Webinar on Liquid Biopsy by BIS Research

Liquid Biopsy –
An Emerging
Frontier in Early
Multicancer
Detection
Emerging Tech Webinar
January 2023

2
Riya Gupta
Research Analyst
BIS Research Inc.
Nitish Kumar Singh
Principal Consultant
BIS Research Inc.
Dr. Azeet Narayan
Yale University School
of Medicine
Speakers
Liquid Biopsy: An Emerging Frontier in Early Multicancer Detection www.bisresearch.com

3
Agenda
▪ Overview
▪ Patient Cases
▪ Study Design
Key Speaker 2 : Ms. Riya Gupta
▪ Liquid Biopsy: Trends and Key Market Developments
Key Speaker 3 : Mr. Nitish Kumar Singh
▪ Liquid Biopsy: Market Dynamics
▪ Extracellular Vesicles (EVs)/Exosomes Based Liquid
Biopsy
Insight Monk and Previous Reports
Q&A
Introduction
Key Speaker 1 : Dr. Azeet Narayan
Liquid Biopsy: An Emerging Frontier in Early Multicancer Detection

Key Speaker
Dr. Azeet Narayan
Yale University School
of Medicine

5
Circulating Tumor DNA as Early Cancer Biomarker
Dr. Azeet Narayan
January 24, 2023

6
Adapted from Vogelstein B et al., Science, (2013)
Cancer is Caused by Altered Genome
Liquid Biopsy: An Emerging Frontier in Early Multicancer Detection Yale School of Medicine

7
• Access to the altered content is limited
• Tumor biopsy is painful, risky and technologically challenging
• Alternative sources: Bodily fluids
Tissue is the Issue

8
Most protein biomarkers:
background
tumor-derived
Circulating mutant tumor DNA:
Virtually zero
physiologic
background
All tumor-derived
Physiologic Background Level
Yale School of Medicine

9
A liquid biopsy may
capture mutation
heterogeneity that a
tissue biopsy could miss.
Can Assess Global Mutation Profile

10
Challenges in Measuring ctDNA
• DNA is highly fragmented.
• Rare mutant copies in wild-type background.

11
Where is the Mutant DNA?

12
Exon 1 Exon 2 Exon 3 .
Mutation
hot-spot or warm-spot
Need to distinguish
Sequencer and PCR errors
Ultra-Deep Sequencing for Rare Variants

13
 Our Approach: Overlapping paired-end sequencing
3’
5’
5’
3’
Sequencing
adapter
75 or 150 bp
Paired-end sequencing:
3’
5’
5’
3’
Mutation-prone region
Clonal overlapping paired-end sequencing:
Narayan et al, Cancer Res. 2012
Suppressing Sequencer Errors

14
Spiked in 0.2% mutant KRAS G12S DNA.
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
G G T G G C
variant
:
wild-type
ratio
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
G G T G G C
Overlapping Paired-End
True
GGT → AGT
variant
variant
:
wild-type
ratio
Narayan et al, Cancer Res, 2012
Deviations from wild-type KRAS
Single-End Sequencing
G C
T
A
Overlapping Paired-End Sequencing

15
Gene Name Chromosome Genomic Position
AKT1 Chr. 14 104780210 - 104780217
APC Chr. 5 112839939 - 112839947
BRAF Chr. 7 140753328 - 140753343
CDKN2A Chr. 9 21971169 - 21971195
21970993 - 21971037
CTNNB1 Chr. 3 41224605 - 41224647
EGFR Chr. 7 55174764 - 55174801
55191819 - 55191836
55181370 - 55181382
EZH2 Chr. 7 148811633 - 148811655
FGFR3 Chr. 4 1806587 - 1806610
1801828 - 1801845
1804358 - 1804377
FLT3 Chr. 13 28018499 - 28018508
FOXL2 Chr. 3 138946309 - 138946329
GNAS Chr. 20 58909358 - 58909370
HRAS Chr. 11 534282 - 534307
533862 - 533875
IDH1 Chr. 2 208248385 - 208248394
JAK2 Chr. 9 5073766 - 5073775
KIT Chr. 4 54733150 - 54733165
54727417 - 54727452
Gene Name Chromosome Genomic Position
KRAS Chr. 12 25245344 - 25245360
25227327 - 25227343
MET Chr. 7 116700202 - 116700217
116783357 - 116783377
MYD88 Chr. 3 38141147 - 38141156
NRAS Chr. 1 114716120 - 114716148
114713887 - 114713909
PIK3CA Chr. 3 179218290 - 179218310
179234288 - 179234305
PPP2R1A Chr. 19 52212716 - 52212734
52213067 - 52213082
PTEN Chr. 10 87933144 - 87933167
87957910 - 87957928
STK11 Chr. 19 1207017 - 1207045
1223101 - 1223128
TP53 Chr. 17 7675124 - 7675162
7675064 - 7675095
7674934 - 7674965
7674871 - 7674896
7674215 - 7674263
7673763 - 7673806
Genes Targeted

