ARID1A Mutations in Endometriosis-: Associated Ovarian Carcinomas

original article
The new engl and journal o f medicine

n engl j med 363;16 nejm.org october 14, 2010
1532
ARID1A Mutations in Endometriosis-
Associated Ovarian Carcinomas
Kimberly C. Wiegand, B.Sc., Sohrab P. Shah, Ph.D., Osama M. Al-Agha, M.D.,
Yongjun Zhao, D.V.M., Kane Tse, B.Sc., Thomas Zeng, M.Sc., Janine Senz, B.Sc.,
Melissa K. McConechy, B.Sc., Michael S. Anglesio, Ph.D., Steve E. Kalloger, B.Sc.,
Winnie Yang, B.Sc., Alireza Heravi-Moussavi, Ph.D., Ryan Giuliany, B.Sc.,
Christine Chow, B.M.L.Sc., John Fee, B.Sc., Abdalnasser Zayed, B.Sc.,
Leah Prentice, Ph.D., Nataliya Melnyk, B.Sc., Gulisa Turashvili, M.D., Ph.D.,
Allen D. Delaney, Ph.D., Jason Madore, M.Sc., Stephen Yip, M.D., Ph.D.,
Andrew W. McPherson, B.A.Sc., Gavin Ha, B.Sc., Lynda Bell, R.T.,
Sian Fereday, B.Sc., Angela Tam, B.Sc., Laura Galletta, B.Sc., Patricia N. Tonin, Ph.D.,
Diane Provencher, M.D., Dianne Miller, M.D., Steven J.M. Jones, Ph.D.,
Richard A. Moore, Ph.D., Gregg B. Morin, Ph.D., Arusha Oloumi, Ph.D.,
Niki Boyd, Ph.D., Samuel A. Aparicio, B.M., B.Ch., Ph.D., Ie-Ming Shih, M.D., Ph.D.,
Anne-Marie Mes-Masson, Ph.D., David D. Bowtell, Ph.D., Martin Hirst, Ph.D.,
Blake Gilks, M.D., Marco A. Marra, Ph.D., and David G. Huntsman, M.D.
From the British Columbia (BC) Cancer
Agency (K.C.W., S.P.S., O.M.A., J.S.,
M.K.M., M.S.A., S.E.K., W.Y., A.H.-M.,
R.G., A.Z., L.P., N.M., S.Y., A.W.M., G.H.,
L.B., D.M., N.B., D.G.H.), the Michael
Smith Genome Sciences Centre (Y.Z., K.T.,
T.Z., A.D.D., A.T., S.J.M.J., R.A.M., G.B.M.,
M.H., M.A.M.), the BC Cancer Research
Centre (J.F., G.T., A.O., S.A.A.), the Ge-
netic Pathology Evaluation Centre (C.C.,
B.G., D.G.H.), and the University of British
Columbia (K.C.W., D.M., G.B.M., S.A.A.,
B.G., M.A.M., D.G.H.) all in Vancouver,
Canada; and Simon Fraser University,
Burnaby, BC (G.B.M., M.A.M.) all in
Canada; Centre de Recherche du Centre
Hospitalier de lUniversit de Montral
Institut du Cancer de Montral Hpital
Notre-Dame (J.M., D.P., A.-M.M.-M.),
McGill University and the Research Insti-
tute of the McGill University Health Cen-
tre (P.N.T.), and Universit de Montral
(D.P., A.-M.M.-M.) all in Montreal; Pe-
ter MacCallum Cancer Centre (S.F., L.G.,
D.D.B.) and the University of Melbourne
(D.D.B.) both in Melbourne, VIC, Aus-
tralia; and Johns Hopkins University
School of Medicine, Baltimore (I.-M.S.).
Address reprint requests to Dr. Huntsman
at the Centre for Translational and Applied
Genomics, Rm. 3427, BC Cancer Agency,
600 W. 10th Ave., Vancouver, BC, Canada,
or at dhuntsma@bccancer.bc.ca.
This article (10.1056/NEJMoa1008433) was
published on September 8, 2010, at NEJM
.org.
N Engl J Med 2010;363:1532-43.
Copyright 2010 Massachusetts Medical Society.
ABSTRACT
Background
Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but
the molecular events involved in this transformation have not been described.
