age many cell-free dna (cfdna) mutations are derived from ... · dnmt3a r882c dnmt3a r882h dnmt3a...
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Improved LOD Increases Detection of Clonal Hematopoiesis Variants• NEJM 2014 study (Genovese 2014) identified 217 unique candidate drivers in WES.
- Disruptive (nonsense, frameshift, splice-site) mutations in DNMT3A, TET2, ASXL1 and PPM1D; missense in DNMT3A. - Recurrent (≥ 7 patients) in hematopoietic and lymphoid cancers in COSMIC.
• 212 of 217 previously identified drivers are covered by cfDNA panel.• 37 unique drivers (77 events) were detected in quality filtered WBC variants (total = 1072) among 198 subjects.
Blood(Streck BCT)
Plasma/Buffy CoatSeparation
Optimized single-tube library prepUnique Molecular Identifiers for error suppression
Error suppressionVariant callingAnnotationFiltering
508-gene cancer panel (2.1 Mb)
>60,000x raw target coverage for both cfDNA and gDNA
High-intensity Sequencing
DNA Extraction Library Prep Target Capture Sequencing Analysis
cfDNA assayPlasma cfDNA
WBC gDNA
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
Many cell-free DNA (cfDNA) mutations are derived from clonal hematopoiesis: Implications for interpretation of liquid biopsy tests Pedram Razavi1, Bob T. Li1, Chenlu Hou7, Ronglai Shen2, Oliver Venn7, Raymond S. Lim4, Earl Hubbell7, Ino De Bruijn4, Qinwen Liu7, Ravi Vijaya Satya7, Hui Xu7, Ling Shen7, Amy Sehnert7, Tara Maddala7, Michael F. Berger1, 4, 5, Alex Aravanis7, Jorge S. Reis-Filho4, Mark Lee7, Howard Scher1, 6, David B. Solit1, 3, 5, Jose Baselga1, 3
1. Department of Medicine, Memorial Sloan Kettering Cancer Center (MSKCC), 1275 York Avenue, New York, NY, USA. 2. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center. 3. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center. 4. Department of Pathology, Memorial Sloan Kettering Cancer Center. 5. Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center. 6. Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center. 7. GRAIL, Menlo Park, CA 94402 USA.
Background• A large fraction of cell-free DNA (cfDNA) fragments are
known to be derived from hematopoietic cells (Lui 2002).• Sequencing of genomic DNA (gDNA) from white blood
cells (WBC) in circulation has revealed an age-dependent presence of clones carrying somatic variants—a phenomenon now termed clonal hematopoiesis (Genovese 2014).
• Somatic alterations in cfDNA can be tumor-derived but also could represent somatic changes associated with clonal hematopoiesis.
• We applied a high-intensity sequencing approach (ultra-deep sequencing with broad genomic coverage) to characterize both cfDNA and WBC gDNA from cancer and non-cancer subjects.
Objective• To evaluate the contribution of clonal hematopoiesis to
the variants observed in cfDNA through high-intensity sequencing of both plasma cfDNA and matched WBC gDNA.
Methods: Patient PopulationsCancer Patient Population:• Metastatic breast, lung, or prostate cancer, either de novo
or with progressive disease on current therapy.• All patients have provided written informed consent to
an MSK institutional protocol (NCT01775072) allowing research of cfDNA and clinical tumor sequencing.
• Two tubes of blood collected in Streck Cell-Free DNA BCT.
Healthy Donor Population:• Enrolled through San Diego Blood Bank. • Self reported as non-smokers and cancer-free.• Two tubes of blood collected in Streck Cell-Free DNA BCT.
Methods: AnalysiscfDNA and WBC gDNA variant calling pipeline• The variant calling pipeline includes the following steps:
- Read alignment, error correction (consisting of read collapsing and stitching of mate pairs), de novo assembly, variant calling, and variant filtering.
- Variants are filtered using two approaches (1) heuristics applied based on the surrounding sequence context and type and quality of reads supporting the variant and (2) empirical noise levels observed in a set of healthy samples:
• Blood samples from an independent cohort of 24 non-cancer (self-reported) individuals (vendor sourced), were used for noise modeling for the 151 cancer patients in this study.
• The 47 healthy subjects were processed with a slightly different assay protocol and UMI set and therefore had 20 additional non-cancer subjects used to establish a noise model for that assay protocol.
- Variants are further filtered based on dbSNP presence and restricted to protein coding variants.
