of Digital

PCR
Denise Ho, PhD
Regional Product Support

1
 About Us
Suzhou, China
● Founded in 2017

● Life sciences and in vitro diagnostics

● Strong management and R&D team from

Brown, Carnegie Mellon and Cornell

Medical Device
Manufacturing FDA EUA CE-IVD
Licence ISO 13485 NMPA
 One Stop Solution
Technical
04
Support

Sales and Marketing,

03
Global Supply

02 Manufacturing, Production

 Instruments  Reagents 01 R&D, Design

 Consumables
 Recognitions

• Distinguished • Exceptional Enterprise of the Top Private The most

High-Tech Talent Year Enterprise promising IVD
Enterprise companies in
• Most
• Technologically Promising China year 2020
Innovative SME Enterprise

4
 Our Partners
● Harvard University
● Princeton University
● Virginia Tech
● University of Michigan
● University of Florida
● Stony Brook University
● Gachon University
● University of California
● Rochester Institute of
Technology
● Louisiana State
University
● Institute of Hematology, Chinese
Academy of Medical Science
● Shanghai East Hospital of Tongji
University
● National Institute of Metrology
● Ruijin Hospital Shanghai Jiaotong
University School of Medicine
● California Institute of ● China-Japan Friendship Hospital
Technology
● Nanjing Drum Tower Hospital of
● University of Illinois
● Huashan Hospital of Fudan
Nanjing University Medical
School
University
● Monterey Bay Aquarium
Research Institute ● PLA Rocket Force General
● Cancer Hospital, Chinese Hospital
Academy of Medical Science
● Complete Genomics
● Wuhan Huo Shen Shan Hospital
5
Part I: Introduction to Digital PCR

6
 Digital PCR • End-point detection

Increased sensitivity, precision and accuracy

• Time-consuming

EQUAL QUALITATIVE
PCR
ARM
BALANCES
• Real-time detection
• Needs standard curve

SPRING SEMI QUANTITATIVE

qPCR
BALANCES

End-point detection

QUANTITATIVE
DIGITAL SCALE dPCR

7
 Digital PCR (dPCR)
• DPCR uses limiting dilutions and sample partitioning into sub-microlitre reactions to achieve
sensitive, precise, accurate, reliable and reproducible quantification of nucleic acids.
• This digital format is achieved either by a microfluidic-based approach, where a reaction is
divided into hundreds or thousands of chambers on a single plate or array, or by a droplet-
based approach, where a reaction is separated into thousands or millions of droplets.
• After the PCR, each partition is individually assayed, giving a 0 (neg) or 1 (pos) result if any
molecules are present.
• The ratio of positive to negative partitions can then be related to the number of molecules in
the sample, using Poisson statistics.

8
 Benefits of Partitioning
1. Concentration effect 20

- Small reaction volume

2. Enrichment effect 10X
- Purification 10 µL

The concentration effect scales

inversely with sample volume, while
the enrichment effect scales with the 20
number of partitions.
3. Superior precision and linearity 1
10
4. Extends the dynamic range
10X

MODIFIED FROM BASU, A. S., 2017 9

Partitioning Methods
1. Arrays of physically isolated chambers or wells
2. Droplet emulsions where the reaction is conducted in water droplets separated by a
continuous oil phase

- As with qPCR 96-well plates, the chambers are non-reusable plastic consumables.
- The number and size of chambers per device is fixed.
- The number of partitions and reactions per run is often lower than in droplet-based
platforms.
- Emulsions are cheaper to create.

10
 Assumptions
Estimating the number of targets based on a Poisson distribution is based on the following
assumptions:
• Target molecules are randomly distributed over the total number of partitions under analysis
• Presence of the target leads to a positive classification of the partition
• Absence of the target leads to a negative classification of the partition
• All partitions have the same volume

11
 Understanding Poisson Correction
Ideally,
If we could count the individual
molecules we would not need to use
Poisson statistics or dPCR.