Methods
We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovar-
ian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-
rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes
BAF250a, a key component of the SWISNF chromatin remodeling complex. We se-
quenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-
cell carcinoma cell line and measured BAF250a expression by means of immunohis-
tochemical analysis in an additional 455 ovarian carcinomas.
Results
ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33
endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carci-
nomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a
protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid
carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A
mutations and loss of BAF250a expression were evident in the tumor and contiguous
atypical endometriosis but not in distant endometriotic lesions.
Conclusions
These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in
ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of
BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early
event in the transformation of endometriosis into cancer. (Funded by the British Co-
lumbia Cancer Foundation and the Vancouver General HospitalUniversity of British
Columbia Hospital Foundation.)
The New England Journal of Medicine
Downloaded from nejm.org on April 16, 2014. For personal use only. No other uses without permission.
Copyright 2010 Massachusetts Medical Society. All rights reserved.
ARID1A Mutations in Ovarian Carcinomas
1533
I
n the United States, ovarian cancer
ranks as the fifth deadliest cancer among wom-
en.
1
Of the several subtypes of epithelial ovar-
ian cancer, high-grade serous carcinomas are the
most common, accounting for approximately 70%
of all cases of epithelial ovarian cancer in North
America.
2
Although ovarian clear-cell carcinoma is
the second most common subtype in North Amer-
ica (accounting for 12% of cases and an even
higher percentage in Japan
3
) and is the second lead-
ing cause of death from ovarian cancer,
2
it is rela-
tively understudied. Ovarian clear-cell carcinoma
is defined on the basis of histopathological find-
ings, including a predominance of clear cells and
hobnail cells.
4
Ovarian clear-cell carcinomas have
a low mitotic rate,
5,6
are genetically stable, and
do not exhibit the complex karyotypes or chro-
mosomal instability of high-grade serous carci-
nomas
5,7-9
that may contribute to their lack of
sensitivity to platinum-based chemotherapy.
10-12

Although ovarian clear-cell carcinoma does not re-
spond well to conventional platinumtaxane che-
motherapy for ovarian carcinoma, this remains
the adjuvant treatment of choice, because effec-
tive alternatives have not been identified. Both
ovarian clear-cell and endometrioid carcinomas
are associated with endometriosis.
13,14
The genet-
ic events associated with the transformation of en-
dometriosis into ovarian clear-cell carcinoma and
endometrioid carcinoma are unknown.
The SWISNF chromatin remodeling complex,
present in all eukaryotes, is involved in the regu-
lation of many cellular processes, including de-
velopment, differentiation, proliferation, DNA re-
pair, and tumor suppression.
15
The complex uses
ATP to mobilize nucleosomes, thereby modulating
the accessibility of promoters to transcriptional
activation or repression. BAF250a, the protein en-
coded by ARID1A (the AT-rich interactive domain
1A [SWI-like] gene),
16,17
is one of the accessory
subunits of the SWISNF complex believed to con-
fer specificity in regulation of gene expression.
Mutations or other aberrations in ARID1A have
not been described in ovarian carcinomas; how-
ever, an ARID1A rearrangement has been found in
a breast-cancer cell line, an ARID1A deletion has
been identified in a lung-cancer cell line, and it has
been suggested that ARID1A is a tumor-suppressor
gene.
18
We used data derived from the whole-transcrip-
tome sequencing (RNA sequencing) of 18 ovarian
clear-cell carcinomas and an ovarian clear-cell
carcinoma cell line to identify variants in ARID1A,
as previously described.
19,20
We then studied this
gene in a larger cohort of patients with ovarian
carcinoma and associated endometriosis. The re-
sults suggest that ARID1A is a tumor suppressor
in ovarian clear-cell and endometrioid carcinomas.
Methods
Patients and Samples
Ovarian clear-cell carcinomas from 18 patients,
obtained from the OvCaRe (Ovarian Cancer Re-
search) frozen-tumor bank, and 1 ovarian clear-
cell carcinomaderived cell line (TOV21G)
21
were
selected as the discovery cohort for RNA sequenc-
ing. Approval from the hospitals institutional
review board was obtained to permit the use of
these samples for RNA-sequencing experiments.
