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Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes.
Jaiswal, Siddhartha; Fontanillas, Pierre; Flannick, Jason; Manning, Alisa; Grauman, Peter V;
Mar, Brenton G; Lindsley, R Coleman; Mermel, Craig H; Burtt, Noel; Chavez, Alejandro;
Higgins, John M; Moltchanov, Vladislav; Kuo, Frank C; Kluk, Michael J; Henderson, Brian;
Kinnunen, Leena; Koistinen, Heikki A; Ladenvall, Claes; Getz, Gad; Correa, Adolfo; Banahan,
Benjamin F; Gabriel, Stacey; Kathiresan, Sekar; Stringham, Heather M; McCarthy, Mark I;
Boehnke, Michael; Tuomilehto, Jaakko; Haiman, Christopher; Groop, Leif; Atzmon, Gil;
Wilson, James G; Neuberg, Donna; Altshuler, David; Ebert, Benjamin L
Published in:
New England Journal of Medicine
DOI:
10.1056/NEJMoa1408617
2014
Link to publication
Citation for published version (APA):
Jaiswal, S., Fontanillas, P., Flannick, J., Manning, A., Grauman, P. V., Mar, B. G., Lindsley, R. C., Mermel, C. H.,
Burtt, N., Chavez, A., Higgins, J. M., Moltchanov, V., Kuo, F. C., Kluk, M. J., Henderson, B., Kinnunen, L.,
Koistinen, H. A., Ladenvall, C., Getz, G., ... Ebert, B. L. (2014). Age-Related Clonal Hematopoiesis Associated
with Adverse Outcomes.
New England Journal of Medicine
,
371
(26), 2488-2498.
https://doi.org/10.1056/NEJMoa1408617
Total number of authors:
34
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original article
The
new england journal
of
medicine
n engl j med 371;26 nejm.org december 25, 2014
2488
Age-Related Clonal Hematopoiesis
Associated with Adverse Outcomes
Siddhartha Jaiswal, M.D., Ph.D., Pierre Fontanillas, Ph.D., Jason Flannick, Ph.D.,
Alisa Manning, Ph.D., Peter V. Grauman, B.A., Brenton G. Mar, M.D., Ph.D.,
R. Coleman Lindsley, M.D., Ph.D., Craig H. Mermel, M.D., Ph.D., Noel Burtt, B.S.,
Alejandro Chavez, M.D., Ph.D., John M. Higgins, M.D., Vladislav Moltchanov, Ph.D.,
Frank C. Kuo, M.D., Ph.D., Michael J. Kluk, M.D., Ph.D., Brian Henderson, M.D.,
Leena Kinnunen M.Sc., Heikki A. Koistinen, M.D., Ph.D., Claes Ladenvall, Ph.D.,
Gad Getz, Ph.D., Adolfo Correa, M.D., Ph.D., Benjamin F. Banahan, Ph.D.,
Stacey Gabriel, Ph.D., Sekar Kathiresan, M.D., Heather M. Stringham, Ph.D.,
Mark I. McCarthy, M.D.,* Michael Boehnke, Ph.D.,* Jaakko Tuomilehto, M.D., Ph.D.,
Christopher Haiman, Sc.D., Leif Groop, M.D., Ph.D., Gil Atzmon, Ph.D.,
James G. Wilson, M.D., Donna Neuberg, Sc.D., David Altshuler, M.D., Ph.D.,*
and Benjamin L. Ebert, M.D., Ph.D.†
The authors’ affiliations are listed in the
Appendix. Address reprint requests to
Dr. Ebert at the Department of Medicine,
Division of Hematology, Brigham and
Women’s Hospital, Harvard Medical
School, 1 Blackfan Cir., Karp 5.210, Boston,
MA 02115, or at bebert@partners.org.
* Dr. McCarthy is listed on behalf of the
Type 2 Diabetes (T2D) Genetic Explora-
tion by Next-Generation Sequencing in
Multi-Ethnic Samples (T2D-GENES)
study investigators; Dr. Boehnke, on be-
half of the Genetics of Type 2 Diabetes
(GoT2D) study investigators; and Dr.
Altshuler, on behalf of the SIGMA T2D
study investigators.