In reality,

HAYNES, R., 2012 12

 Ideally,
100 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
λ=
100 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤 𝑒𝑒 −λ λ𝑘𝑘
= 1 target molecule/well 𝑃𝑃 𝑋𝑋 = 𝑘𝑘 =
𝑘𝑘!
Probability of a well
k= 0, P(X=0) = 𝑒𝑒 −λ
containing k number of
target molecules λ = −ln 𝑃𝑃
P(X=0) = 36.788%
#𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
P(X=1) = 36.788% = − ln
𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 #𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
−λ 𝑘𝑘
𝑒𝑒 λ
𝑃𝑃 𝑋𝑋 = 𝑘𝑘 = P(X=2) = 18.394% Each well contains V volume,
𝑘𝑘!
P(X=3) = 6.131%
λ= average rate of occurrence Concentration (copies/µL)
P(X=4) = 1.533%
#𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
− ln
P(X=5) = 0.307% = 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 #𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
𝑉𝑉
…

#𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤 37
−ln −ln
Concentration= 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 #𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤
= 100
𝑉𝑉 0.2 µ𝐿𝐿

= 4.9713 copies/µL = 99.4253 copies/20 µL

HAYNES, R., 2012 13
 dPCR vs qPCR
dPCR qPCR
May still require a titration step when dealing Requires calibration with standard curve and
with unknown concentrations. generating a standard curve requires sample
dilutions over a 5 log range, with multiple
replicates at each concentration.
Linear and digital quantification that is based Amplification signal is logarithmic and the
only on the number of positive and negative quantification is based on external calibration.
reactions, with the Poisson distribution taken
into account.
More tolerant to some PCR inhibitors, Different PCR components are known to have
sequence variations and different types of significant influences on the efficiency of
DNA templates, PCR assays and master mixes. qPCR, which can lead to different Cq values.
HAYNES, R., 2012 14
Part II: RainSure Droplet Digital PCR

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 Flexible Configuration

8-channel SPS 16-channel SPS 32-channel SPS

3-color CS DropX-2130 DropDx-2030 DropX-2230

4-color CS DropDx-2140 DropDx-2044 DropDx-2240

5-color CS DropX-2150 DropDx-2050 DropX-2250

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 Excellent Performance
Concordance Detection Limit Droplet Count

10.00%
8.00%
6.00%
4.00%
2.00%
0.00%
0.00% 5.00% 10.00% 0.01% 25,000
99%
Fluorescence Channels Retrieval Testing Time

PCR products recovery;

Cy5, FAM, HEX, ROX repeated scanning of cartridge; 2.5 hrs
droplet images 17
NMPA, FDA, CE Approved

CE-IVD
NMPA FDA EUA

18
More Than 30 Publications

19
 Wide Coverage ddPCR Panels BCR-ABL 210 Fusion
BCR-ABL T315I Mutation
IDH1 R132C Mutation
IDH1 R132G Mutation
IDH1 R132H Mutation
Epstein-Barr Virus (BAMHI- JAK2 V617F Mutation
W/ EBNA1) MYD88 L265P Mutation
Influenza A Virus (H1N1-2009/ TP53 R175H Mutation
H3N2/ H7N9) Leukemia TP53 R248L Mutation
Respiratory
Influenza B Virus (BV/ BY) TP53 R273C Mutation
Viruses Cytomegalovirus TP53 R273H Mutation
Novel Coronavirus (SARS-CoV-2)
NPM1 Type A RNA
NPM1 Type B RNA
Hepatitis B Virus NPM1 Type D RNA
Sepsis Pathogenic Microorganisms
ALK G1269A Mutation
ESR1 Y537S Mutation BRAF V600E Mutation
ESR1 D538G Mutation Solid EGFR G465R Mutation
KRAS G12A Mutation Solid Tumor EGFR G719A Mutation
KRAS G12C Mutation Tumor EGFR G719S Mutation
KRAS G12D Mutation EGFR E746_A750delELREA (COSM6223) Mutation
KRAS G12S Mutation EGFR E746_A750delELREA (COSM6225) Mutation
KRAS G12V Mutation EGFR E747_A750delinsP (COSM2338) Mutation
KRAS G13D Mutation EGFR E747_A750delinsP (COSM2339) Mutation
NRAS G12V Mutation EGFR L747_P753>S Mutation
NRAS Q61R Mutation EGFR S768I Mutation
PIK3CA E542K Mutation EGFR V769_D770insASV Mutation
PIK3CA E545K Mutation Organ EGFR D770_N77insG Mutation
PIK3CA H1047R Mutation
NIPT Transplantation EGFR T790M Mutation
PIK3CA Mutation Multiplex EGFR C797S (COSM6493937) Mutation
HER2 Copy Number Variation EGFR C797S (COSM5945664) Mutation
EGFR L858R Mutation
EGFR L861Q Mutation 20
Simple Workflow

