Download PIK3CA and PTEN Mutations in Uterine Endometrioid Carcinoma

Human Cancer Biology
PIK3CA and PTEN Mutations in Uterine Endometrioid Carcinoma
and Complex Atypical Hyperplasia
Monica Prasad Hayes,1,2 Hong Wang,1 Rosanny Espinal-Witter,1 Wayne Douglas,1
Garron J. Solomon,1 Suzanne J. Baker,3 and Lora Hedrick Ellenson1
Abstract
Purpose: The tumor suppressor PTEN gene and the PIK3CA oncogene are frequently mutated
in uterine endometrioid carcinoma (UEC). PTEN mutations are also common in complex atypical
hyperplasia (CAH), the precursor lesion of UEC. The status of PIK3CA has not yet been explored
in CAH. In this study, we evaluated both CAH and UEC for PTEN and PIK3CA mutations.
Experimental Design: Neoplastic tissue was microdissected, and DNA was extracted from
29 cases of CAH. DNA was available from 44 UEC cases previously characterized for PTEN
mutations. Direct DNA sequencing of exons 9 and 20 of the PIK3CA gene was done on all 73
cases. In addition, CAH cases were analyzed for PTEN mutations. Statistical analyses were done
using the Fisher’s exact test.
Results: Two (7%) of 29 CAH and 17 (39%) of 44 UEC cases contained a PIK3CA mutation
(P = 0.003). Fourteen (48%) of 29 CAH cases had a PTEN mutation, but none contained both
a PTEN and PIK3CA mutation. Twenty-five (57%) of 44 UEC cases had a PTEN mutation, and
12 (48 %) of these 25 cases also contained a PIK3CA mutation. Coexistent PIK3CA and
PTEN mutations were significantly correlated with UEC compared with CAH (P = 0.002),
but the association in UEC did not reach statistical significance (P = 0.21).
Conclusions: PIK3CA is the most commonly mutated oncogene in UEC; however, mutations
are uncommon in CAH. Thus, mutations in PIK3CA, unlike PTEN mutations, are associated with
invasion.These findings suggest that mutations in PIK3CA may serve as a marker of invasion with
potential clinical use. Furthermore, PIK3CA and PTEN mutations may play distinct roles in
endometrial tumorigenesis.
Within
the last 10 years, our knowledge of the molecular
genetics of endometrial cancer has expanded with the
discovery of mutations in genes, such as PTEN, K-RAS,
CTNNB1, and TP53. These discoveries have led to a better
understanding of the pathogenesis of endometrial cancer and
its distinct histologic subtypes, broadly categorized as type I
and II cancers, which are thought to arise via different
pathogenetic pathways. Uterine endometrioid carcinoma
(UEC) is the most common histologic subtype, representing
>85% of cases. Not only is PTEN the most commonly mutated
Authors’ Affiliations: 1Department of Pathology and Laboratory Medicine, Weill
Medical College of Cornell University; 2Division of Gynecologic Oncology, The
Mount Sinai School of Medicine, New York, New York ; and 3 Department of
Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis,
Tennessee
Received 6/7/06; revised 7/26/06; accepted 8/3/06.
Grant support: NIH grant CA095427 (L.H. Ellenson), Alice Bohmfalk Charitable
Trust (L.H. Ellenson), NIH (S.J. Baker), and American Lebanese and Syrian
Associated Charities (S.J. Baker).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Lora Hedrick Ellenson, Department of Pathology and
Laboratory Medicine,Weill Medical College of Cornell University, Room Starr 1015,
1300 York Avenue, New York, NY 10021. Phone: 212-746-6447; Fax: 212-7468079; E-mail: lora.ellenson@ med.cornell.edu.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-1375
Clin Cancer Res 2006;12(20) October 15, 2006
gene in UEC, the mutations also occur early in its pathogenesis
as they are present at approximately the same frequency in
complex atypical hyperplasias (CAH), the precursor lesion of
UEC (1, 2).