† A complete list of investigators in the
T2D-GENES, GoT2D, and SIGMA T2D
studies is provided in the Supplemen-
tary Appendix, available at NEJM.org.
This article was published on November 26,
2014, at NEJM.org.
N Engl J Med 2014;371:2488-98.
DOI: 10.1056/NEJMoa1408617
Copyright © 2014 Massachusetts Medical Society.
ABSTRACT
Background
The incidence of hematologic cancers increases with age. These cancers are associ-
ated with recurrent somatic mutations in specific genes. We hypothesized that such
mutations would be detectable in the blood of some persons who are not known to
have hematologic disorders.
Methods
We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells
of 17,182 persons who were unselected for hematologic phenotypes. We looked for
somatic mutations by identifying previously characterized single-nucleotide vari-
ants and small insertions or deletions in 160 genes that are recurrently mutated in
hematologic cancers. The presence of mutations was analyzed for an association
with hematologic phenotypes, survival, and cardiovascular events.
Results
Detectable somatic mutations were rare in persons younger than 40 years of age but
rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to
89 years of age, and 90 to 108 years of age, these clonal mutations were observed
in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respec-
tively. The majority of the variants occurred in three genes: DNMT3A, TET2, and
ASXL1. The presence of a somatic mutation was associated with an increase in the
risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to
32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and
increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI,
1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8).
Conclusions
Age-related clonal hematopoiesis is a common condition that is associated with
increases in the risk of hematologic cancer and in all-cause mortality, with the lat-
ter possibly due to an increased risk of cardiovascular disease. (Funded by the Na-
tional Institutes of Health and others.)
The New England Journal of Medicine
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Age-Related Clonal Hematopoiesis and Adverse Outcomes
n engl j med 371;26 nejm.org december 25, 2014
2489
C
ancer is thought to arise through
the stepwise acquisition of genetic or epi-
genetic changes that transform a normal
cell.
1
Hence, the existence of a premalignant state
bearing only the initiating lesions may be detect-
able in some persons who have no other signs of
disease. For example, multiple myeloma is fre-
quently preceded by monoclonal gammopathy of
unknown significance,
2
and chronic lymphocytic
leukemia is commonly preceded by monoclonal
B-cell lymphocytosis.
3
Several lines of evidence have suggested that
clonal hematopoiesis resulting from an expansion
of cells that harbor an initiating driver mutation
might be an aspect of the aging hematopoietic
system. Clonal hematopoiesis in the elderly was
first demonstrated in studies that showed that ap-
proximately 25% of healthy women over the age of
65 years have a skewed pattern of X-chromosome
inactivation in peripheral-blood cells,
4,5
which in
some cases is associated with mutations in TET2.
6
Large-scale somatic events such as chromosomal
insertions and deletions (indels) and loss of het-
erozygosity also occur in the blood of approxi-
mately 2% of persons older than 75 years of age.
7,8
Preleukemic hematopoietic stem cells harboring
only the initiating driver mutation have been found
in the bone marrow of patients with acute my-
eloid leukemia (AML) that is in remission.
9-11
Sequencing studies have identified a set of re-
current mutations in several types of hematologic
cancers.
12-24
However, the frequency of these so-
matic mutations in the general population is un-
known. We tested the hypothesis that somatically
acquired single-nucleotide variants (SNVs) and
small indels might be detectable in the blood of
older persons who are not known to have any
hematologic abnormalities.
Methods
Sample Ascertainment
The study sample was selected from 22 popula-
tion-based cohorts in three consortia (see Table S1
in the Supplementary Appendix, available with
the full text of this article at NEJM.org). The pro-
tocols for these studies were approved by the eth-
ics committees at all involved institutions; written
informed consent was obtained from all partici-
pants. Persons with missing data on age (116 per-
sons) or with cell lines as the source of DNA (492
persons) were excluded.
Whole-Exome Sequencing and Targeted
Amplicon Sequencing
DNA was obtained from individual cohorts, and
further processing was performed at the Broad
Institute of Harvard and the Massachusetts Insti-
tute of Technology. In brief, genomic DNA was
subject to hybrid capture, sequencing, and align-
ment with the use of the Broad genomics platform
and Picard pipeline. We analyzed BAM files for
SNVs using MuTect with OxoG filtering and for
indels using Indelocator.
