Profiling tumor circulating cell-free DNA with an enhanced whole-exome to enable sensitive assessment of somatic mutationsSimo V. Zhang*, Mengyao Tan*, Fabio C. P. Navarro*, Josette M. Northcott, Shuyuan Ma, Christopher S. Nelson, Devayani P. Bhave, L. Gordon Bentley, Manju Chinnappa, Dan Norton, Gabor Bartha, Jason Harris, John Lyle, Sean M. Boyle, John West, Richard Chen
Personalis, Inc. | 1330 O’Brien Dr., Menlo Park, CA 94025* Co-first author
#29
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 247 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 built from a panel of normal individual plasma samples, and custom filters including a dedicated blacklist that is tailored to our NeXT LB technology.
Results100% sensitivity achieved for SNVs with AF≥1%Our NeXT LB platform successfully and consistently detected all 25/25 (100%) known SNVs across all 2% and 1% dilution replicates, and missed only one or two events (94.6% on average) in the 0.5% dilution SeraCare® samples. We also achieved 100% sensitivity in 5%, 2.5%, and 1% Horizon dilution samples. The observed AFs in SeraCare and Horizon sample are well correlated with the expected AFs.
Introduction The analysis of tumors using circulating cell-free DNA (cfDNA) is beginning to transform cancer diagnosis, prognosis, response to therapy and to enable disease progression monitoring. Most cfDNA assays are centered around the identification of therapeutically actionable mutations and only cover a limited number of genes, and as such cannot inform on all genetic alterations present in the tumors. 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 20,000 genes. The NeXT LB platform generates comprehensive sequencing data derived from the cfDNA (plasma), the tumor (e.g. FFPE) and the patient’s germline (e.g. blood cells), and therefore enable to closely discover and monitor clonal and subclonal evolution of mutations in plasma, and novel genetic mutations following surgery, and/or treatment therapies.
Our NeXT LB platform detected 1334 somatic SNVs in a cohort of 9 head and neck cancer plasma samples, 31 of which were in clinically-relevant immuno-oncology genes. The amount of detected plasma SNVs would be significantly less if intersecting with genes included across all commerically available targeted panels. With NeXT LB, we could gain additional insights on IO pathways, such as innate and adaptive immune responses.
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. We developed cell-free media samples from two breast cancer cell lines, HCC1143 and HCC1954, from which curated sets of high-quality known somatic mutations. As demonstrated in Figure 3B and 3C, 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% across co-culture cell media dilutions.
Figure 2: NeXT LB technology sensitivity with Seracare (A) and Horizon (B) cfDNA reference standards.
ConclusionWe have developed a whole-exome scale NeXT LB technology that enables sensitive monitoring and novel detection of so-matic SNVs from cfDNA. The NeXT LB platform generates a much broader view of the tumor mutational landscape from the plasma than typical commercially available targeted-based liquid biopsy platforms. The platform enables broader monitor-ing of changes in response to cancer therapy, mechanisms of drug resistance, and intra- and inter-tumor heterogeneity.
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We next applied NeXT LB to assess tumor-plasma concordance and cfDNA cancer driver mutations in two independent col-orectal cancer and head and neck cancer cohorts. NeXT LB consistently detected both tumor concordant and plasma dis-tinct mutations from cancer patient samples, both of which have implications for non-invasive monitoring of patient re-sponse 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 3: NeXT LB technology whole-exome scale sensitivity with HCC1143 (A) and HCC1954 (B) breast cancer cell media system.
Figure 4: NeXT LB technology detects significantly more SNVs at whole-exome (A) or ImmunoOncology related genes (B, C) outside of footprint of targeted panels.
Figure 5: 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 B
A
BPatient 12: non-responder
Figure 6:(A)-(B) Frequently mutated genes in HNSCC, such as TP53, GNAQ, HRAS were also detected in plasma samples. Patient 12 who is defined as non-responder by clinical measurement also demonstrated increased AF of HRAS mutation post treatment.
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%
1334
930
500
1000
NeXT LB Targeted Panels
Nu
mb
er o
f P
lasm
a SN
Vs
Plasma SNVs detected by different platforms(Whole-Exome)
A
31
16
0
10
20
30
NeXT LB Targeted Panels
Nu
mb
er
of
Pla
sma
SNV
s
Plasma SNVs detected by different platforms(ImmunoOncology related genes)
B
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
C
A
B
●●
● ● ●
●
●
● ●● ●
0.75
0.80
0.85
0.90
0.95
1.00
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
HCC1143
● ●●
● ●
●
● ●
●●
●
0.75
0.80
0.85
0.90
0.95
1.00
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
Tumor SNVsPlasma SNVs
Figure 1: The NeXT LB technology complements the ImmunoID NeXT platform by surveying the mutation landscape and immune status at baseline, as well as the following timepoints.
NeXT LB detected more SNVs outside of footprint of targeted panels
Non-invasively profiling tumor heterogeneity and clinical relevant somatic mutations
Whole-exome scale high sensitivity achieved using cell media
0%
20%
40%
60%
80%
Pt1 Pt2 Pt3 Pt4 Pt5
% S
NVs
det
ecte
d in
Plas
ma/
Tum
or
PlasmaTumor
ImmunoID NeXT Platform®
T=0 T=1 T=2 T=N
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
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
Missense_MutationNonsense_Mutation
Multi_Hit
Mutational landscape detected in plasma samples
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