0%

20%

40%

60%

80%

Pt1 Pt2 Pt3 Pt4 Pt5

% S

NVs

det

ecte

d in

Plas

ma/

Tum

or Cat

PlasmaTumor

Checkpoint Modulators

Antigen Presenting Machinery

Chemokines

Cytokines

Innate Immune Response

Adaptive Immune Response

0 1 2 3 4Number of Genes

IO genes detected by NeXT LBoutside of targeted panel

panel genes, 17 of which are in immuno-oncology related genes. These observations highlight the extent of somatic evidence observable through an exome-scale cell free approach. In addition to IO pathways, NeXT LB provides other important insights across the entire exome, such as innate and adaptive immune responses.

1334

930

500

1000

NeXT LB Targeted Panels

Num

ber o

f Pla

sma

SNVs

Plasma SNVs detected by differ−ent platforms (Whole−Exome)

31

16

0

10

20

30

NeXT LB Targeted Panels

Num

ber o

f Pla

sma

SNVs

Plasma SNVs detected by differ−ent platforms (IO related genes)

Enhanced Whole Exome Profiling of Tumor Circulating Cell-Free DNA Enables Sensitive Assessment of Tumor MutationsSimo V. Zhang*, Mengyao Tan*, Josette M. Northcott, Shuyuan Ma, Christopher S. Nelson, L. Gordon Bentley, Manju Chinnappa, Devayani P. Bhave, Dan Norton, Jason Harris, Sean M. Boyle, John West, Richard Chen

Personalis, Inc. | 1330 O’Brien Dr., Menlo Park, CA 94025* Co-first author

#1989

MethodsWhole-Exome NeXT LB assay and Silencer

To enable sensitive detection across the exome, we developed an enhanced exome assay and chemistry that augments hard to sequence genomic regions such as regions of high GC content, to enable more uniform coverage across the exome. Additionally, we achieve a high average depth of approximately 2000X for the entire exome, with additional boosted depth (5000X) for 248 clinically relevant oncogenic and tumor suppressor genes to further enhance sensitivity. For analysis, we developed a computational algorithm, Silencer, that enables accurate somatic mutation detection without compromising sensitivity in the plasma. Silencer is empowered by an error suppression model estimated from a panel of normal individual plasma samples, and ad hoc filters including a dedicated blacklist that is tailored to our NeXT LB technology.

Sensitivity Evaluation

Sensitivity of the NeXT LB platform was evaluated using three approaches. First, Horizon reference materials at 5%, 2.5%, and 1% allele frequency (AF) dilutions were evaluated. Second, Seracare reference materials at 2%, 1% and 0.5% AF dilutions were analyzed. Both evaluations used 50ng input and have 3 replicates at each AF dilutions. To further demonstrate our sensitive detection at the whole exome scale, we developed a cell culture media system that models the shed and degraded tumor DNA fragments seen in human plasma samples. Two breast cancer cell lines, HCC1143 and HCC1954, were selected as a set of high-quality goldset SNVs were already established as part of the tissue somatic verification. The two cell lines and their corresponding B-lymphoblast cells were cultured in serum-free media. Cell media was collected after 48-72hr culture, and cfDNA was extracted for the downstream NeXT LB assay. Here, sensitivity is defined as the percentage of detected goldset SNVs.

Clinical Application

Lastly, we demonstrated the power of our NeXT LB technology in providing a much broader view in mutation landscape profiling, tumor-plasma concordance evaluation, and disease monitoring using matched tumor, plasma and matched normal samples from nine head and neck cancer patients (with both pre- and post-treatment samples), as well as five late stage colorectal cancer patients.

Results

100% sensitivity achieved for SNVs with AF≥1%

Our NeXT LB platform successfully and consistently detected all 25/25 (100%) known SNVs across each of the 2% and 1% dilution replicates, and detected 24/25 (96%) events in the majority of the 0.5% dilution Seracare sample replicates. Further, we achieved 100% sensitivity in each of the 5%, 2.5%, and 1% Horizon dilution samples. Additionally, the observed AFs were well correlated with the expected AFs in both of the Seracare and Horizon samples.