Sample preparation Sample loading Droplet generation and PCR Chip reading and
data analysis

21
Part III: Application of ddPCR in the Early Diagnosis
and Management of Sepsis

22
Sepsis- cause of 1 in 5 deaths worldwide
Sepsis: Life-threatening organ dysfunction secondary to a dysregulated host response to
infection.
-- International Guidelines for the Management of Sepsis and Septic Shock 2021

Rapid onset
High incidence
01 03
 For every six-hour delay in treatment,
 Global: 49 million/year (WHO, 2020) the risk of death increases by 58%
 China: About 6.11 million/year  A two-day delay in treatment increases
the risk of death by 38.8 times

Post-sepsis syndrome
High mortality  Only half will completely recover (WHO,

02  Global: 11 million/year (WHO, 2020)

 China: 11 million in 2017 (Liu, Y. et al.,
04 2020)
 The rest will either die within 1 year or be
2022) burdened by long-term disabilities (WHO,
2020)
23
Blood culture remains the gold standard for diagnosing
sepsis

Wang, H., & Wang, C. B., 2022 24

 Three major pain points of blood culture analysis
need to be addressed: #1 Low sensitivity
The detection rate of blood cultures in patients with sepsis is only 10%.
 Low bacterial content in the bloodstream
 Dependent on the blood volume drawn and the timing of the blood draw
 Misuse of antibiotics
 Unstandardized clinical practice of blood culture

Thus, a negative test for bacteremia is both (1)

common and (2) relatively noninformative as to
whether the patient requires antibiotics.

Antibacterial Restriction Order: Notice on Further Strengthening the

Obtaining blood cultures after initiation of empirical treatment reduces sensitivity
by approximately 50% when preantimicrobial cultures are positive. (Cheng, M. P.
et al., 2019)
Management of Clinical Application of Antibacterial Drugs to Curb
Bacterial Drug Resistance
 Inpatients treated with special use grade antimicrobial drugs
 The rate of microbiological examination before the use of
antimicrobial drugs should not be less than 80%
25
Three major pain points of blood culture analysis
need to be addressed: #2 Delayed turnaround time
Blood Gram Stain Standard Antimicrobial
Blood Pathogen ID Susceptibility Testing
Draw Positive Testing
Culture

12-72 h 5 min 24-72 h

Often lead to unnecessarily long courses of empiric broad-

spectrum antibiotics (i.e., antibiotics given without
knowledge of the etiologic agent), exposing patients to
unnecessary risk of adverse events, and leading to selection
pressure for downstream resistance.

<10% of patients have access to precise 26

medication
Three major pain points of blood culture analysis
need to be addressed: #2 Delayed turnaround time

International Guidelines for the Management

of Sepsis and Septic Shock, 2021

27
Three major pain points of blood culture analysis
need to be addressed: #3 False positives

20 to 56% of all positive blood cultures are found to be contaminated, with overall contamination
rates of 0.6 to 12.5%. (Dargere, S. et al., 2018)
 Poor technique by individuals obtaining blood cultures
 Insufficient disinfection of the skin
 Collection of blood through indwelling vascular catheters
 Type of broth medium used for blood cultures
 Change in the standard of practice for obtaining blood cultures by venipuncture

“Even when optimal blood specimen collection protocols are used, completely eliminating blood culture contamination
may be impossible. However, laboratories should still be able to achieve blood culture contamination rates substantially
lower than 3%. When best practices are followed, a target contamination rate of 1% is achievable”