17
Pre-treatment ~5 months post
▪ Stage I Adenocarcinoma of right lung
▪ Tumor measured 2.9 x 2.5 cm
▪ Had KRAS Gly13Arg mutation
▪ Treated with stereotactic body RT
0 50 100 150
0
10
20
30
40
Undetectable
KRAS Gly13Arg
Days
Mutant
copies
per
mL
measurement
before radiation SBRT
Early Stage Clinical Case – 75 y/o Female

18
Mutant
molecules
per
mL
• Had stage IIIB non-small cell lung cancer.
• Received chemotherapy followed by radiation to the chest.
• Collected blood samples after completing treatment.
• Developed widespread metastases, passed away on day 73.
Narayan et al., Cancer Res. 2012
Clinical Case 2 – 72 y/o Male

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•Patient with metastatic EGFR-mutated lung adenocarcinoma.
•Treated with erlotinib for ~1 year.
•At progression, T790M was found in plasma and Bx.
0 50 100 150 200
0
20
40
60
80
100
Mutant
copies
per
mL
Erlotinib
Undetectable
Days
EGFR Exon 19 Del
EGFR T790M
Liver Bx confirmed
T790M and exon 19 del
CT showed
progression

20
•Presented with locally advanced, unresectable pancreatic cancer.
•Treated with chemo/radiation therapy.
•PET scan after ~9 weeks showed no evidence of disease.
•PET scan after ~3.5 months showed recurrence in liver and pancreas.
0 50 100
0
200
400
600
800
1000
Mutant
copies
per
mL Chemo/RT
Undetectable
Days
Scan 1 Scan 2
KRAS Gly12Leu
9 weeks 3.5 months

21
Can monitoring tumor cell death in real time by
quantifying changes in ctDNA levels enable early
assessment of immunotherapy efficacy for
patients with NSCLC?

22
• Predictive Biomarkers are imperfect (PD-L1 expression, mutation load)
• Immunotherapy can produce unique radiographic response patterns.
• Slow tumor shrinkage
• Prolonged tumor stability without clear response
• Pseudo progression
Hodi et al, JCO, 2016
Challenges in Accessing Immunotherapy Efficacy

23
Metastatic NSCLC
Patients (N = 49)
Patients with mutant
ctDNA detected in
baseline plasma
(N = 28)
Obtain baseline
radiographic
and ctDNA
measurement
Begin
immunotherapy
Collect serial plasma
samples for ctDNA
analysis
(every ~2-3 weeks)
Radiographic
assessment by CT
(every ~6-12 weeks)
Assess for radiographic
response
(RECIST v1.1 criteria)
Assess for ctDNA response
(50% drop from baseline)
Study Outline

24
Transient
spike as
many tumor
cells die
Goldberg, Narayan et al, Clin Cancer Res, 2018
ctDNA Trajectory in Radiographic
Responders vs. Progressors

25
Derived clinical benefit
despite lack of response on
imaging:
• One remained on tx for
386 days until death due
to unrelated event
• One remained on tx until
data cut-off date (>152
days)
ctDNA Decreases Drastically in Radiographic Responders

26
(tumor burden decreased by at least 30%)
(tumor burden increased by at least 20% or new
lesion)
(does not meet criteria for PR or SD)
3 discordant results
ctDNA findings
reflected clinical
course
Correlation of Radiographic and ctDNA Responses

27
10 patients with both
ctDNA and radiographic
response
Timing of Radiographic vs. ctDNA Response

28
10 patients with both ctDNA
and radiographic response
First ctDNA response:
Median 24.5 days
First radiographic response:
Median 72.5 days
Timing of Radiographic vs. ctDNA Response

29
Group Treatment duration (median)
ctDNA Responders (n=14) 205.5 days
ctDNA Non-responders (n=14) 69 days
P < 0.001
ctDNA Responders Derive Longer-term Treatment Benefit

