The role of ctDNA in drug
development across solid tumors

Alexey Aleshin, MD, MBA
June 12th, 2021

AgendaMRD testing technologies
Clinical dataApplications in clinical development
Closing thoughts2

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ctDNA MRD Testing is focused on residual disease detection
and applications in cancer monitoring
Uses of ctDNA testing

Asymptomaticcancer screening

Molecular residual disease (MRD) status
Surveillance for early recurrencemonitoring
Treatment response monitoring3

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Cancertherapy selection

Signatera FDA designation and Medicare coverage
BreakthroughDevice3 breakthrough device
designations by the FDA

Final coverage for colorectal
cancer (CRC)

Draft immunotherapy
coverage

May 2019, March 2021

September 2020

September 2020

• As a lab-developed test (LDT),●

Signatera does not need FDA
approval for clinical use• Breakthrough helps clear
regulatory pathway to support
biopharma studies

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Finalized a local coveragedetermination (LCD) to provide
Medicare benefits for serial use of
Signatera in patients with stage IIor III CRC●Draft LCD proposes coverage ofSignatera for immunotherapy
response monitoring in allclinically validated solid tumors

Why personalized and tumor-informed?
Limit ofdetection

TechniqueApproach

Candidate gene
analysisqPCR, dPCR, ddPCR

Single-locus or
multiplex assays

Tumor-informed NGS
CAPP-seq, PCM assay,
Signatera™️, RaDaRⓇ

Personalized,multiplex PCRtargets<0.01% to 1.0%

Tumor-naïve NGS
SAFE-SeqS, TEC-seq,
Guardant360Ⓡ,
FoundationOneⓇLiquid

Panel-based,targetedsequencing

0.01% to >0.1%

0.01% to 1%

Advantages/Limitations• Can only query small number of specific variants ormutations concurrently• Only able to monitor known mutations• Highly sensitive and specific to detect small traces of ctDNA• Quantitative measurement, ideal for monitoring MRD over time
• Requires WES of tumor tissue to design personalized assay• Does not require a priori knowledge of the molecular alteration• Not designed for monitoring MRD in solid tumors

Sources: 1. Siravegna G, Marsoni S, Siena S. et al. Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol 2017;14:531–548 (2017). https://doi.org/10.1038/nrclinonc.2017. 2. Chin RI, Chen K. Usmani A, et al. Detection of Solid TumorMolecular Residual Disease (MRD) Using Circulating Tumor DNA (ctDNA). Mol Diagn Ther 2019;23:311–331. https://doi.org/10.1007/s40291-019-00390-5. 3. Abbosh C, et al. Phylogenetic tracking and minimal residual disease detection using ctDNA in earlystage NSCLC: A lung TRACERx study. AACR Annual Meeting 2020 Virtual Meeting I, CT023. 4. Marsico G, et al. Analytical development of the RaDaR assay, a highly sensitive and specific assay for the monitoring of minimal residual disease. AACR AnnualMeeting 2020 Virtual Meeting, Poster no. 3097.

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Signatera™ residual disease test (MRD)
The personalized and tumor-informed approach

Sequence tumor tissue
to identify unique signatureof tumor mutations

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Custom design and manufacture
personalized mPCR assay foreach patient, targeting top clonal
mutations found in tumor

Use personalized assay to test
patient’s blood for presence ofcirculating tumor DNA (ctDNA)

What is needed in an AV study| High sensitivity, high specificity• Sensitivity of at least 0.01% VAF

Proportion of positive results (PPR)
for estimating analytical sensitivity• LOD confirmation with clinical samples• Specificity needs to be > 99%• Well designed AV studies• Consistent performance across multiple
patient samples

Intended MTM
per mL plasma

IntendedVAF

Empirical PPR
pos/[pos+neg]

0.15

0.0075%72/76 (94.7%)

0.2

0.0100%75/76 (98.7%)

0.3

0.0150%76/76 (100%)0.4

0.0200%76/76 (100%)

Source: Natera internal data from Signatera analytical validation

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Optimizing sensitivity and specificity: a delicate balance
Expected sample sensitivity andspecificity by variants tracked• Adding more targets above 16:– Negative impact to specificity– Unclear impact to sensitivity– Higher failure rate in certain
histologies• 16 ensures stable algorithm andworkflow across histologies– Variable targets per patientcomplicates development,validation and commercialization

1. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545(7655):446-451. 2. Natera internal analysis.

8Not for reproduction or further distribution.