- M47 Principles and Procedures for Blood Cultures, 2nd Edition 2022 28
Three major pain points of blood culture analysis
need to be addressed: #3 False positives
Isolated Organisms
Brucella, Francisella tularensis, Histoplasma capsularis etc.
Staphylococcus aureus, Streptococcus pneumoniae,
Escherichia coli, Enterobacteriaceae sp., Pseudomonas
aeruginosa, Candida albicans, and other common
community or nosocomial pathogens
Clostridium perfringens, filamentous fungi etc.
Propionibacterium, Bacillus (except Bacillus anthracis),
most Corynebacterium, Aerococcus, Micrococcus,
Streptococcus viridans, Coagulase-negative Staphylococcus
(except pediatric and catheter) etc.
Diagnostic value may be affected when a microorganism of questionable evidence is isolated. (Wang, H., & Wang, C. B., 2022)
Making a correct diagnosis of pathogenicity (vs. contamination) is challenging.
Interpretation
True pathogen
Usually true pathogen, but in rare
cases may be contaminating bacteria
Contamination, but could also be
disease-causing
Common skin-colonizing bacteria,
most of which are contaminants
Management
Observe biosafety protection
Even if only one bottle of blood culture is positive, the laboratory should
consider it as pathogenic and indicate this in the report
Comprehensive analysis should be performed with patient’s clinical
presentation and other laboratory findings to rule out its pathogenicity. If
Clostridium perfringens was found, hemoglobin and creatinine levels should
be monitored; If filamentous fungi was identified, it should be combined
with glycan and galactomannan test results etc.
Contact clinician to determine its diagnostic value even if it is only a single
positive. For example, a single bottle of Streptococcus viridans-positive
culture should also raise an alarm of the risk of infective endocarditis
29
 an imperfect
• Blood culture is the gold standard.

30
 RainSure Sepsis Pathogenic Microorganisms ddPCR
Detection Kit

• Simultaneously detects 15/ 21 pathogenic bacteria and fungi

• LoD 3-5 copies/µL

• Lab on a chip; no sample transfer, no cross contamination

• Highly automated; prevents laboratory-acquired infection

• High amplification speed (1.5-3h)

• Sample in results out in less than 5h

31
RainSure Sepsis Pathogenic Microorganisms ddPCR Detection
Kit – Designed for high clinical prevalence bacteria and fungi
FAM HEX ROX Cy5
Panel 1 Bacteroides fragilis Staphylococcus epidermidis Enterococcus faecium1 Streptococcus pneumoniae

Panel 2 Acinetobacter baumannii1 Enterobacter cloacae1 Enterococcus faecalis Staphylococcus aureus1

Panel 3 Internal Control Staphylococcus haemolyticus Klebsiella pneumoniae1 Pseudomonas aeruginosa1

Panel 4 Escherichia coli Staphylococcus capitis Stenotrophomonas maltophilia Staphylococcus spp.

1 ESKAPE pathogens
FAM HEX ROX Cy5
Panel 5 Candida tropicalis - Cryptococcus neoformans Internal Control

Panel 6 Candida krusei Candida parapsilosis Candida glabrata Candida albicans

Have been listed as antibiotic resistance threats in the United States 2019 (CDC, 2019)
Have been listed as fungal priority pathogens list by WHO (WHO, 2022)
32
Germany, 2015-2019 Korea, 2017-2019

Ethiopia, 2019-2020

Pakistan, 2019

Global Burden of Bacterial Antimicrobial Resistance, 2019

33
RainSure Sepsis Pathogenic Microorganisms ddPCR
Detection Kit is sensitive, fast and accurate
Traditional blood culture + drug sensitivity

Antimicrobial
Blood Blood Gram Isolation Pathogen
Sensitivity Test
Average
Collection Culture Stain Culture Treatment
Identification 57.4 hrs
1 2 3 4 5 6 7

Blood culture + FilmArray LoD 5x103-105 CFU/mL

Blood Blood Gene Average

Treatment
Collection Detection
Culture 17.2 hrs
1 2 3 4

RainSure 2-4 mL blood

Blood Sample Gene

Collection Prep Detection Treatment
Average 3-5 hrs
Oeschger, T. et al., 2019
11 22 3 4
4
LoD 50-100 CFU/mL The goal is to use drugs effectively! 34
RainSure Sepsis Pathogenic Microorganisms ddPCR
Detection Kit enables testing of different specimens
such as blood, pleural fluid, ascites, sputum, etc.