Quality filtering on cfDNA and WBC gDNA variant calls• Analyses focus on matched variants in cfDNA and WBC
gDNA meeting the following criteria: - Non-synonymous. - Allele frequency (AF) >= 0.1% and a minimum of 3 supporting reads.
- In order to exclude germline, AF < 15% in cfDNA. - In order to exclude germline, AF < 35% in WBC gDNA. - Indels not in homopolymer >= 3. - Variants must be found in <=10 samples to avoid recurrent artifacts.
- Unique coverage >=500 to exclude low coverage regions.
Statistical analysis of variants by age• A Poisson regression model was used for analysis of the
association between age, cohort, and mutation count (dependent variable).
Patient Disposition
Distribution of Age by CohortHealthy subjects and breast cancer patients tend to be younger than lung cancer and prostate cancer patients
WBC Variants Contain Previously Identified Drivers• Matched variants in cfDNA and WBC contain 77 driver variants (37 unique variants).
- Drivers supported by a median of 12 unique reads in WBC gDNA (3 - 533 ). - Drivers supported by a median of 18 unique reads in cfDNA (3 - 617).
Summary and Conclusions• This high-intensity sequencing approach applied
to both plasma cfDNA and WBC gDNA reveals that many cfDNA variants arise from clonal hematopoiesis:
- The most common variants matched between cfDNA and WBC gDNA are in genes previously implicated in clonal hematopoiesis, including DNMT3A, TET2, PPM1D, and TP53. - As with previous WBC gDNA sequencing studies, the number of cfDNA variants attributable to clonal hematopoiesis increases with age.
• The limit of detection of our sequencing approach suggests that clonal hematopoiesis variants may be present at low allele frequencies and possibly prevalent in more patients than previously reported. This hypothesis will require further evaluation.
• Accurate interpretation of cell-free DNA assays for detection of circulating tumor DNA must account for the confounding signals arising from clonal hematopoiesis, and may require deep sequencing of WBC gDNA (Abstract #11528, Poster #228, Razavi et al).
References
Lui, Y.Y., Chik, K.W., Chiu, R.W., Ho, C.Y., Lam, C.W., Lo, Y.M. (2002). Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem, 48, 421-7.
Genovese, G., Kahler, AK., Handsaker, R.E., Lindberg, J., Rose, S.A., Bakoum, S.F., Chambert, K., Mick, E., Neale, B.M, … McCarroll, S.A. (2014). Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA Sequence. NEnglJMed, 371, 2477-87. https://doi.org/10.1056/NEJMoa1409405
Matched Variants in cfDNA and WBC Are Associated with Age• Positive association with age: p-val <0.0001. • The association of number of matched variants with age does not appear to be different between healthy subjects and those with breast/lung/prostate
cancer in this cohort. Interaction of age with cancer not significant: p = 0.08.
Patient Characteristics
Sample Workflows and Assays
Patient Characteristics (Cancer Characteristics)
Analysis
EnrollmentAssigned pt ID (N = 282)
Breast80
Lung82
Prostate70
EligibleEligibleClinically Eligible and Lab Evaluable (N = 161)
NBreast
53
Lung
53
Prostate
55
EligibleClinically Eligible and Lab Evaluable (N = 211)
Breast53
Lung53
Prostate55
cfDNA Assay Evaluable (N = 198)
Breast48
Lung49
Prostate54
Excluded subjects (N = 71)
Clinical exclusion* Lab exclusions for insufficient sampleTissue or cfDNA assay unevaluable
Breast
27
Lung
29
Prostate
15
** Includes 2 pseudoprogressionsHealthy
50
Healthy50
Healthy
0
9 20* 7 010 8 0 08 1 8 0
Healthy47