To determine the frequency of ARID1A muta-
tions in ovarian clear-cell carcinoma and other
subtypes of ovarian cancer, we performed targeted
exon resequencing in the discovery cohort, as well
as in a mutation-validation cohort, consisting of
210 samples: samples of ovarian clear-cell carci-
noma from 101 patients (independent of the 19
samples used in RNA sequencing for the discov-
ery cohort), samples of endometrioid carcinoma
from 33 patients, samples of high-grade serous
carcinoma from 76 patients, and the ovarian clear-
cell carcinomaderived cell line ES2.
22
Ten spec-
imens of ovarian clear-cell carcinoma came from
Johns Hopkins University, 29 from the Centre
Hospitalier de lUniversit de MontralHpital
Notre-Dame, and 42 from the Australian Ovarian
Cancer Study (AOCS); all others were obtained
from the OvCaRe frozen-tumor bank. All patients
from both the discovery and mutation-validation
cohorts provided written informed consent to
have their tumors and germ-line DNA used for
research, including genomic studies. Details re-
garding the consents and other approvals by
the institutional review boards are supplied in the
Supplementary Appendix (available with the full
text of this article at NEJM.org).
Pathological Review
All tumor samples were reviewed independently
by a gynecologic pathologist before mutational
analysis was performed. In cases in which the re-
view diagnosis differed from the diagnosis at the
source institution, the samples were further re-
viewed by another gynecologic pathologist, who
1534
acted as an arbiter. Both review pathologists were
unaware of the results of the genomic studies.
Paired-End RNA Sequencing and Analysis
RNA sequencing and analysis were performed as
previously described.
19,20
For details, see the Meth-
ods section in the Supplementary Appendix.
Targeted Exon Resequencing of ARID1A
and Mutation Validation
Genomic DNA from samples in both the discov-
ery and mutation-validation cohorts were subject-
ed to targeted exon resequencing. Selected ARID1A
variants (those with truncating changes or radical
missense mutations
23
with an allele frequency
of >10%) detected by means of exon resequenc-
ing were validated in tumor DNA by means of
Sanger sequencing. In most cases, germ-line DNA
(from formalin-fixed paraffin-embedded sections,
blood samples, or cultured fibroblasts) was also
analyzed by means of Sanger sequencing (see Ta-
ble 3 in the Supplementary Appendix). Full details
are provided in the Supplementary Appendix.
Immunohistochemical Analysis of BAF250a
Immunohistochemical staining for BAF250a was
performed in all samples except 42 ovarian clear-
cell carcinoma specimens from the AOCS and
4 from Johns Hopkins University. A total of 455
additional ovarian-carcinoma samples includ-
ing 132 ovarian clear-cell carcinomas, 125 endo-
metrioid carcinomas, and 198 high-grade serous
carcinomas from a previously described tissue
microarray
6
were used for an immunohistochem-
ical validation cohort and were analyzed for
BAF250a expression. All normal gynecologic tis-
sues showed moderate or intense nuclear immu-
noreactivity for BAF250a. Tumors were scored pos-
itive for BAF250a if tumor cells showed definite
nuclear staining and negative if tumor nuclei had
no immunoreactivity but endothelial and other
nontumor cells from the same samples showed
immunoreactivity. Cases in which neither normal
cells in the stroma nor tumor cells were immu-
noreactive were considered to be the result of tech-
nical failure. Details of the staining protocol are
provided in the Supplementary Appendix. Addi-
tional immunohistochemical staining for hepa-
tocyte nuclear factor 1 (HNF-1) and estrogen
receptor was performed on whole sections for two
tumors with contiguous atypical endometriosis,
as previously described.
24
Laser-Capture Microdissection,
DNA Isolation, and Cloning
In two tumors with identified ARID1A mutations,
sections of atypical (contiguous) and distant en-
dometriosis were identified by a gynecologic pa-
thologist. Laser-capture microdissection was used
to isolate endometriotic epithelium. DNA extract-
ed from these cells was analyzed by means of
sequencing for the mutations seen in the tumor
(see the Supplementary Appendix).