Patient Ascites：High expression of

Patient whole blood Patient pleural fluid
16SRNA, suggesting severe bacterial
EB virus infection Enterococcus faecalis infection
infection

35
 Bacteroides fragilis Staphylococcus epidermidis Acinetobacter baumannii Enterobacter cloacae

Recommended
threshold:
Enterococcus faecium Streptococcus pneumoniae Enterococcus faecalis Staphylococcus aureus Pseudomonas
aeruginosa ≥ 4
pos droplets
Others ≥ 3 pos
droplets
Staphylococcus haemolyticus Escherichia coli Staphylococcus capitis

Klebsiella pneumoniaePseudomonas aeruginosa Staphylococcus spp. 36

Stenotrophomonas maltophilia
 Successful detection of sepsis using RainSure Sepsis
Pathogenic Microorganisms ddPCR Detection Kit
mNGS vs
Adjudication
Sample # Sample type IC RPM (mNGS) Q-mcfDNA seq (copy number) RainSure (copy number) RainSure Conventional test
Classification
results
Acinetobacter baumannii (34.5) Klebsiella
Acinetobacter baumannii (157.72) Partially Blood culture negative, other
N20200691 7760.22 pneumoniae (241.25) Staphylococcus aureus Definite
Klebsiella pneumoniae (277.82) Agreed culture negative
(51.75)
Klebsiella pneumoniae (13.20) Klebsiella pneumoniae (11.8) Acinetobacter Blood culture negative, other
N20200693 12868.91 Agreed Definite
Acinetobacter baumannii (53.00) baumannii (35.375) culture negative
Blood culture negative, negative
Mycobacterium fragilis (18.72) Mycobacterium fragilis (45) Pseudomonas venous catheter culture, positive
N20200708 16906.54 Pseudomonas aeruginosa (162.79) aeruginosa (25.25) Klebsiella pneumoniae Agreed bile culture for Klebsiella Definite
mNGS: plasma
Klebsiella pneumoniae (224.12) (190) pneumoniae and Pseudomonas
w/o blood
aeruginosa
culture; dPCR:
Blood culture negative, pus culture
N20200714 whole blood w/o 401.69 Klebsiella pneumoniae (9427.90) Klebsiella pneumoniae (55875) Agreed Definite
positive for Klebsiella pneumoniae
blood culture
Blood culture negative, sputum
N20200719 4350.46 Pseudomonas aeruginosa (397.26) Pseudomonas aeruginosa (338.75) Agreed culture positive for Pseudomonas Definite
aeruginosa
Pseudomonas aeruginosa (69.28) Pseudomonas aeruginosa (101.125) Blood culture negative, other
N20200746 4431.43 Agreed Definite
Klebsiella pneumoniae (241.21) Klebsiella pneumoniae (260) culture negative
Blood culture positive of
Pseudomonas aeruginosa (1.26) Partially
N20200698 55567.05 Klebsiella pneumoniae (45.25) Enterococcus faecalis, Klebsiella Definite
Klebsiella pneumoniae (6.39) Agreed
pneumoniae

Huashan Hospital, Shanghai Shanghai East International Medical Centre

Blood culture mNGS
N=144 N=525
Positive Negative Positive Negative
Positive 57 0 Positive 40 4
RainSure RainSure
Negative 0 87 Negative 4 477 37
Part IV: ddPCR and Liquid Biopsy

38
Cancer Today

GLOBOCAN, 2020 39
Cancer Tomorrow

IARC, 2022 40
Cancer Tomorrow

IARC, 2022 41
 Early
Late detection/
presentation Cancer
screening
Reduced
cancer-
Challenges
related
mortality
Delayed Quality
cancer care treatment