Introduction An increasing number of studies have demonstrated the potential use of circulating cell-free DNA (cfDNA) for diagnosis, prognosis, and disease progression monitoring. However, many of these studies utilize assays covering a limited set of well known genes, and therefore can miss biologically and clinically important genetic alterations in DNA repair pathways, immuno-modulatory pathways, mechanisms of resistance and changes in neoantigen status. To address this, we have developed a whole-exome scale cfDNA platform, NeXT Liquid Biopsy, that enables sensitive detection and tracking of mutations in approximately 20000 genes. The NeXT LB platform sequences a triplet of tumor, normal, and plasma samples from a patient, allowing 1) discovery of novel genetic mutations, 2) non-invasively close monitoring of clonal and subclonal evolution in response to treatment of curative intent, and 3) broad interrogation of inter- and intra-tumor heterogeneity.

NeXT LB detected a substantial number of distinct variants outside of the footprint of targeted panels in a head and neck cancer patient cohort

NeXT LB technology was applied to profile somatic events in a cohort of 9 head and neck cancer patients. NeXT LB detected 1,334 plasma SNVs across the platform, 31 of which were in immuno-oncology related genes. Application of our exome-scale approach detected 1,241 somatic events which are outside of commercially available targeted cfDNA

High sensitivity acheived at the whole-exome scale using cell media system

Our internally developed cell media system is whole-exome scale and built upon well-characterized cancer cell lines, providing a far broader assessment of accuracy than the commercially available reference standards. Using this system to measure accuracy across 137 (HCC1143) and 185 (HCC1954) variants, our NeXT LB platform achieved >95% sensitivity for variants with AF>=2%, and between 85% to 92% sensitivity for mutations with AF between 1% to 2%.

Figure 1: NeXT LB technology sensitivity with Seracare (A) and Horizon (B) ctDNA reference standards.

ConclusionWe have developed a whole-exome scale NeXT LB technology that enables sensitive monitoring and detection of somatic SNVs from cfDNA. The NeXT LB platform generates a much broader view of the tumor mutational landscape from the plasma than typical targeted liquid biopsy platforms. The platform enables broader monitoring of changes in response to cancer therapy, ac-quired mechanisms of drug resistance, and intra- and inter-tumor heterogeneity.

simo.zhang@personalis.commengyao.tan@personalis.com

Contact:

Non-invasively profiling tumor heterogeneity and clinical relevant somatic mutations on NeXT LB

We next applied NeXT LB to assess tumor-plasma concordance and cfDNA cancer driver mutations in two independent colorectal cancer and head and neck cancer cohorts. NeXT LB consistently detected both tumor concordant and plasma distinct mutations from cancer patient samples, both of which have implications for non-invasive monitoring of patient response to treatment and profiling tumor heterogeneity. Further, we observed dynamic allele frequency changes in cancer driver genes in checkpoint blockade non-responders, detecting increasing allele frequency over the course of therapy, highlighting NeXT LB’s utility in non-invasive response monitoring.

101 63121 70 4574 83 2796

187 3023 198 2934

Figure 2: NeXT LB technology whole-exome scale sensitivity with HCC1143 (A) and HCC1954 (B) breast cancer cell media system.

Figure 3: NeXT LB technology detects significantly more SNVs at whole-exome (A), ImmunoOncology related genes (B), and ImmunoOncology related pathways (C) outside of footprint of targeted panels.

Figure 4: The number (A) and the percentage (B) of tumor-plasma concordant mutations identified from 5 late-stage colorectal cancer patients.

Colorectal Cancer Patients

Patient 4 Patient 5

Patient 1 Patient 2 Patient 3

Head and Neck Cancer Patients

A B

A

BPatient 12: non-responder

Figure 5: Frequently mutated genes in HNSCC, such as TP53, GNAQ, HRAS were also detected in plasma samples (A). Patient 12 who is defined as non-responder clinically also demonstrated increased AF of HRAS mutation post treatment (B).