Results
ARID1A Mutations
The RNA-sequencing data, including the number
of mapped sequencing reads and potential non-
synonymous sequence variants, are summarized
in Table 1 in the Supplementary Appendix. RNA
sequencing of the 19 samples in the discovery
cohort resulted in the detection of the following
nucleotide mutations (and corresponding amino
acid mutations) (also shown in Table 1 and Fig. 1):
three somatic nonsense mutations C4201T
(Q1401*), C5164T (R1722*), and C1680A (Y560*)
(stars denote a stop codon); two somatic indels
(insertiondeletion) 6018-6020delGCT and
5541insG; one somatic missense mutation
T5953C (S1989P) (found in the same sample as
the 5541insG mutation); and one gene rearrange-
ment involving ARID1A and the neighboring gene
ZDHHC18 (encoding the zinc-finger DHHC do-
main-containing protein 18). The fusion ends of
this rearrangement map to a homozygous dele-
tion involving most of the ARID1A gene (Fig. 1 in
the Supplementary Appendix).
All predicted variants of ARID1A were validated
with the use of Sanger sequencing of DNA from
the source tumors, except for the deletionrear-
rangement, which was validated with the use of
microarray data (Affymetrix SNP 6.0) (Table 3 in
the Supplementary Appendix). The finding of mul-
tiple types of mutations in a single gene, ARID1A,
in ovarian clear-cell carcinoma led us to further
explore ARID1A in this cancer type. Since muta-
tions in PIK3CA (the phosphoinositide-3-kinase,
catalytic, alpha polypeptide gene), CTNNB1 (the
catenin beta-1 gene), KRAS (the v-Ki-ras2 Kirsten
rat sarcoma viral oncogene homologue gene), and
TP53 (the tumor protein p53 gene) are recurrent
in ovarian clear-cell carcinoma,
25
we analyzed the
RNA-sequencing data and performed a poly-
merase-chain-reaction assay for the presence of
1535
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1537
variants in these genes (Table 1). Whole-transcrip-
tome sequence data for the 19 samples of the
discovery cohort have been deposited at the Eu-
ropean GenomePhenome Archive (accession num-
ber, EGAS00000000075).
ARID1A mutation frequency in ovarian clear-cell
carcinomas and other ovarian-cancer subtypes was
established through targeted exon resequencing
of the mutation-validation cohort of 210 samples
of various subtypes of ovarian carcinomas and
1 ovarian clear-cell carcinoma cell line, along with
the original discovery cohort of 18 samples of
ovarian clear-cell carcinoma and 1 ovarian clear-
cell carcinoma cell line. ARID1A mutations were
identified in 55 of 119 (46%) ovarian clear-cell
carcinomas, 10 of the 33 (30%) endometrioid car-
cinomas, and none of the 76 high-grade serous
carcinomas (Table 2, and Table 3 in the Supple-
mentary Appendix). A total of 17 samples (12 of
ovarian clear-cell carcinoma and 5 of endometri-
oid carcinoma) each had two validated ARID1A
mutations. In addition, the ovarian clear-cell car-
cinoma cell line TOV21G had a truncating muta-
tion in ARID1A (1645insC).
We analyzed germ-line DNA from 55 samples
(47 ovarian clear-cell carcinomas and 8 endometri-
oid carcinomas) in the discovery and mutation-
validation cohorts for the presence of 65 truncat-
ing mutations (53 found in ovarian clear-cell
carcinomas and 12 found in endometrioid carci-
nomas). In all 55, the mutations were found to
be somatic. On this basis, we made the assump-
tion that 12 subsequent truncating mutations (10
in ovarian clear-cell carcinoma and 2 in endo-
metrioid carcinoma) would be somatic (i.e., pre-
dicted to be somatic without germ-line DNA test-
ing) (Table 3 in the Supplementary Appendix). The
presence of ARID1A mutations showed a strong
association (P<0.001 by Fishers exact test) with
the two ovarian-cancer subtypes associated with
endometriosis (ovarian clear-cell carcinoma and
endometrioid carcinoma).