30
ctDNA Decline is Associated with Better PFS and OS

31
Study Summary and Conclusion
Research Question
• Can monitoring tumor cell death
in real time by quantifying
changes in circulating tumor
DNA (ctDNA) levels enable early
assessment of immunotherapy
efficacy for patients with
NSCLC?
Results
• Treatment response with ctDNA
correlates with radiographic
response
• Radiographic responders show
a substantial decrease in ctDNA
levels
• ctDNA response is seen more
rapidly than radiographic
response
• Longer-term treatment benefit is
seen among ctDNA responders
• ctDNA response is associated
with improved progression-free
and overall survival
Conclusion
• A drop in ctDNA level is an early
marker of therapeutic efficacy
and predicts prolonged survival
in patients treated with immune
checkpoint inhibitors for NSCLC

32
Acknowledgements
Lab Members
Abhijit A Patel
Chaitanya Kantak
Trinh Thi
Dennis Vu
Collaborators
Roy Herbst
Roy Decker
Howard Hochster
Sarah Goldberg
Scott Gettinger
Katie Politi
Daniel J. Boffa
Juliane Juergensmeier
Stacey Stein
Bryan Chang
Jill Lacy
Melinda Irwin
Brenda Cartmel
Elena Ratner
Specimen Lab
Rocco Carbone
Cindy Ostravage
Bio-informatics
Nicholas Carriero
Sameet Mehta
Oligo Synthesis
Joseph DeLuca
Next-Gen Sequencing Core
Shrikant Mane

33
Thank You

Key Speaker Riya Gupta
Research Analyst
BIS Research Inc.
Nitish Kumar Singh
Principal Consultant
BIS Research Inc.

Liquid Biopsy:
Trends and Key
Market
Developments

36
Liquid Biopsy: Product Definition
What is Liquid Biopsy:
Liquid biopsy is a non-invasive medical procedure that
holds the ability to detect the presence of molecular cancer
biomarkers in biological fluids. It has a critical role in the
precision medicine approach, as it confirms the safe and
effective application of targeted therapeutics. A liquid biopsy
helps physicians analyze tumor-related information through
a simple blood test. It can be used as a better alternative
for treatment and recurrence monitoring as it overcomes
invasive biopsy challenges of taking a tumor sample again
and again during the course of the treatment.
Advantages of Liquid Biopsy Include Non-invasive, Rapid, Precise, and Real-time
Source: JCO Precision Medicine Journal

37
60.8 90.1
219.3 383.6
2020 2022 2027 2032
$Million
North America Europe
Asia-Pacific Latin America and Middle East
CAGR (2022-2032): 19.83% North
America
Europe
Asia-pacific
Latin
America and
Middle East
Rest-of-the-
World
0.00%
40.00%
80.00%
0.00% 15.00% 30.00%
Market
Share
%
(2021)
CAGR (2022-2032)%
Low
Share
High
Share
Low
Growth
High
Growth
Regional Market Landscape
Current market adoption rates across regions indicate a high-growth market
Source: BIS Research Analysis Source: BIS Research Analysis

38
35.15%
57.55%
5.14%
2.15%
2021
Polymerase Chain Reaction
Next Generation Sequencing
Other Technologies
Emerging Technologies
34.72%
58.31%
4.98%
1.99%
2032
The technologies powering liquid
biopsies are in a state of rapid
evolution to overcome challenges
associated with the utilization of liquid
biopsy tests, including the
improvement of their underlying clinical
utility and validity.
Other Technologies include
ParsorTix Technology, CELLSEARCH
and DEPArray Technology, ISET
Technology, and CancerSEEK
technology, among others
Emerging Technologies include nano
detection technology, CTC iChip
technology, and others
The global liquid biopsy market (by technology) is presently dominated by next-generation sequencing (NGS), with a value of $1,443.5 million, as of 2021.
Growing at a CAGR of 19.96% during the forecast period 2022-2032, the market is anticipated to be dominated by NGS, with an estimated revenue of
$11,118.3 million in 2032.
Technological Landscape
Liquid Biopsy as an Emerging Frontier with Different Technologies
Source: BIS Research Analysis

39
Market Shares > 7%
Market Shares in the Range of 5%-7%
Market Shares < 5%
Leading Players in the Market
The market is dominated by big players

41
Rising Prevalence of Cancer Increased Research Funding
+75.13%
According to ourworldindata.org,
cancer deaths have increased
75.13% between 1990 and 2019
66.7%
The funding on the liquid biopsy
market increased 66.7% between
2019 and 2021
Need for Early Cancer Detection
>12.4%
Prostate cancer has 100% survival
rate in stage I, II and III, which falls
down to 87.6% at stage IV
Factors Propelling the Demand for Emerging Liquid Biopsy Technology in
Multicancer Screening
The Rising Rate of Cancer has been a Key Catalyst in the Growing Demand