AF0.5% AF1% AF2%

1:115256529:T:C1:43815009:G:T10:43617416:T:C12:25398284:C:T13:28592642:C:A14:105246551:C:T17:7577120:C:T17:7577538:C:T17:7578406:C:T19:3118942:A:T2:209113113:G:A20:57484420:C:T3:138665163:G:C3:178936091:G:A3:178952085:A:G3:41266124:A:G4:1803568:C:G4:55152093:A:T4:55599321:A:T5:112175639:C:T7:140453136:A:T7:55249071:C:T7:55259515:T:G9:5073770:G:T9:80409488:T:G

Dilution

AF_Spectrum

0

0.01

0.02

0.03

AF0p5_50ng_

1

AF0p5_50ng_

2

AF0p5_50ng_

3

AF1_50ng_

1

AF1_50ng_

2

AF1_50ng_

3

AF2_50ng_

1

AF2_50ng_

2

AF2_50ng_

3

NRAS/CSDE1

MPL

RET

KRAS

FLT3

AKT1

TP53

TP53

TP53

GNA11

IDH1

GNAS

FOXL2

PIK3CA

PIK3CA

CTNNB1

FGFR3

PDGFRA

KIT

APC

BRAF

EGFR

EGFR

JAK2

GNAQ

AF S

pect

rum

0%

0.5%

1%

2%

AF1% AF2.5% AF5%

1:115256530:G:T

1:115256536:C:T

12:25398284:C:T

3:178936091:G:A

7:55249071:C:T

7:55259515:T:G

Dilution

AF_Spectrum

0

0.02

0.04

0.06

HD_AF1_50ng_

1

HD_AF1_50ng_

2

HD_AF1_50ng_

3

HD_AF2p5_50ng_

1

HD_AF2p5_50ng_

2

HD_AF2p5_50ng_

3

HD_AF5_50ng_

1

HD_AF5_50ng_

2

HD_AF5_50ng_

3

NRAS

NRAS

KRAS

PIK3CA

EGFR

EGFR

AF S

pect

rum

0%

1%

2.5%

5%

A B C

A

B

Tumor SNVsPlasma SNVs

● ● ● ● ●

●● ●

● ● ●

0.750.800.850.900.951.00

0.5_1pct

0.5_5pct

1_2pct

1_5pct

2_3pct

2_5pct

3_4pct

3_5pct

4_5pct

5_10pct

AF bins

Sens

itivi

ty

HCC1954

● ●● ● ●

●●

● ● ● ●

0.750.800.850.900.951.00

0.5_1pct

0.5_5pct

1_2pct

1_5pct

2_3pct

2_5pct

3_4pct

3_5pct

4_5pct

5_10pct

AF bins

Sens

itivi

ty

HCC1143A B

Tumor-Pre Plasma-Pre Plasma-Post

0

302

NA

NA

TRIM51TMEM37

POTEELILRA4

KRTAP10−11EEF1A1

ARHGAP23APOBRAGAP6

ZNF814ZNF705G

PPM1EPDE4DIP

MUC12HRAS

CYP21A2CD163L1NBPF10

GNAQTP53

12%12%12%12%12%12%12%12%12%18%18%18%18%18%18%18%18%24%24%35%

0001

_pre

0001

_pos

t

0002

_pre

0002

_pos

t

0008

_pre

0008

_pos

t

0009

_pre

0009

_pos

t

0011

_pre

0011

_pos

t

0012

_pre

0012

_pos

t

0013

_pre

0013

_pos

t

7140

_pre

0014

_pre

0014

_pos

t

0 6

Mutational landscape detected in plasma samples

0.764

0.05

0.032

HRAS:p.Gly13Arg

ANKRD18B:p.Gly294Arg

BTN2A2:p.Gln426Pro

0.015 0.058

0.04 0.045

NA 0.009

Tumor AF

00.20.40.60.8

Plasma AF

00.020.040.06

Missense_MutationNonsense_MutationMulti_Hit