42
Liquid
Biopsy vs
Tissue
Biopsy

LONE, S. N. ET AL., 2022

43
Main Detection Techniques

GATTUSO, G. ET AL., 2022

44
 ctDNA
qPCR ddPCR NGS Microarray ELISA Biosensors LOC MS

Quantify the expression of Absolute allele Analyze small and non- Evaluation of gene Gold standard for the Detect several biological Allow for the detection of Desorption/ ionization
specific mRNAs and quantification, ctDNA coding RNAs, post- expression to DNA detection of various molecules including CTCs, ctDNA, miRNAs, and and measurement of
miRNAs somatic mutations, DNA transcriptional methylation and non- circulating tumor markers nucleic acids, enzymes as proteins proteins extracted from
methylation, and gene modifications as well as coding RNA expression such as PSAs and CEAs well as antibodies and body fluids
rearrangements changes in gene profile; identification of antigens
expression single-nucleotide
polymorphisms, gene
mutations, and genes
related to drug resistance
in tissue biopsies
Relative quantification is Based on a water–oil Sequencing by the Complementarity and in Antigen-antibody binding, Consists of a receptor able Integrates several Associated with SELDI-TOF
based on the comparison emulsion of the reaction synthesis of millions of situ hybridization, which which allows for the to bind the target and a analytical laboratory and LC-MS
between the mix, in which the nucleic DNA fragments in a short allows for the evaluation detection and transducer, which allows procedures on a single
concentration of the acid sample is amount of time of thousands of genes in quantification of the conversion of chip, providing a
target gene and the fractionated into a single assay. DNA antibodies, antigens, the biochemical signal miniaturized and
concentration of the thousands of droplets fragments are collected proteins, and other into an electrical signal automated system for the
standard gene, while (~20,000). Then, PCR on a solid surface where peptides such as detection of cellular and
absolute quantification is amplification is performed they can bind with probes, glycoproteins and molecular elements.
performed using a within each resulting in the emission Hormones Among the various
calibration curve with portion and of a fluorescence signal. devices developed, the
known concentrations of positive/negative Regarding RNA, reverse- microfluidic-based system
the target fluorescent signals transcription into cDNA is represents the most used,
emitted by specific probes mandatory to detect and as characterized by the
or dyes are quantify the targets. easy manipulation and
detected from each suitability of cell
droplet. separation.
Low-expressed High sensitivity (0.01%) High sensitivity in the High throughput and Specific and cost- Short time using small
biomarkers or a slight and specificity detection of a wide board sensitivity effective, are a valuable amounts of biological
variation in their of known and unknown tool for the diagnosis of fluid samples, low-cost
expression may not be cancer-related mutations various diseases such as devices guarantee a high
correctly detected by by analyzing the whole diabetes mellitus, sensitivity and specificity
qPCR sequence of target genes cardiovascular diseases, in clinical settings
(mutant allele fraction and viral infections
<1%)

GATTUSO, G. ET AL., 2022 45

 FDA Approved ctDNA Tests
BRACAnalysis CDx® cobas® EGFR Mutation Test FoundationOne® Liquid Guardant360® CDx
v2 CDx

PCR, multiplex PCR and Real-time PCR NGS NGS

Sanger sequencing

Germline BRCA1/2 Identifies 42 mutations in Comprehensive genomic Tumor mutation profiling in

mutations exons 18, 19, 20 and 21 of profiling in 311 genes 55 genes
the EGFR gene, including
the T790M resistance
mutation

Pancreatic cancer, breast NSCLC Advanced cancer patients Advanced cancer patients
cancer, ovarian cancer, and with solid tumors with any solid malignant
prostate cancer neoplasm

FDA, 2022 46
(Digital PCR + ctDNA) Clinical Trials

NIH, 2022 47
RainSure EGFR Mutation Detection Kits
G465R / Panitumumab

G719A / Afatinib, Capmatinib, Crizotinib, Dacomitinib,

Erlotinib, Gefitinib, Osimertinib, and
G719S / / Pembrolizumab

E746_A750del / / / Poziotinib

L747_A750delinsP / Afatinib, and Gefitinib

L747_P753delinsS / Afatinib, Erlotinib, and Gefitinib

Afatinib, Capmatinib, Cetuximab, Crizotinib,
S768I / Dacomitinib, Erlotinib, Gefitinib, Osimertinib, and
Pembrolizumab
V769_D770insASV / Poziotinib