BAF250a Protein Expression
The correlation between ARID1A mutations and
BAF250a expression was evaluated by means of
immunohistochemical staining for BAF250a in
182 tumors for which formalin-fixed, paraffin-
embedded sections were available in the discovery
cohort and the mutation-validation cohort: 73 ovar-
ian clear-cell carcinomas, 33 endometrioid carci-
nomas, and 76 high-grade serous carcinomas. The
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1538
presence of mutations was significantly associat-
ed with BAF250a loss in endometriosis-associat-
ed cancers (P<0.001 by Fishers exact test). A total
of 27 of 37 samples (73%) and 5 of 10 samples
(50%) of ovarian clear-cell carcinoma and endo-
metrioid carcinoma, respectively, with an ARID1A
mutation showed a loss of BAF250a expression, as
compared with 4 of 36 samples (11%) and 2 of 23
samples (9%), respectively, without an ARID1A
mutation (Fig. 2A and Table 2). Loss of BAF250a
expression was strongly associated with the endo-
metriosis-related ovarian cancers with 31 of 73
samples (42%) of ovarian clear-cell carcinoma and
7 of 33 samples (21%) of endometrioid carcino-
ma showing a loss of expression as compared
with high-grade serous carcinomas, for which
1 of the 76 samples (1%) had loss of expression
(P<0.001 by Fishers exact test) (Fig. 2B). ARID1A
mutations were not significantly associated with
the presence of endometriosis in 86 ovarian clear-
cell carcinomas and 33 endometrioid carcinomas
(Table 5 in the Supplementary Appendix).
The immunohistochemical validation cohort
was also assessed for BAF250a expression (Fig. 2B).
This analysis revealed that 55 of the 132 samples
(42%) of ovarian clear-cell carcinoma, 39 of the
125 samples (31%) of endometrioid carcinoma,
and 12 of the 198 samples (6%) of high-grade se-
rous carcinoma lacked BAF250a expression.
These findings are in agreement with the pro-
portions observed in the discovery and muta-
tion-validation cohorts. No significant associa-
tions with absence of BAF250a expression were
noted on the basis of age of presentation, stage
of disease (low or high), or disease-specific sur-
vival within any of the cancer subtypes, as as-
sessed by means of Welchs analysis of variance,
Fishers exact test, and the log-rank statistic, re-
spectively (P>0.05 for all analyses).
Analysis of ARID1A in Endometriosis
Associated with Ovarian Cancer
Two patients with ovarian clear-cell carcinomas
(samples CCC13 and CCC23) carrying ARID1A mu-
tations had contiguous atypical endometriosis
(Fig. 3, and Fig. 3 in the Supplementary Appendix).
For one of the two patients, the specimen was
heterozygous for an ARID1A truncating mutation
(G6139T [E2047*]) in exon 20. This mutation was
also found in 17 of 42 clones derived from atypi-
cal endometriosis but in none of 52 clones from
a distant endometriotic lesion (P<0.001 by Fishers
exact test) (Fig. 3C). Epithelial samples of both the
ovarian clear-cell carcinoma and atypical endo-
metriosis had loss of BAF250a expression, whereas
expression was maintained in the distant endo-
metriotic lesion (Fig. 3B). HNF-1 was expressed
in the ovarian clear-cell carcinoma but not in the
73%
(27/37)
11%
(4/36)
50%
(5/10)
9%
(2/23)
0
1%
(1/76)
6%
(12/198)
1%
(1/76)
21%
(7/33)
31%
(39/125)
42%
(31/73)
42%
(55/132)
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(
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90
70
60
40
30
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0
CCC EC HGS carcinoma
Cancer Subtype
B
A
ARID1A mutation No ARID1A mutation
L
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80
90
70
60
40
30
10
50
20
0
CCC EC HGS carcinoma
Cancer Subtype
Discovery and mutation-
validation cohorts
Immunohistochemical
validation cohort
Figure 2. Results of Immunohistochemical Analyses of BAF250a Expression.
The percentages of tumors (with number and total number in parentheses)
from three subtypes of ovarian cancer clear-cell carcinoma (CCC), endo-
metrioid carcinoma (EC), and high-grade serous (HGS) carcinoma from
the discovery and mutation-validation cohorts that showed loss of BAF250a
expression are shown in Panel A for samples with and samples without
ARID1A mutations and in Panel B for samples in the discovery and muta-
tion-validation cohorts and samples in the immunohistochemical valida-
tion cohort. The rate of BAF250a loss was higher among CCC specimens
with an ARID1A mutation than among those without an ARID1A mutation
(P<0.001); the same was true for EC specimens (P = 0.02). The loss of ex-
pression was also consistently more common in CCC and EC (the two en-
dometriosis-associated carcinomas) than in HGS carcinoma when assessed
in the discovery and mutation-validation cohorts and again in the immuno-
histochemical validation cohort (Panel B), with P<0.001 for all compari-
sons. All P values were calculated with the use of Fishers exact test.
1539
contiguous atypical or distant endometriosis, and
estrogen receptor was expressed in both the con-
tiguous and distant endometriosis but not in the
ovarian clear-cell carcinoma, as was expected.