Recently, mutations of a novel oncogene (PIK3CA) were
discovered in multiple human epithelial cancers, including
UEC (3, 4). PIK3CA encodes the catalytic p110-a subunit of
phosphatidylinositol 3-kinase (PI3K), a lipid kinase that
generates phosphatidyl inositol-3,4,5-triphosphate by phosphorylating phosphatidyl inositol-3,4-diphosphate. This in
turn activates the AKT/mammalian target of rapamycin
oncogenic pathway, directly opposing the actions of the lipid
phosphatase PTEN (5). It is unclear what role mutations in
PIK3CA play during endometrial tumorigenesis and what, if
any, additional role they confer over PTEN mutations. Recent
literature has suggested that PIK3CA mutations not only
promote cell growth, but that they also promote invasion
and are oncogenic in vitro and in vivo (6 – 8).
Given the importance of the PI3K pathway and the early
inactivation of PTEN in the development of UEC, we
investigated the status of the PIK3CA gene and its association
with PTEN mutations in CAH and UEC. We found a high
frequency of PIK3CA mutations in UEC similar to the only
reported study thus far and, furthermore, show that mutations
are infrequent in CAH. Our findings suggest that PIK3CA
mutations, unlike PTEN mutations, are associated with
invasion in the development of UEC.
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PIK3CA and PTEN Mutations in Uterine CAH and UEC
Table 1. CAH and UEC cases with PIK3CA and
PTEN mutations
PIK3CA
Uterine CAH (n = 29)
UEC (n = 44)
2 (7%)
17 (39%)
PTEN
PTEN + PIK3CA
14 (48%)
25 (57%)
0
12 (27%)
Table 2. PIK3CA mutations in CAH and UEC
Materials and Methods
Specimen collection. Twenty-nine cases of uterine CAH were
retrospectively identified from an institutional database at the Weill
Medical College of Cornell University. In addition, 44 cases of UEC
that have been previously molecularly characterized in our lab for
mutations in PTEN were evaluated for PIK3CA mutations (9). Of the
UEC cases, 36 had early stage I/II disease, and eight had stage III/IV
disease. Histologic grade of the UEC tumors was as follows: 23 grade 1,
14 grade 2, and 7 grade 3 tumors. A gynecologic pathologist (L.H.E.)
reviewed the H&E-stained slides of all cases to confirm the diagnoses.
All cases were anonymized, and Institutional Review Board approval
was obtained.
Microdissection and DNA extraction. Five-micrometer tissue sections
were prepared from formalin-fixed, paraffin-embedded endometrial
curettings or hysterectomy specimens of both hyperplastic and normal
tissue. Slides were stained with hematoxylin, and areas of hyperplasia
were microdissected with a 26-gauge needle under direct light
microscopic guidance to a concentration of >70% tumor cells. Tissue
was then placed in TE-9 [500 mmol/L Tris, 20 mmol/L EDTA,
10 mmol/L NaCl (pH 9)] with 1% SDS at 60jC overnight. DNA was
extracted with phenol-chloroform and precipitated with ethanol as
previously described (9).
PIK3CA and PTEN mutational analysis. The exons that are known
to be most commonly mutated in PTEN (exons 3, 4, 5, 7, and 8) were
evaluated using exon-specific PCR amplification and direct DNA
sequencing. In addition, exons 9 and 20 of the PIK3CA gene are the
most common sites of mutations in this gene, and PCR amplification
of each exon was done with subsequent DNA sequencing using the
Applied Biosystems Automated 3730 DNA Analyzer (Foster City, CA).