D770_N771insG / Osimertinib
Afatinib, Capmatinib, Cetuximab, Crizotinib,
T790M / / / / Dacomitinib, Erlotinib, Gefitinib, Osimertinib, and
Pembrolizumab
C797S / Osimertinib

L858R / / Afatinib, Capmatinib, Cetuximab, Crizotinib,

Dacomitinib, Erlotinib, Gefitinib, Osimertinib, and
L861Q / / / Pembrolizumab
48
FDA, 2022 NCCN, 2022
RainSure ESR1 and IDH1 Mutation Detection
Kits
ESR1

Y537S / /
Anastrozole, Exemestane, and Letrozole
D538G / /

IDH1

R132C / /

R132G / Ivosidenib

R132H / / /

49
FDA, 2022 NCCN, 2022
RainSure KRAS and NRAS Mutation Detection
Kits
KRAS

Afatinib, Cetuximab, Dacomitinib, Erlotinib,

G12A / / /
Gefitinib, Osimertinib, and Panitumumab
Afatinib, Cetuximab, Dacomitinib, Erlotinib,
G12C / / / / / Gefitinib, Osimertinib, Panitumumab, and
Sotorasib
G12D / / / / / / /

G12S / / /
Afatinib, Cetuximab, Dacomitinib, Erlotinib,
Gefitinib, Osimertinib, and Panitumumab
G12V / / /

G13D / / /

NRAS

G12V /
Binimetinib, Cetuximab, and Panitumumab
Q61R / / / /

50
FDA, 2022 NCCN, 2022
 RainSure TP53, ALK & BRAF Mutation
Detection Kits

TP53

R175H / / / / / Doxorubicin

R248L / /

R273C / / / / Cisplatin, Etoposide, and Mitomycin

Doxorubicin, Methotrexate,
R273H / / /
Tamoxifen, and Temozolomide

ALK

G1269A / Crizotinib

BRAF
Binimetinib, Cemiplimab,
Cetuximab, Cobimetinib,
V600E / / / / / / / Dabrafenib, Encorafenib, Imatinib,
Panitumumab, Pembrolizumab,
51
Trametinib, and Vemurafenib

FDA, 2022 NCCN, 2022

 RainSure EGFR Mutation Detection Kits
Showed Concordance with NGS & Bio-Rad
ddPCR Fractional Abundance
1.20%
RainSure
Mutation NGS 1.00% y = 0.9786x - 0.0006
Replicate 1 Replicate 2
0.80% R² = 0.9591
7.70% 8.4% 8.6%

Bio-Rad
0.60%
T790M 1.90% 1.3% 1.4%
RainSure 0.40%
0.96% 0.8% 0.9% Mutation NGS
Replicate 1 Replicate 2 0.20%
L858R 4.4% 3.4% 3.9% 0.00%
-0.20%0.00% 0.50% 1.00% 1.50%
L858R RainSure
Hi Mut
Copy Number
Hi Mut 6000.00
5000.00 y = 0.9518x - 224
4000.00 R² = 0.9647

Bio-Rad
Med Mut 3.4% 3.9%
3000.00
2000.00
1000.00
0.00
0.00 2000.00 4000.00 6000.00 8000.00
Lo Mut
RainSure
52
RainSure EGFR Mutation Detection Kits are
Useful for Monitoring Therapy Response in
NSCLC
T790M
0.60%

WT copy number Mutant copy number Mutation

Sample Mutation
(copies/20μL） （copies/20μL） frequency

1980 7.8 0.39%

T790M
1960 3.6 0.18% L858R
2920 9.6 0.33% 0.39%
L1 L858R
3080 16.0 0.53%
2040 12.2 0.60%
19DEL
2060 11.6 0.56%

19DEL
Real-time monitoring from post first-line 0.33%
erlotinib therapy to disease progression.