24

Thus, atypical endometrium could be distinguished
from the distant endometrium only on the basis
of loss of BAF250a expression, which correlated
with the presence of an ARID1A mutation.
For the other patient, the sample of ovarian
clear-cell carcinoma had two somatic mutations
in ARID1A (and loss of BAF250a expression): both
these mutations, along with a CTNNB1 missense
mutation, were present in the tumor and the ad-
jacent atypical endometriosis but not in a distant
endometriotic lesion (Fig. 3B in the Supplemen-
tary Appendix).
Discussion
Overall, 46% of patients with ovarian clear-cell
carcinoma and 30% of those with endometrioid
carcinoma had somatic truncating or missense
mutations in ARID1A; no ARID1A mutations were
found in any of the 76 specimens of high-grade
serous carcinoma analyzed. Loss of ARID1A ex-
pression was also specific to the subtype of ovar-
ian cancer, with loss of nuclear BAF250a expres-
sion seen in 36% of ovarian clear-cell carcinomas
and endometrioid carcinomas, but only 1% of high-
grade serous carcinomas. Our initial mutation-
screening assays involving RNA sequencing in the
discovery cohort identified seven somatic muta-
tions in ARID1A in the 19 samples; four additional
mutations were subsequently identified when these
samples were analyzed by means of amplicon-
exon resequencing. Most likely, the additional mu-
tations had not been seen in the RNA-sequencing
data owing to their transcripts being rapidly tar-
geted for nonsense-mediated decay
26
or the in-
herently decreased sensitivity of the assay to mu-
tations at the 5 end of transcripts. Thus, although
RNA sequencing is a useful tool for discovery,
targeted exon resequencing may be more appro-
priate for the determination of true mutation fre-
quency.
ARID1A is located at 1p36.11.
27
This chromo-
somal region is commonly deleted in tumors,
and it has been suggested that deletion regions
encompassing 1p36 could contain tumor-suppres-
sor genes.
28,29
Rearrangements and deletions in
ARID1A have been identified in a primary breast-
cancer cell line and a lung-cancer cell line, re-
spectively,
18
and the loss of BAF250a has also been
observed in cervical- and breast-carcinoma cell
lines.
30
In a study by Wang and colleagues,
31
the
screening of 241 tumors revealed that ARID1A
transcript levels are decreased in approximately
6% of cancers in general and in 30% of renal
carcinomas and 10% of breast carcinomas, spe-
cifically; however, none of the 14 ovarian can-
cers showed loss of expression, probably be-
cause they were predominantly the high-grade
serous subtype.
The ARID1A mutations identified in our study
were mostly truncating mutations, which were
evenly distributed across the gene. The presence
of mutations is strongly correlated with the
loss of BAF250a protein (Table 2 and Fig. 2A).
Loss of BAF250a expression was seen in 73% and
50% of samples of ovarian clear-cell carcinoma
and endometrioid carcinoma with an ARID1A
mutation, respectively, and in only 11% and 9%
of samples without an ARID1A mutation, respec-
tively. Seventeen of the mutation-positive sam-
ples had two ARID1A mutations; in all but one of
the specimens with two mutations for which im-
munohistochemical data were available, BA-
F250a expression was not detected. That single
exception (an endometrioid carcinoma) had both
a C-terminal truncating mutation and a mis-
sense mutation; either of these changes could
produce a detectable protein. A single sample of
ovarian clear-cell carcinoma had ARID1A loss and
rearrangement resulting in the homozygous dele-
tion of the gene. Three other cases of ovarian
clear-cell carcinoma also appear to be character-
ized by loss of heterozygosity, on the basis of the
frequency of mutant alleles and wild-type alleles
(Table 3 in the Supplementary Appendix) and
subsequent loss of BAF250a expression. However,
the majority of cancers with somatic ARID1A mu-
tations and loss of BAF250a expression appear
to have a wild-type allele present. Data from
exon resequencing and RNA sequencing show
excellent agreement between the fraction of mu-
tant and wild-type alleles at both the DNA and
RNA levels (Table 1), suggesting that epigenetic
silencing is not a significant factor. Post-tran-
scriptional or post-translational regulation or
dominant negative effects of the mutations are
possible, albeit untested, explanations for the
lack of protein expression in these heterozygous
cases.