25,26
A clinically validated,
targeted amplicon assay was used for sequencing
95 genes in select samples.
Variant Calling
We searched the literature and the Catalog of So-
matic Mutations in Cancer (COSMIC; http://cancer
.sanger.ac.uk/cancergenome/projects/cosmic) (see
Table S2 in the Supplementary Appendix) and
compiled a list of pathogenic variants associated
with human hematologic cancers in 160 genes.
As a negative control, we also searched for vari-
ants that were recurrently seen in nonhemato-
logic cancers (see Table S4 in the Supplementary
Appendix).
27
Statistical Analysis
All the statistical analyses were performed with
the use of the R statistical package (www.r-project
.org). Full details of the statistical analysis are
provided in the Methods section in the Supple-
mentary Appendix.
Results
Identification of Candidate Somatic
Mutations
To determine the extent of clonal hematopoiesis
with somatic mutations, we analyzed whole-exome
sequencing data from DNA in the peripheral-
blood cells of 17,182 persons who were selected
without regard to hematologic characteristics. Of
these, 15,801 were case patients and controls as-
certained from 22 cohorts in type 2 diabetes as-
sociation studies, and the remaining 1381 were
previously unsequenced participants in the Jack-
son Heart Study, a population-based cohort study
(Table S1 in the Supplementary Appendix). The
median age of the persons included in our study
at the time DNA was obtained was 58 years (range,
19 to 108); 8741 were women, and 7860 had type 2
diabetes.
The New England Journal of Medicine
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The
new england journal
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2490
The identification of somatic driver mutations
in cancer has come largely from studies that have
compared differences in DNA sequence between
tumor and normal tissue obtained from the same
person. Once mutations are identified, investiga-
tors may genotype samples for these somatic vari-
ants without relying on matched normal tissue.
Because we had DNA from only one source
(blood), we limited our examination to variants
that had been described previously in the litera-
ture in 160 recurrently mutated candidate genes
in myeloid and lymphoid cancers (Table S2 in the
Supplementary Appendix). We removed potential
false positive variants by using variant-calling
algorithms that had filters for known artifacts
such as strand-bias and clustered reads, as well as
by performing additional filtering for rare error
modes using a “panel of normals” (sequence data
from a panel of normal persons).
25
The lower
limit of detection for variants depended on the
depth of coverage. The median average sequenc-
ing depth for exons from the 160 genes was 84
reads (range, 13 to 144). At a sequencing depth
of 84 reads, the limit of detection for SNVs was
at a variant allele fraction of 0.035; the limit of
detection for indels was 0.070.
With this approach, we identified a total of
805 candidate somatic variants (hereafter referred
to as mutations) in 73 genes from 746 persons
(Table S3 in the Supplementary Appendix). As a
negative control, we searched for previously de-
scribed, nonhematologic cancer-associated vari-
ants in 40 genes (Table S4 in the Supplementary
Appendix)
27
and found only 10 such variants in
these genes (Table S5 in the Supplementary Ap-
pendix), indicating that the rate of false discovery
due to technical artifacts was low. We also verified
a subset of the variants using amplicon-based,
targeted sequencing; 18 of 18 variants were con-
firmed, with a correlation coefficient of 0.97 for
the variant allele fraction between the two meth-
ods (Fig. S1 in the Supplementary Appendix).
Increase in the Frequency of Clonal Somatic
Mutations with Age
Hematologic cancers, as well as other cancers
and premalignant states, increase in frequency
with age. Mutations were very rare in samples
obtained from patients younger than 40 years of
age but rose in frequency with each decade of life
thereafter (Fig. 1). Mutations in genes implicated
in hematologic cancers were found in 5.6% (95%
confidence interval [CI], 5.0 to 6.3) of persons 60
to 69 years of age, 9.5% (95% CI, 8.4 to 10.8) of
persons 70 to 79 years of age (219 of 2300 per-
sons), 11.7% (95% CI, 8.6 to 15.7) of persons 80
to 89 years of age (37 of 317), and 18.4% (95% CI,
12.1 to 27.0) of persons 90 years of age or older
(19 of 103). These rates greatly exceed the inci-
dence of clinically diagnosed hematologic cancer
in the general population.