53
Current and Future Burden of Breast Cancer

GLOBOCAN, 2020
54
IARC, 2022
Current Practice
Grading Staging and Prognosis

Grades Imaging
• Chest X-ray
Early Detection and Diagnosis Ploidy & cell proliferation • CT scan
• Ki-67 test • MRI scan
Imaging • S-phase fraction • Ultrasound
• Mammograms • PET scan
• Breast ultrasound Hormone receptor status
• Bone scan
• Breast MRI • Immunohistochemistry (IHC)
• Others e.g. CT scans, bone scans, PET Stages
HER2 status
scans, breast tomosynthesis (3D • IHC
mammography), fast breast MRI, Survival rates
• Fluorescence in situ hybridization
radionuclide imaging, contrast-
enhanced mammography, elastography, Gene expression
optical imaging tests, electrical • Oncotype DX Treatment
impedance tomography • MammaPrint
• Prosigna Local
Biopsy • Breast Cancer Index • Surgery
• Fine needle aspiration • Radiation
• Core needle biopsy Other genes, proteins & blood tests
• Surgical biopsy • Hormone receptor proteins, HER2 protein, PD-L1 protein Systemic
• Lymph node biopsy • Tissue/ liquid biopsy: BRCA1 & BRCA2 mutations, MSI & • Adjuvant & neoadjuvant chemotherapy (Oncotype DX,
MMR testing, tumor mutational burden, NTRK fusion ECHO, MUGA scan)
genes • Hormone therapy (Breast Cancer Index)
• Complete blood count, blood chemistry tests, tumor • Targeted drug therapy (HER2, hormone receptor, BRCA)
markers • Immunotherapy (PD-1)

ESMO, 2019
55
American Cancer Society, 2022
Challenges

ASCO & CAP, 2018 56

 FDA Approved ctDNA Testing
BRACAnalysis CDx® FoundationOne® Liquid CDx QIAGEN therascreen PIK3CA RGQ
PCR Kit

PCR, multiplex PCR and Sanger NGS Real-time PCR

sequencing

Germline BRCA1/2 mutations Comprehensive genomic profiling in 11 mutations in the exons 7, 9, and 20
311 genes of PIK3CA gene

Pancreatic cancer, breast cancer, Advanced cancer patients with solid Breast cancer
ovarian cancer, and prostate cancer tumors

FDA, 2022 57
 RainSure HER2 CNV Detection Kit

Ado-Trastuzumab
Emtansine, Aromatase
inhibitor, Capecitabine,
Carboplatin, Cisplatin,
Docetaxel, Endocrine
therapy, Fam-
Trastuzumab Deruxtecan,
ERBB2 Fluorouracil,
Hyaluronidase, Lapatinib,
Margetuximab, Neratinib,
Paclitaxel,
Pembrolizumab,
Pertuzumab,
Trastuzumab, and
Tucatinib

FDA, 2022 NCCN, 2022

ASCO & CAP, 2018

RainSure HER2 CNV

Detection Kit is a 4-color
assay
58
 RainSure PIK3CA Mutation
Detection Kits
C420R

E542K

E545A

E545D

E545G

E545K Alpelisib and fulvestrant

Q546E

Q546R

H1047L

H1047R

H1047Y

FDA, 2022 NCCN, 2022

59
RainSure HER2 CNV Detection Kit
Detected HER2 in Reference Standard
and FFPE Sample
Expected HER2 (FAM) RPP30 (HEX)
CNV
Primer/Probe Sample CNV Concentration Concentration
(Copies)
(Copies) (Copies/µL) (Copies/µL)
Reference
7.60-9.40 750 158 8.74
Standard
HER2+RPP30
FFPE
12.96-14.61 247 40 13.00
Sample

Reference Standard FFPE Sample

FAM (HER2)

FAM (HER2)
HEX (RPP30) HEX (RPP30) 60
 Thank you
Contact us at:
Address Suzhou PreciGenome Ltd, Co., 218 Sangtian Street, BioBay II,
Building 1, 202-1, Suzhou Industrial Park, Suzhou, China.

61