High performance and consistent results across multiple tumor
types

Positive Signatera result, without further treatment, has predicted relapse with overall PPV >98%.1-4
1. Reinert T, Henriksen TV, Christensen E, et al. Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer. JAMA Oncol. 2019. 2. Coombes RC, Page K, Salari R, et al. Personalized Detection of Circulating Tumor DNAAntedates Breast Cancer Metastatic Recurrence. Clin Cancer Res. 2019;25(14):4255-4263. 3. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545(7655):446-451. 4. Christensen E, BirkenkampDemtroder K, Sethi H, et al. Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma. J Clin Oncol. 2019;37(18):1547-1557.

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Decrease from baseline or clearance of ctDNA is predictiveof outcomes
INSPIRE Trial

94 patients with various solid tumors in the Phase IIINSPIRE trial treated with single agent pembrolizumab(200 mg IV q3W).

98%of patients had detectablebaseline ctDNA

1. Bratman SV, Yang SMC, lafolla MAJ, et al. Personalized circulating tumor DNA analysis as a predictive biomarker in solid tumor patients treated with pembrolizumab. Nat Cancer. 2020; 1:873-881. doi:10.1038/s43018-020-0096-5.

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IMvigor010 data demonstrated the predictive power ofSignatera ctDNA for treatment benefit
809 patients in IMvigor010 ITT population• Atezolizumab (n=406)• Observation (n=403)
190 with insufficient tumour, matchednormal or plasma sample

Key results
41% increase in OS benefit for Signatera ctDNA-positive
patients treated with atezolizumab, while no treatment
benefit was observed in the ctDNA-negative population
1.00

4 failed tumour library prep

3 failed quality control checks
581 ctDNA-evaluable patients(72% of ITT population)Observation (n=281)
ctDNA(+): 98 (35%)
ctDNA(–): 183 (65%)

Atezolizumab (n=300)
ctDNA(+): 116 (39%)
ctDNA(–): 184 (61%)

ctDNA-negative (63%)

Overall survival

31 did not have C1D1 data

0.75

HR, 1.31
P=0.320.50

ctDNA-positive: (37%)
HR, 0.59
P=0.0059

0.25▬ ▬ Atezolizumab▬ ▬ Observation
0.00

MIBC = muscle invasive bladder cancer

1. Powles T, Assaf ZJ, Davarpanah N, et al. Clinical outcomes in ctDNA-positive urothelial carcinoma patients treated with adjuvant immunotherapy. Nature. 2021. (Accepted)

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0102030Months

4050

ctDNA clearance was associated with improved outcomes in theatezolizumab arm
ctDNA clearance at C3D1 occurs at
higher rate in treatment arm vs. obs

ctDNA clearance associated with improved
DFS and OS outcomes in the treatment arm
Atezolizumab arm (DFS)1.00

Overall survival

0.75

200.50100.25

0POS ➔ NEG

ctDNA(+) → (‒) (n=18)
ctDNA(+) → (+) (n=81)

0.00
Atezolizumab Observation
n=99
n=7918 (18.8%)

3 (3.8%)010

2030Months

Assessed using Fisher exact test.
1. Powles T, Assaf ZJ, Davarpanah N, et al. Clinical outcomes in ctDNA-positive urothelial carcinoma patients treated with adjuvant immunotherapy. Nature. 2021. (Accepted)

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Atezolizumab arm (OS)HR, 0.26 (95% CI: 0.12, 0.56)

Disease-free survival

Signatera-POS patients (%)

P=0.004

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4050

1.00
0.75
0.50

0.25

HR, 0.41 (95% CI: 0.10, 1.70)

0.00

ctDNA(+) → (‒) (n=18)
ctDNA(+) → (+) (n=81)0102030

Months4050

IMvigor011 | Adjuvant atezolizumab vs placebo in MIBC patients
who are ctDNA positive following cystectomy
Postcystectomy forhigh-risk
urothelialcancer

20 weeks
Positive

Serial ctDNA testing

Negative

PFS
Primary endpoint

atezolizumab
Rplacebo

1. NCT04660344. Clinicaltrials.gov. Accessed April 27, 2021.

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OS
2nd endpoint

CIRCULATE Japan| Escalation and de-escalation in Stage II/III CRC
CIRCULATE Japan study schema

1. Yukami H, Mishima S, Kotani D, et al. Trial in progress: Prospective Observational Study Monitoring Circulating Tumor DNA in Resectable Colorectal Cancer Patients Undergoing Radical Surgery: GALAXY Study in CIRCULATE-Japan. Poster (P-120) presented at:
ESMO: GI – World Congress on Gastrointestinal Cancer. July 1-4, 2020; Virtual Meeting.