28
Though we searched for mutations in genes
implicated in many different hematologic can-
cers, we primarily identified genes that were most
frequently mutated in AML and the myelodys-
plastic syndrome. The most commonly mutated
gene was DNMT3A (403 variants) (Fig. 2A, and
Fig. S2 in the Supplementary Appendix), fol-
lowed by TET2 (72 variants) and ASXL1 (62 vari-
ants). In TET2, only exon 3 was obtained by exon
capture (corresponding to approximately 50% of
the coding region), and the portion of exon 12
of ASXL1 that accounts for approximately 50% of
the mutations in this gene had poor coverage
depth. Thus, mutations in TET2 and ASXL1 are
probably underrepresented in this study. Other
frequently mutated genes included TP53 (33 vari-
ants), JAK2 (31 variants), and SF3B1 (27 variants).
In sequencing studies of the myelodysplastic
syndrome and AML, most patients have mutations
in two or more driver genes (the median number
of recurrently mutated genes in patients with de
Frequency
0.5
0.3
0.4
0.2
0.1
0.0
20–29
30–39
40–49
50–59
60–69
70–79
80–89
90–99
100–108
Age (yr)
No. with Mutation
Total
0
240
5
17
14
86
37
317
219
2300
282
5002
138
5441
50
2894
1
855
Figure 1. Prevalence of Somatic Mutations, According to Age.
Colored bands, in increasingly lighter shades, represent the 50th, 75th,
and 95th percentiles.
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Age-Related Clonal Hematopoiesis and Adverse Outcomes
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2491
novo AML is five
17
). In this study, we found that
693 of 746 persons with a detectable mutation
had only one mutation in the set of genes we
examined (Fig. 2B, and Fig. S2 in the Supplemen-
tary Appendix), a finding that was consistent with
the hypothesis that these persons had clones har-
boring only an initiating lesion.
The most common base-pair change in the
somatic variants was a cytosine-to-thymine (C→T)
transition (Fig. 2C), which is considered to be a
somatic mutational signature of aging.
16,29
The
median variant allele fraction for the identified
mutations was 0.09 (Fig. 2D), suggesting that the
variants are present in only a subset of blood cells
and supporting their somatic rather than germ-
line origin.
Persistence of Somatic Mutations over Time
Blood-cell DNA obtained 4 to 8 years after the
initial DNA collection was available for targeted
sequencing in 13 persons with 17 somatic muta-
tions (4 persons had 2 mutations). In all cases,
No.
400
300
200
100
0
DNMT3A
TET2
ASXL1
TP53
JAK2
SF3B1
GNB1
CBL
SRSF2
GNAS
Gene
Proportion of Variants
Density
0.6
0.5
0.4
0.3
0.2
0.1
0.0
C→A C→G C→T T→A T→C T→G
6
4
2
0
0.0 0.2 0.4 0.6 0.8 1.0 0.8 1.0
6
4
88
2
0
0.0 0.2 0.4 0.6
Density
6
4
2
0
0.0 0.2 0.4 0.6 0.8 1.0 0.8 1.0
6
4
88
2
0
0.0 0.2 0.4 0.6
Allele Fraction
Nonsense Variants Indel Variants
Splice-Site Variants Missense Variants
C D
A
403
72
62
33
31
27
22
12
11
8
693
49
2 2
No. of Patients
800
600
400
200
0
1 2 3 4
Mutations
B
Mean AF=0.12
Median AF=0.09
Mean AF=0.14
Median AF=0.10
Mean AF=0.12
Median AF=0.10
Mean AF=0.14
Median AF=0.09
Figure 2. Characteristics of Candidate Somatic Variants.
Panel A shows the 10 most frequently mutated genes implicated in hematologic cancers. Panel B shows the number of persons with 1,
2, 3, or 4 candidate variants. Panel C shows the distribution of the types of single-nucleotide base-pair changes seen in the candidate
variants. Panel D shows the allele fractions (AFs) of candidate somatic variants. The allele fraction was calculated as the number of vari-
ant reads divided by the number of variant-plus-reference reads. For variants on the X chromosome in men, this number was divided by
2. Indel denotes insertions and deletions.
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