The presence of BAF250a immunoreactivity in
15 samples positive for an ARID1A mutation (all
but 1 of which had truncating mutations) may in-
1540
Original Specimen
Distant Endometriosis
Clear-Cell Carcinoma
H&E
BAF250a
Expression
HNF-1
Expression ER Expression
Contiguous Atypical
Endometriosis
Contiguous Atypical
Endometriosis
Contiguous Atypical Endometriosis
B
C
A
G G T T T T T G G G G G G C C A A A A A A
G G T T K T T G G G G G G C C A A A A A A
G G T T T T G G G G G G G C C A A A A A A
1541
dicate that haploinsufficiency is pathogenic, as has
been reported in mice.
32
Alternatively, immuno-
histochemical detection of a truncated but non-
functional BAF250a protein may account for the
immunostaining results. The antibody used in the
assay targets a region of 111 amino acids (amino
acids 1216 through 1326) in the middle of the
protein, and 7 of the 15 specimens that were posi-
tive for loss of BAF250a expression had mutations
that would result in truncation distal to the
epitope.
The mutations are common in ovarian carci-
nomas that are associated with endometriosis
(ovarian clear-cell carcinoma and endometrioid
carcinoma) but not in the unrelated high-grade
serous carcinoma. This suggests that the muta-
tions may be pathogenic, rather than random,
events. Mutations in the PTEN gene (encoding the
phosphatase and tensin homologue) have been
described in 20% of endometriotic cysts,
33
and
conditional expression of either oncogenic Kras or
deletion of the Pten tumor suppressor in the ovar-
ian surface epithelium in mice was found to induce
endometriosis.
34
Expression of oncogenic Kras ac-
companied by simultaneous loss of Pten resulted
in widely metastatic ovarian carcinoma; however,
KRAS mutations are not seen in human cases of
endometriosis and are uncommon in endometri-
osis-associated ovarian cancers in humans. By
comparing ovarian clear-cell carcinomas to their
contiguous atypical endometriotic lesions in two
patients, we show that the same mutations may
be present in the putative precursor lesions and
in the tumors. In contrast, the distant endometri-
otic lesions do not have ARID1A mutations. In the
case of ovarian clear-cell carcinoma described in
Figure 3, the mutation (G6139T [E2047*]) was
present before the atypical endometriosis resulted
in the development of the immunophenotype as-
sociated with the cancer (estrogen-receptornega-
tive, HNF-1positive
24
), suggesting that the
mutation is an early event in neoplastic transfor-
mation. Taken together, these data suggest that
ARID1A is a classic tumor-suppressor gene. Unlike
BRCA or TP53 mutations, which can be found in
the germ-line DNA, all truncating ARID1A muta-
tions were somatic. Deletion of ARID1A on one
allele results in embryonic lethality in mice.
32
Mutations in ARID1A and loss of BAF250a ex-
pression were seen preferentially in ovarian clear-
cell carcinomas and endometrioid carcinomas,
cancers that do not feature the chromosomal in-
stability, nearly ubiquitous TP53 mutations, and
frequent abnormalities in BRCA (associated with
early breast cancer) seen in high-grade serous car-
cinomas.
5,35
It is possible that defects in genes
that alter the accessibility of transcription factors
to chromatin, such as ARID1A, in addition to mu-
tations in the WNT and PI3 kinase pathways,
25

will help to define ovarian clear-cell carcinomas
and endometrioid carcinomas. If such a model is
correct, other abnormalities affecting the ARIDIA
locus or dysregulation of other chromatin-remod-
eling genes may be found in ovarian clear-cell and
endometrioid carcinomas that are negative for an
ARID1A mutation. This idea is supported by the
clinical similarities between ovarian clear-cell car-
cinomas positive for and those negative for an
ARID1A mutation.
The mechanism by which somatic mutations
in ARID1A enable the progression of benign en-
dometriosis to carcinoma is unclear; however, our
findings are consistent with a critical role for
Figure 3 (facing page). Analysis of Ovarian Clear-Cell
Carcinoma and Associated Endometriosis in a Study
Patient.