42
NSCLC or SCLC 2 Year Survival Rate 5 Year Survival Rate
Stage IA1 97% 90%
Stage IA2 94% 85%
Stage IA3 92% 80%
Stage IB 89% 73%
Stage IIA 82% 65%
Stage IIB 76% 56%
Stage IIIA 65% 41%
Stage IIIB 47% 24%
Stage IIIC 30% 12%
Stage IVA 23% 10%
Stage IVB 10% 0%
Note: NSCLC = Non-Small Cell Lung Cancer and SCLC = Small Cell Lung Cancer
Source: Web-MD
Non-Small Cell Lung Cancer or Small Cell Lung Cancer Survival Rates
Depending on the Stage of Diagnosis
Source: Clinicaltrials.gov
Need for Early Cancer Detection
Early Multicancer Detection With Liquid Biopsy is a Game Changer
There are currently 327 active liquid biopsy clinical trials going on
according to clinicaltrials.gov. They can be classified according to cancer
types as shown in table below.
20%
12%
9%
5%
4%
4%
4%
42%
Lung Cancer
Breast Cancer
Rectal/Colorectal Cancer
Head and Neck Squamous Cell
Carcinoma
Prostate Cancer
Pancreatic Cancer
Ovarian Cancer
Other Cancer Types

43
Increased Research Funding
Funding in Liquid Biopsy and Cancer Research has increased tremendously
Source: National Cancer Institute
514.70
69.40
403.20
155.60
178.50
209.40
43.30
211.60
0.0
100.0
200.0
300.0
400.0
500.0
600.0
Breast
Cancer
Cervical
Cancer
Lung
Cancer
Melanoma Pancreatic
Cancer
Prostate
Cancer
Multiple
Myeloma
Colorectal
Cancer
Total
Investment
($Million)
Research Funding for Different Cancer Types, 2020
Source: Cancer.gov
4,952.6
5,206.2
5,636.4
5,927.7
6,440.4 6,440.4
2015 2016 2017 2018 2019 2020
NCI Fiscal Budget
Year-on-Year Fiscal Budget of NCI, $Million, 2015-2020

Extracellular
Vesicles (EVs)
/Exosomes
Based Liquid
Biopsy

45
Market
Size
and
Growth
Key Trends:
• Exosome Diagnostics Inc. developed an exosome-based liquid biopsy platform called ExoLution, and a point-of-care instrument, Shaky, that
captures protein for analysis. Both technologies assess RNA, DNA and proteins in biofluids to raise the sensitivity of the assay.
• A partnership between the Atlantic Cancer Research Institute, Canada, and New England Peptide, USA., created Excipio Technologies in 2013 to
commercialize the patented Vn96 EV isolation technology that employs the high selectivity of the Vn96 peptide for heat shock proteins that are
present on the surface of exosomes and EVs. This allows EVs to be analyzed for pathological biomarkers associated with cancers.
• Focus on early cancer detection: The ability of EVs to detect cancer at an early stage is an area of research, several companies are focusing on
developing liquid biopsy tests that can detect cancer at its earliest stages, when it is most treatable.
Growing Number of Publications
50.3 62.7
116.9
385.1
2021 2022 2025 2032
Market Size ($ Mn)
EVs Based Liquid Biopsy: Size, Growth, Trends

46 Liquid Biopsy: An Emerging Frontier in Early Multicancer Detection
Key Developments
Company Development
Key Massage: Most of the liquid biopsy tests based on EVs are still in clinical trials and the approval process is ongoing. Additionally, while liquid biopsy tests based
on EVs show promising results, they are not yet widely adopted in clinical practice, and more research is needed to establish their clinical utility and reliability.
In 2022, Biotechne announced the launch of Sjögren's Syndrome diagnostic and monitoring tool by Exosome Diagnositcs that leverages
extracellular vesicles (EVs) to monitor the RNA transcriptome for Sjögren's Syndrome diagnosis and monitoring
In 2021, Biocept, a liquid biopsy company, announced the launch of its new liquid biopsy test for lung cancer using EVs
In 2021, Biotechne announces CE-IVD certification for Exodx prostate test for exosomal liquid biopsy-based diagnostic assays.
In 2020, Natera, a company that develops genetic testing products, acquired Inivata, a liquid biopsy company that develops non-
invasive liquid biopsy tests for cancer using EVs.
In 2020, the FDA granted Breakthrough Device Designation to Biofluidica, a liquid biopsy company that develops EV-based liquid biopsy
tests for cancer.
In 2019, Exosome Diagnostics (Biotechne), a company that develops liquid biopsy tests based on EVs, received clearance from the FDA for
its exosome-based liquid biopsy test for lung cancer
In 2019, the FDA granted Breakthrough Device Designation to Exosome Diagnostics (Biotechne) for its liquid biopsy test for prostate cancer
In 2018, Janssen Biotech, a subsidiary of Johnson & Johnson, acquired Veracyte, a company that develops liquid biopsy tests for thyroid and other
head and neck cancers using EVs.
www.bisresearch.com