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Signatera ctDNA detection rates pre- and post-surgery

Yukami et al
ASCO 2021*History of chemotherapy prior to surgery within 6 months

Across multiple studies in CRC, Signatera has shown to have pre-surgical detection 89-94%1-2
1. Reinert T, Henriksen TV, Christensen E, et al. Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer. JAMA Oncol. 2019. 2. CIRCULATE data on file.

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Prospective trials to assess association of MRD-positivity withtreatment response
Trial/ NCT#MERMAID-1
NCT04385368
MERMAID-2
NCT04642469
CATHAYA
NCT04611776
IMvigor011
NCT04660344
ISPY-2
NCT01042379
c-TRAK-TN
NCT03145961
LEADER
NCT03285412
DARE
NCT04567420
CIRCULATEJapan
UMIN000039205
MGH 18-397
NCT03803553
COBRA
NCT04068103
TAPISTRY
NCT04589845
NCT04510285

Stage / Tumor type

Description

Stage II/III NSCLC

Durvalumab + chemo vs chemo for MRD+ after resection

3

Stage II/III NSCLC

Durvalumab vs placebo for MRD+ after resection and possible neoadj/adjuv treatment

3

Stage IB-IIIB NSCLC

Atezolizumab + chemo vs placebo + chemo for MRD+ after resection

2

Muscle-invasive bladder cancer

Atezolizumab vs placebo for MRD+ after resection

3

Neoadjuvant breast cancer

ctDNA dyanamics pre- and post-neoadjuvant chemotherapy prior to resection

2High risk, early stage TNBC

Pembrolizumab vs placebo for MRD+ after resection and possible neoadj/adjuv tx

2Ribociclib + endocrine therapy vs endocrine therapy for MRD+ patients after possible neoadj/adjuv tx

2

Palbociclib + fulvestrant vs SoC endocrine tx for MRD+ patients treated with adjuvant aromatase inhibitor/tamoxifen

2Stage II-III CRC

Treatment escalation with experimental therapies in MRD+ patients after surgery, and treatment de-escalation (no chemo)
in MRD- patients after surgery

3

Stage III CRC

Nivolumab, Encorafenib/Binimetinib/Cetuximab, FOLFIRI, or active surveillance as appropriate for MRD+

3

Stage IIA colon cancer

FOLFOX6 or CAPOX vs surveillance for resected MRD+

2/3

Stage I-III breast cancer(ER+, HER2-)
Stage II-III breast cancer(ER+, HER2-)

TMB-high, advanced solid
TKIs vs atezolizumab vs ipatasertib vs trastuzumab vs idasanutlin vs GDC-0077
tumors
HER2+ esophagogastric tumors Trastuzumab + placebo vs trastuzumab + pembrolizumab in MRD+Sources: 1. Clinicaltrials.gov. Accessed April 27, 2021; 2. Yukami H, Mishima S, Kotani D, et al. Trial in progress: Prospective Observational Study Monitoring Circulating Tumor DNA in Resectable Colorectal Cancer Patients Undergoing Radical Surgery: GALAXY Studyin CIRCULATE-Japan. Poster (P-120) presented at: ESMO: GI – World Congress on Gastrointestinal Cancer. July 1-4, 2020; Virtual Meeting.

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Phase22

Using ctDNA to bring life-saving therapeutics to patients❖ Clinical studies and data shows strong correlation for quantification of ctDNA levels
and clinical outcomes❖ ctDNA can serve as a biomarker in various manners including prognostic, predictive,monitoring, and treatment response❖ ctDNA status has the potential to be used as:• A surrogate endpoint of treatment efficacy to accelerate clinical trial results• A stratification method to identify the subset of patients who may still benefit fromtherapies studied in trials that failed to meet their primary endpoint17

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Open questions| Guidance needed from FDA / PMDA
❖ Pathway for treatment-related ctDNA dynamics to become a surrogate endpoint for
drug approvals❖ Pathway for updating label based on MRD data using retrospective banked samples• Complementing other surrogate endpoints with ctDNA, or work through regulatory
paradigms like accelerated approvals?

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Thank you!
Questionsaaleshin@natera.com

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The test described has been developed and its performance characteristics determined by the CLIA-certified laboratory performing the test. The test has notbeen cleared or approved by the US Food and Drug Administration (FDA). Although FDA is exercising enforcement discretion of premarket review and otherregulations for laboratory-developed tests in the US, certification of the laboratory is required under CLIA to ensure the quality and validity of the tests. CAP
accredited, ISO 13485 certified, and CLIA certified. © 2021 Natera, Inc. All Rights Reserved.
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