Panel A shows a section (hematoxylin and eosin
[H&E]) on which a clear-cell carcinoma (black arrow)
has arisen in an endometriotic cyst (white arrow). The
same section, viewed at a higher magnification, shows
regions of the clear-cell carcinoma and contiguous
atypical endometriosis. A region of distant endome-
triosis from the same patient is also shown. Panel B
shows the results of immunohistochemical staining
of the epithelial portions of tissue specimens shown
in Panel A for expression of BAF250a, hepatocyte nu-
clear factor 1 (HNF-1), and estrogen receptor (ER).
BAF250a immunoreactivity is lost in both the clear-cell
carcinoma and the contiguous atypical endometriosis
but is maintained in the distant endometriosis. Both
regions of endometriosis differ from the carcinoma in
their lack of HNF-1 expression (with weak expression
in the contiguous atypical endometriosis) and mainte-
nance of estrogen-receptor expression. Panel C shows
sequencing chromatograms for the clear-cell carcino-
ma and polymerase-chain-reaction (PCR) clones of
microdissected material from the contiguous atypical
endometriosis and distant endometriosis, from which
DNA was extracted. The carcinoma and contiguous
atypical endometriosis show nucleotide variation cor-
responding to G6139T (as indicated with the dashed
box); the tumor shows a heterozygous peak at that
location, whereas the atypical endometriosis is homo-
zygous for the substitution (in 17 of 42 clones). In con-
trast, the distant endometriosis shows wild-type se-
quence (in all 52 clones analyzed). None of the PCR
clones from the distant endometriosis showed varia-
tion from the wild-type sequence.
1542
ARID1A mutations in the genesis of a substantial
fraction of ovarian clear-cell and endometrioid
carcinomas.
Supported by grants from the British Columbia (BC) Cancer
Foundation and the Vancouver General Hospital (VGH)Univer-
sity of British Columbia Hospital Foundation (to the OvCaRe
ovarian cancer research team in Vancouver) and the Canadian
Institutes of Health Research (CIHR). The Michael Smith Genome
Sciences Centre (MSGSC), with which many of the authors are
affiliated, is funded by Genome Canada, and OvCaRe and the
MSGSC are also funded by the Michael Smith Foundation for
Health Research (MSFHR). Salary support is provided by MSFHR
to Drs. Shah, Marra, Jones, and Huntsman; by the CIHR Training
Program for Clinician Scientists in Molecular Oncologic Patholo-
gy to Drs. Al-Agha and Turashvili (STP-53912); by the CIHR Bioin-
formatics Training Program for Health Research to Mr. McPher-
son and Mr. Ha; by the Canadian Breast Cancer Foundation to Dr.
Shah; and a Canada Research Chair in Molecular Oncology to Dr.
Aparicio. The Genetic Pathology Evaluation Centre, which con-
structed the tissue microarrays, has received nondirected research
grants from Sanofi-Aventis, Canada. The contributing tumor
banks were supported by OvCaRe and Ovarian Cancer Canada
(VGH, Banque de Tissus et de Donnes of the Rseau de Recher-
che sur le Cancer of the Fonds de la Recherche en Sant du Qu-
bec, affiliated with the Canadian Tumor Repository Network),
and by a grant (DAMD17-O1-1-0729) from the U.S. Army Medical
Research and Materiel Command; grants from the Cancer Coun-
cil Tasmania, the Cancer Foundation of Western Australia, and
the National Health and Medical Research Council of Australia to
AOCS; and grants from the National Cancer Institute, National
Institutes of Health (RO1CA103937 and RO1CA129080).
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
This article is dedicated to the memory of OvCaRe research
administrator Cecelia Suragh.
We thank all members of the OvCaRe ovarian cancer research
team (www.ovcare.ca) for their enthusiastic support of this proj-
ect. We also thank the AOCS management group (D. Bowtell, G.
Chenevix-Trench, A. Green, P. Webb, A. deFazio, and D. Gertig)
and the AOCS study nurses and research assistants for their
contributions. The full AOCS study group is listed at www
.aocstudy.org. We thank all the women who donated the sam-
ples used in this study. In addition, we thank Christian Steidl for
technical consultation; Coco Yu for help in preparation of the
manuscript; Robert Bartusiak, Sylvia Lee, and Julie Lorette for
technical assistance; and the fellows of the University of British
Columbia Gynecologic Oncology Program for obtaining consent
from patients for data in our tumor bank.
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Copyright 2010 Massachusetts Medical Society.
Dubrovnik, Croatia Albert R. Frederick, Jr., M.D.

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