97.99% 2.01%
2.01%
Global Liquid Biopsy Market Size in 2021:
$2,508.2 Million
Other Circulating Biomarkers Exosomes and Extracellular Vesicles
(CTC, cf-DNA, cf-RNA)
Major Players Vs Emerging Players
Key Message:
▪ Other emerging companies working on Evs based liquid biopsy include Acuamark Diagnostics, Clara Biotech, Nodality and SomaLogic
▪ Emerging players are working on new or improved methods for isolating and purifying EVs from bodily fluids, developing new assays for analyzing the genetic
material contained within EVs, or seeking new partnerships or funding to support the development and commercialization of their assays.
Emerging Players
▪ Offer nProbe for prostate cancer
▪ nProbe is based on a proprietary isolation
method that can detect and quantify
specific EV subpopulations in urine.
▪ Test is under clinical validation
▪ XCF COMPLETE Exosome and cfDNA
Isolation Kit and ExoBiopsy
▪ Marrketed as RUO
▪ Focusing on developing liquid biopsy
tests for prostate and breast cancer
▪ The product is in Pre-clinical stage
▪ Developing liquid biopsy assays for PoC
settings
▪ Exo-MIR platform uses exosomes as the
source of genetic material for analysis
Major Players
www.bisresearch.com

Major Applications
▪ Oncology is one of the main areas of focus for EVs-based
liquid biopsy, as EVs can contain genetic material that
reflects the state of cancer cells. This makes them a useful
source of information for detecting, diagnosing, and
monitoring cancer.
▪ Researchers are studying the use of EVs-based liquid
biopsy for a variety of cancer types, including lung cancer,
breast cancer, prostate cancer, and others.
▪ EVs-based liquid biopsy is also being studied for other
diseases, such as neurological disorders, cardiovascular
diseases and infectious diseases. For example, in
neurodegenerative disorders such as Alzheimer's disease,
researchers are studying the use of EVs-based liquid
biopsy as a means of detecting and monitoring the disease
in its early stages.
▪ Similarly, in cardiovascular diseases, researchers are
studying the use of EVs-based liquid biopsy as a means of
identifying biomarkers that can be used to predict or
diagnose disease.
Source: Clinical Trial Analysis
28.47%
23.57%
18.65%
13.89%
5.52%
7.74%
2.16%
EVs Liquid Biopsy Adoption
Lung Cancer
Breast Cancer
Prostate Cancer
Colorectal Cancer
Melanoma
Other Types of Cancer
Non-Oncology Disorder
www.bisresearch.com

Diagnostic
Roadblocks in Market Adoption (Diagnostic Vs. RUO)
Isolation and purification of EVs
▪ Isolating and purifying EVs from bodily fluids,
such as blood, can be a challenging task as
EVs are present in low concentrations and
are often highly heterogeneous. Researchers
need to develop efficient and selective
methods for isolating and purifying EVs,
which can be costly and time-consuming.
Standardization
▪ Developing a standardized method for
isolating, purifying and analyzing EVs is
critical for the reproducibility, comparability,
and reliability of the tests. This is a critical but
challenging task as different groups of
researchers may use different methods for
isolating, purifying and analyzing EVs, which
can lead to variability in the results.
Uncertain Reimbursement and Regulatory
Policies
▪ Even if a liquid biopsy assay is approved and
validated, it still needs to be reimbursed by
the health system. Given the high cost of the
technology and the uncertainty surrounding
its clinical utility, this can be a major
challenge to its adoption.
RUO
Lack of consensus on biomarkers:
▪ Another challenge is that there is no consensus on the best biomarkers
to use for liquid biopsy assays. This lack of consensus makes it difficult
to compare results and to develop assays that can be used in different
settings.
Analysis of EVs
▪ Once EVs are isolated and purified, researchers need to develop
methods for analyzing the genetic material within the EVs. This can be
challenging as EVs are small and contain a limited amount of genetic
material. Researchers need to use highly sensitive and specific methods
for analyzing the genetic material within EVs, which can be technically
challenging and expensive.
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50 www.bisresearch.com I All right reserved
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52
Liquid Biopsy: An Emerging Frontier in
Early Multicancer Detection
Speaker Contact Information
Dr. Azeet Narayan
Reach out to him at
azeet.narayan@gmail.com
Nitish Kumar Singh
Reach out to him at
nitish.kumar@bisresearch.com

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