Two to 100 ng of genomic DNA were amplified using previously
described exon-specific primers for PTEN (9) and PIK3CA (3). For exon
20 of the PIK3CA gene, new primer sequences were constructed for
optimal PCR conditions. A forward primer of 5¶-CATTTGCTCCAAACTGACCA-3¶ and a reverse primer of 5¶-TGTGGAATCCAGAGTGAGCTT-3¶
were used with the following PCR conditions to produce a 353-bp PCR
product: 5 minutes at 95jC for one cycle, 40 cycles at 95jC for
1 minute, 60jC for 1 minute, and 72jC for 1 minute, followed by
5 minutes at 72jC for one cycle. Purification of PCR fragments was
done with the QIAquick PCR Purification kit (Qiagen, Valencia, CA)
and submitted for direct sequence analysis. All potential mutations
were verified by reamplification from genomic DNA and direct
sequencing of the mutated exon. In addition, insertion and deletion
mutations were reamplified from genomic DNA and sequenced a third
time. Novel alterations were confirmed to be somatic mutations by
comparison of normal and neoplastic DNA sequences.
Statistical analysis. The Fisher’s exact test was used for a comparison
of frequencies between two groups. All tests were two sided, and
P < 0.05 was considered statistically significant.
PIK3CA mutation
Frequency
Uterine CAH (n = 29)
H1047R
2
UEC (n = 44)
E545K
E545D
Q546K
Q546R
Q546H
H1047R
H1047L
Y1021C
T1025A
M1043V
I1058F*
4
1
1
1
1
4
1
1
1
1
1
*Novel PIK3CA mutation not previously reported in other human
cancers.
PIK3CA mutation had identical missense mutations in exon
20, an A3140G transition mutation converting histidine to
arginine (H1047R). In the UEC cases, the 17 PIK3CA
mutations were evenly distributed between exons 9 and 20,
with eight and nine cases mutated in each exon, respectively.
The most commonly mutated codons in PIK3CA were 1047 in
exon 20 and 545 in exon 9. H1047R was found in four UEC
cases and H1047L (A3140T) in one case, whereas E545K
(G1633A) was discovered in four cases and E545D (G1635T)
in one case (Table 2). In addition, a novel mutation that has
not previously been reported in the literature was confirmed in
exon 20 of an UEC tumor. This mutation was an A-to-T
transversion, thus replacing isoleucine with phenylalanine
(I1058F; Fig. 1). DNA isolated from the patient’s normal tissue
revealed a wild-type sequence, confirming the somatic nature
of this alteration.
Fourteen (48%) of the 29 CAH cases harbored a PTEN
mutation; however, there were no cases that contained both a
PTEN and PIK3CA mutation. Of the 17 PTEN mutations found
in 14 CAH cases, eight were frameshift mutations; three were
nonsense mutations that coded for a truncated protein
product; and six were missense mutations. The frequency of
PTEN mutations was not significantly different in CAH
compared with UEC. Twenty-five (57%) of the 44 UEC cases
analyzed had a PTEN mutation, and 12 (48%) of these
Results
Two (7%) PIK3CA mutations were found in 29 uterine CAHs
compared with 17(39%) mutations in 44 cases of UEC
(P = 0.003; Table 1). All PIK3CA mutations identified were
missense mutations in a single allele. The two CAH cases with a
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Fig. 1. Novel PIK3CA mutation. A-to-T transversion, resulting in a transformation
of isoleucine to phenylalanine (I1058F) in UEC.
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Human Cancer Biology
25 cases also contained a PIK3CA mutation (Fig. 2). PIK3CA
mutations were not found to be significantly clustered with
PTEN mutations and were also seen in 26% of UEC tumors
without a PTEN mutation (P = 0.21). An observed difference in
the coexistence of PIK3CA and PTEN mutations between CAH
and UEC (0 of 29 versus 12 of 44) was highly statistically
significant (P = 0.002). The frequency of two or more PTEN
mutations was similar between the two groups and was found
in two CAH and five UEC tumors.