cfDNA assay unevaluable (N = 13)Breast
5Lung
4Prostate
1Healthy
3
* Clinical exclusion includes new systemic therapy, disease progression not confirmed on review, etc)
Patient Characteristics Breast (n=48)
Lung (n=49)
Prostate (n=54)
Healthy (n=47)
Age at enrollment
Mean (SD) 57.0 (10.76) 66.1 (10.86) 67.6 (9.55) 57.8 (16.03)
Median 60 67 68 61
Range 30, 79 33, 86 46, 87 20, 78
Gender, N (%)
Female 48 (100.0%) 32 (65.3%) N/A 24 (51.1%)
# of lines of therapy, N (%)
0 22 (45.8%) 28 (57.1%) 18 (33.3%)
1 2 (4.2%) 12 (24.5%) 15 (27.8%)
2 5 (10.4%) 4 (8.2%) 9 (16.7%)
>=3 19 (39.6%) 5 (10.2%) 12 (22.2%)
Receptor Status, N (%)
HR+/HER2+ 3 (6.3%) N/A N/A
HR+/HER2- 34 (70.8%) N/A N/A
HR-/HER2+ 3 (6.3%) N/A N/A
Triple Negative 8 (16.7%) N/A N/A
Histology N (%)
Metastatic Breast Cancer (n=48)
Breast Invasive Ductal Carcinoma 40 (83.3%)
Breast Invasive Lobular Carcinoma 3 (6.3%)
Breast Mixed Ductal and Lobular Carcinoma 5 (10.4%)
Metastatic Lung Cancer (n=49)
Lung Adenocarcinoma 46 (93.9%)
Lung Non-adenocarcinoma 3 (6.1%)
Metastatic Prostate Cancer (n=54)
Prostate Adenocarcinoma 49 (90.7%)
Prostate Neuroendocrine 5 (9.3%)
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
Matched Variants in cfDNA and WBC Per Subject(after Quality Filter)
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
DNMT3A and TET2 Variants are Most Common in Both Healthy Subjects and Cancer Patients
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts * Prior therapy includes:
chemotherapy, hormonal therapy, target therapy delivered for metastatic disease, or prior neoadjuvant/adjuvant therapy
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
sDrivers in
NEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70%
of S
ubje
cts
with
Driv
ers
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts
Variants Found in Both WBC and cfDNA are Preferentially in Previously Described Clonal Hematopoiesis GenesVariants are in genes previously associated with clonal hematopoiesis (Genovese 2014).
20
40
60
80
Healthy Breast Lung Prostate
Age
0
5
10
15
20
Healthy Breast Lung ProstateCohort
Num
ber o
f var
iant
s pe
r sub
ject
Unique Variants per GeneProportion of Subjects
with Variants
RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6RANBP2DNMT3B
NF1EGFR
GRIN2ACBL
ASXL1FAT1
PTPRTATM
CHEK2TP53
PPM1DTET2
DNMT3A
0.0 0.2 0.4 0.6
N 47 48 49 54Cohort
Variants per Gene
TotalPercent in Top 15
1072 99649% 46%
Total VariantsNumber of Subjects
683 195114 37
19447
Cancer: Prior Therapy Cancer: No Prior Therapy Healthy
Previously Implicated Genes
DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0 50 100 150 200DNMT3BGRIN2A
EGFRCBL
FAT1NF1
ASXL1KMT2CPTPRT
ATMCHEK2
TP53PPM1D
TET2DNMT3A
0.0 0.2 0.4
0
2
4
6
8
DN
MT3
A R
882C
DN
MT3
A R
882H
DN
MT3
A R
736H
DN
MT3
A W
860R
DN
MT3
A Y
735C
DN
MT3
A F
755S
DN
MT3
A P
904L
PP
M1D
R57
2*JA
K2
V61
7FD
NM
T3A
R32
6CD
NM
T3A
R73
6CD
NM
T3A
R77
1*D
NM
T3A
V65
7MA
SX
L1 R
634f
sA
SX
L1 Y
591f
sC
BL
R42
0QD
NM
T3A
G54
3CD
NM
T3A
G55
0RD
NM
T3A
I780
TD
NM
T3A
L34
4PD
NM
T3A
M76
1VD
NM
T3A
M88
0VD
NM
T3A
P77
7RD
NM
T3A
Q81
6*D
NM
T3A
R32
0*D
NM
T3A
R59
8*D
NM
T3A
R68
8HD
NM
T3A
R72
9WD
NM
T3A
R79
2HD
NM
T3A
W30
6*D
NM
T3A
W79
5RP
PM
1D C
478f
sP
PM
1D R
458*
SF3
B1
E62
2DS
F3B
1 K
700E
TET2
R55
0*TP
53 R
248Q
# of
Sub
ject
s w
ith D
river
s
ASXL1CBLDNMT3AJAK2PPM1DSF3B1TET2TP53
0
10
20
30
40
50
<50 >50 and <60 >60 and <70 >70
% o
f Sub
ject
s w
ith D
river
s
Drivers inNEJM, at least 10%GRAIL, at least 10%GRAIL, at least 1%GRAIL, at least 0.1%
Pro
porti
on o
f Sub
ject
s w
ith V
aria
nts