The distribution of PIK3CA mutations was not correlated
with grade or stage of disease in the UEC cases. Of the 23 grade
1 tumors in the UEC cohort, 11 (48%) contained a PIK3CA
mutation, whereas 4 of 14 (29%) grade 2 tumors and 2 of 7
(29%) grade 3 tumors contained a PIK3CA mutation. When
UEC cases with PIK3CA mutations were compared with those
without a PIK3CA mutation, 11 of 17 (65%) cases with
PIK3CA mutations were grade 1 tumors, whereas 12 of 27
(44%) cases without PIK3CA mutations were grade 1 tumors
(P = 0.23). Five of the 44 UEC cases had associated CAH, and
two of these cases contained PIK3CA mutations. Interestingly,
both of these tumors were grade 1 carcinomas that also
contained PTEN mutations.
Discussion
Endometrial cancer remains the most common gynecologic
malignancy in the United States with over 41,000 women
diagnosed each year and 7,350 women expected to die from
their disease in 2006 (10). The most common histologic
subtype, UEC, has been shown to arise from the precursor
lesion CAH. Observational studies have found that up to
29% of women with CAH will eventually develop invasive
carcinoma if left untreated (11). Previous studies have also
shown that inter-observer agreement by gynecologic pathologists is lowest when making the diagnosis of atypical
hyperplasia (12). In addition, a recent study conducted by
the Gynecologic Oncology Group showed that 42.6% of cases
with a diagnosis of CAH on endometrial biopsy had
adenocarcinoma in the uterus when hysterectomy was done
shortly following the biopsy (13). Thus, treatment of uterine
hyperplasia in the thousands of women diagnosed each year
remains a clinical dilemma. Currently, the standard of care for
women diagnosed with CAH is to recommend a hysterectomy.
However, these women are often between the ages of 30 and
50 years, and many may desire future fertility. In this situation,
uterine preservation becomes of critical importance, and
clinicians find that hysterectomy may not be the most
appropriate treatment in this patient population. These studies
stress the importance of identifying those patients with CAH
who will develop UEC, to potentially tailor therapeutic
interventions. Currently, there are no markers that can
determine which women are at risk of developing carcinoma;
thus, a large number of women undergo hysterectomy
unnecessarily.
Previous studies have found mutations of PTEN and K-RAS
as well as the presence of microsatellite instability in CAH
(1, 2, 14, 15), providing evidence that these are early genetic
alterations contributing to the pathogenesis of UEC. Recently, a
high percentage of mutations of the oncogene PIK3CA were
identified in endometrial carcinoma (4). Given the fact that
PTEN functions primarily to counteract the effect of PI3K and
the recently reported role of PIK3CA mutants in invasion, we
were interested in determining the status of PIK3CA in CAH
and its relationship to PTEN mutations.
Our finding of a significantly lower frequency of PIK3CA
mutations in CAH compared with UEC (7% versus 39%)
provides evidence that PIK3CA mutations occur as a late event
in endometrial cancer pathogenesis. This is comparable with
colorectal tumors, in which 2 (3%) of 76 premalignant tumors
were found to have a PIK3CA mutation, both of which were
advanced tubulovillous adenomas, compared with a 32%
prevalence of PIK3CA mutations in colon cancers (3). We
found no significant difference in the frequency of PTEN
mutations between CAH (48%) and UEC (57%; P = 0.64),
indicating that PTEN mutations precede PIK3CA mutations in
endometrial tumorigenesis. Mutations of PIK3CA and PTEN
likely occur as independent events, as PIK3CA mutations were
present in UEC with and without PTEN mutations. The high
Fig. 2. A, H&E stain of CAH containing
a PTEN mutation. B, H&E of UEC containing
PIK3CA and PTEN mutations. C, DNA
sequence of an A-to-T transversion in PTEN,
resulting in a nonsense mutation (K144X)
in CAH. D, A-to-G transition, causing a
PIK3CA missense mutation (H1047R) in
UEC. E, C-to-T transition, resulting in a
nonsense mutation (R130X) of the PTEN
gene in UEC.
Clin Cancer Res 2006;12(20) October 15, 2006
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PIK3CA and PTEN Mutations in Uterine CAH and UEC
frequency of PIK3CA and PTEN mutations in our UEC cohort,
as well as a recent study showing that 36% of the endometrial
cancers studied contained a PIK3CA mutation (4), provide
evidence that the PI3K/AKT pathway can be activated by
alterations in multiple genes. Furthermore, it suggests that
PTEN and PIK3CA mutations may have additional nonoverlapping consequences during endometrial tumorigenesis.
CAH is often associated with progression to grade 1 UEC
rather than higher-grade carcinomas (13). We examined grade
as a possible factor associated with the presence of PIK3CA
mutations and found that the majority of UEC cases with
PIK3CA mutations were grade 1 tumors. It does not seem that
PIK3CA mutations are significantly more associated with highgrade tumors. Thus, the higher rate of PIK3CA mutations in
UEC versus CAH cannot be explained based on tumor
differentiation.
Thirty-nine percent of the UEC cases evaluated contained a
PIK3CA mutation, confirming that PIK3CA is the most
commonly mutated oncogene yet identified in endometrial
carcinoma. Thus far, K-RAS has been thought to be the most
commonly mutated oncogene, with mutations reported in up
to 26% of UEC (15 – 17). Twelve of the 19 PIK3CA mutations
seen in the entire cohort of 73 cases were mutations in codon
545, located in the helical domain, and codon 1047, found in
the kinase domain of the p110-a subunit. These are the two
most commonly mutated codons seen in other epithelial
cancers as well (3, 18, 19), and the mutations E545K and
H1047R, in particular, have been shown to be oncogenic (6, 8,
20, 21). These mutations cause increased lipid kinase activity
and result in elevated PI3K expression, both in vitro in
mammary epithelial cells (21, 22), colorectal cancer cell lines
(8, 20), and chicken embryo fibroblasts (6) as well as in vivo in
chickens (7) and nude mice (8). PI3K is a well-established
enzyme that activates the AKT-mammalian target of rapamycin
pathway and thus promotes tumorigenesis via increased cell
proliferation, cell survival, and motility (23). Recently,
functional studies of the specific mutations H1047R and
E545K in the PIK3CA gene have shown that these mutations
result in constitutive activation of AKT, producing a resistance
to apoptosis and increased cell migration, invasion, and
metastases (8). Our findings are consistent with these studies
in that PIK3CA mutations are significantly associated with UEC
compared with CAH and, thus, seem to serve as a marker for
invasive disease. The clinical implications are apparent, and
diagnostic screening tools for CAH may be developed, in which
cases harboring PIK3CA mutations would be treated as invasive
cancer, whereas those without this gene mutation would be
candidates for a more conservative approach, such as pharmacologic treatment with progestins. In additions, agents that
block the mammalian target of rapamycin, such as rapamycin
and its derivative RAD001, inhibit PIK3CA-induced cellular
transformation in vitro (6) and tumor growth in vivo (7). Our
findings further establish the AKT pathway as a major
mechanism through which uterine endometrioid tumorigenesis occurs. Thus, possible therapeutic interventions using
molecular targeted agents that would regulate this pathway
may play a role in the prevention and treatment of endometrial
carcinoma.
References
1. Levine RL, Cargile CB, Blazes MS, van Rees B,
Kurman RJ, Ellenson LH. PTEN mutations and
microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid
carcinoma. Cancer Res 1998;58:3254 ^ 8.
2. Maxwell GL, Risinger JI, Gumbs C, et al. Mutation of
the PTEN tumor suppressor gene in endometrial
hyperplasias. Cancer Res 1998;58:2500 ^ 3.
3. Samuels Y, Wang Z, Bardelli A, et al. High frequency
of mutations of the PIK3CA gene in human cancers.
Science 2004;304:554.
4. Oda K, Stokoe D, Taketani Y, McCormick F. High frequency of coexistent mutations of PIK3CA and PTEN
genes in endometrial carcinoma. Cancer Res 2005;
65:10669 ^ 73.
5. Shayesteh L, LuY, Kuo WL, et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 1999;
21:99 ^ 102.
6. Kang S, Bader AG, Vogt PK. Phosphatidylinositol
3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A 2005;102:802 ^ 7.
7. Bader AG, Kang S, Vogt PK. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proc Natl Acad
Sci U S A 2006;103:1475 ^ 9.
8. Samuels Y, Diaz LA, Jr., Schmidt-Kittler O, et al.
Mutant PIK3CA promotes cell growth and invasion of
human cancer cells. Cancer Cell 2005;7:561 ^ 73.
www.aacrjournals.org
9. Tashiro H, Blazes MS,Wu R, et al. Mutations in PTEN
are frequent in endometrial carcinoma but rare in other
common gynecological malignancies. Cancer Res
1997;57:3935 ^ 40.
10. Jemal A, Siegel R, Ward E, et al. Cancer statistics,
2006. CA Cancer J Clin 2006;56:106 ^ 30.
11. Kurman RJ, Kaminski PF, Norris HJ. The behavior
of endometrial hyperplasia. A long-term study of
‘‘untreated’’ hyperplasia in 170 patients. Cancer 1985;
56:403 ^ 12.
12. Kendall BS, Ronnett BM, Isacson C, et al. Reproducibility of the diagnosis of endometrial hyperplasia,
atypical hyperplasia, and well-differentiated carcinoma. Am J Surg Pathol 1998;22:1012 ^ 9.
13. Trimble CL, Kauderer J, Zaino R, et al. Concurrent
endometrialcarcinomain women withabiopsydiagnosis of atypical endometrial hyperplasia: a Gynecologic
Oncology Group study. Cancer 2006;106:812 ^ 9.
14. EnomotoT, Inoue M, Perantoni AO, et al. K-ras activation in premalignant and malignant epithelial lesions
of the human uterus. Cancer Res 1991;51:5308 ^ 14.
15. Mutter GL, Wada H, Faquin WC, Enomoto T. K-ras
mutations appear in the premalignant phase of both
microsatellite stable and unstable endometrial carcinogenesis. Mol Pathol 1999;52:257 ^ 62.
16. Lax SF, Kendall B, Tashiro H, Slebos RJ, Hedrick L.
The frequency of p53, K-ras mutations, and microsa-
5935
tellite instability differs in uterine endometrioid and
serous carcinoma: evidence of distinct molecular
genetic pathways. Cancer 2000;88:814 ^ 24.
17. Prasad M,Wang H, DouglasW, Barakat RR, Ellenson
LH. Molecular genetic characterization of tamoxifenassociated endometrial cancer. Gynecol Oncol 2005;
96:25 ^ 31.
18. Campbell IG, Russell SE, Choong DY, et al. Mutation
of the PIK3CA gene in ovarian and breast cancer.
Cancer Res 2004;64:7678 ^ 81.
19. Levine DA, Bogomolniy F, Yee CJ, et al. Frequent
mutation of the PIK3CA gene in ovarian and breast
cancers. Clin Cancer Res 2005;11:2875 ^ 8.
20. Ikenoue T, Kanai F, Hikiba Y, et al. Functional analysis of PIK3CA gene mutations in human colorectal
cancer. Cancer Res 2005;65:4562 ^ 7.
21. Isakoff SJ, Engelman JA, Irie HY, et al. Breast cancer-associated PIK3CA mutations are oncogenic in
mammary epithelial cells. Cancer Res 2005;65:
10992 ^ 1000.
22. Zhao JJ, Liu Z, Wang L, Shin E, Loda MF, Roberts
TM. The oncogenic properties of mutant p110a and
p110h phosphatidylinositol 3-kinases in human mammary epithelial cells. Proc Natl Acad Sci U S A 2005;
102:18443 ^ 8.
23. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655 ^ 7.
Clin Cancer Res 2006;12(20) October 15, 2006
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PIK3CA and PTEN Mutations in Uterine Endometrioid
Carcinoma and Complex Atypical Hyperplasia
Monica Prasad Hayes, Hong Wang, Rosanny Espinal-Witter, et al.
Clin Cancer Res 2006;12:5932-5935.
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