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Vol. 4, 2577-2583, November 1998 Clinical Cancer Research 2577
Advances in Brief
Loss of Heterozygosity and Mutational Analysis of the
PTEN/MMAC1 Gene in Synchronous Endometrial
and Ovarian Carcinomas1
w. Michael Lin, Eva Forgacs, David P. Warshal,
I. Tien Yeh, Joilyn S. Martin, Raheela Ashfaq,
and Carolyn Y. Muller�Department of Obstetrics and Gynecology [W. M. L., C. Y. Ml,Hamon Center for Therapeutic Oncology Research [W. M. L., E. F.,C. Y. Ml, and Departments of Pathology [R. A.], University of
Texas Southwestern Medical Center, Dallas, Texas 75235, and
Departments of Obstetrics and Gynecology [D. P. W., J. S. M.] andPathology [I. T. Y.], Abington Memorial Hospital, Abington,
Pennsylvania 19001
AbstractMutations of the human putative protein tyrosine phos-
phatase (PTEN/MMACJ) gene at chromosome 10q23 have
been found frequently in type I endometrial carcinomas.
Endometrioid adenocarcinoma is the most frequent histol.
ogy seen in patients with clinically determined synchronous
endometrial and ovarian carcinomas. We report a high
incidence of PTEN/MMACJ mutations and 10q23 loss of
heterozygosity (LOH) in patients with synchronous endome.
trial and ovarian carcinomas. Paraffin-embedded precision
microdissected tumors were analyzed for 10 matched syn-
chronous endometrial and ovarian cancers and 1 1 matched
control metastatic endometrial cancers. Single-stranded
conformation polymorphism analysis was used to screen for
mutations in all tumors and corresponding normal lympho-
cyte DNA. LOH was determined using a panel of four
microsatellite markers within the PTEN/MMACJ locus.
PTEN/MMACJ mutations were found in 43% (9 of 21) of the
endometrial cancers studied, similarly represented in the
clinically synchronous group (5 of 10 or 50%) and the
advanced metastatic group (4 of 11; 36%; P = 0.53). In two
of the five cases of clinically synchronous cancers, identical
or progressive PTEN mutations were found in both the
Received 6/17/98; revised 8/19/98: accepted 9/2/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.I Supported in part by the Reproductive Scientist Development Programthrough NIH Grant K12HD00849, the American Association of Obste-trician Gynecologist Foundation, and Cancer Research Foundation of
North Texas. Dr. Muller is an AAOGF-NICHD Fellow of the Repro-ductive Scientist Development Program. This study was presented at the
27th Annual Meeting of the Western Association of Gynecologic On-cologists, Napa, CA, May 20-23, 1998.
2 To whom requests for reprints should be addressed, at Hamon Centerfor Therapeutic Oncology Research, University of Texas SouthwesternMedical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9032.Phone: (214) 648-3026; Fax: (214) 648-8404; E-mail: [email protected].
endometrial and ovarian cancers, suggesting that the ovar-
ian tumor is a mestastasis from the endometrial primary.
PTEN/MMACJ mutations in the advanced endometrial can-
cers were similar in the corresponding metastases. In one
case, the mutation was seen in only one of two metastatic
lymph nodes. The LOH analysis demonstrated 55% LOH in
at least one PTEN/MMACI marker. These findings suggest
that the putative tumor suppressor gene PTEN/MMACJ
may be a viable molecular marker to differentiate synchro-
nous versus metastatic disease in a subset of clinically syn-
chronous endometrial and ovarian carcinomas.
IntroductionSynchronous primary neoplasms of the female reproduc-
tive tract occur in 0.7%-l.7% of all gynecological malignancies
with the majority involving both the endometnum and the ovary
( 1 , 2). Synchronous endometrial cancers have been seen in
12-50% of patients with newly diagnosed endometrioid ovarian
cancer (3, 4). Although most clinical reviews support a good
prognosis for early stage synchronous ovarian and endometrial
tumors (2-8), confidence of the correct staging can impact
significantly on recommendations for or against adjuvant treat-
ment. Women with synchronous endometrial and ovarian cancer
are significantly younger (5, 6, 9) than women with either
primary endometrial or primary ovarian cancers alone. This
suggests that perhaps a specific germ-line genetic predisposition
or sporadic gene target may play a role in this small subgroup of
cancers.
Recently, a new candidate tumor suppressor gene,
also called MMACI, was identified and localized to chromo-
some region l0q23.3 (10, 1 1). PTEN/MMACJ shares sequence
homology with the protein tyrosine phosphatase family as well
as with the cytoskeletal proteins, tensin and auxilin. PTEN/
MMACJ is found frequently mutated in gliomas, prostate can-
cers, melanomas, and endometrial cancers (10, 12, 13), with less
frequent mutations in breast and lung cancers (14, 15), and may
be associated with a metastatic phenotype (1 1). Germ-line mu-
tations were found in the PTEN/MMACJ gene for affected
individuals with Cowden disease, a hereditary autosomal dom-
inant cancer syndrome associated with a predisposition to thy-
roid, breast, endometrial, and skin cancers and in the Bannayan-
Zonana syndrome (16-18). Endometrial cancers due to
Cowden’s syndrome are rare, and ovarian cancers have not been
associated with either hereditary syndrome.
3 The abbreviations used are: PTEN, phosphatase and tensin homologuedeleted on chromosome ten; MMAC, mutated in multiple advancedcancers; SSCP, single-stranded conformation polymorphism: LOH. lossof heterozygosity; GDB, Genome Database; MA, microsatellite alter-ation; RER, replication error.
a Adenoacanthoma, endometrioid tumor with benign squamous differentiation.b ROy, right ovary.C LOV, left ovary.d Pap serous, papillary serous carcinoma.e Adenosquamous, endometrioid tumor with malignant squamous differentiation.
2578 PTEN Gene in Synchronous Endometrial and Ovarian Cancers
Table 1 Clinical features of 10 synchronous tumors and 1 1 metastatic tumors
Two recent studies have found the overall PTEN/MMACJ
mutation frequency to be 34-50% in endometrial carcinoma
(13, 19). PTEN/MMACJ mutations seem to represent the most
common defined genetic alteration identified to date in endo-
metrial cancer and strongly suggest that mutations in PTEN/
MMACJ play a significant role in the pathogenesis of the most
common type of endometrial adenocarcinoma. In primary ovar-
ian carcinomas, PTEN/MMACI mutations have not been de-
scribed in serous or mucinous epithelial ovarian cancers to date
(13, 20). However, PTEN/MMACJ mutations have been re-
ported in 21% of primary endometrioid ovarian carcinomas
(21).
In an attempt to identify a novel genetic marker for differ-
entiating synchronous endometrial and ovarian carcinomas from
metastatic endometrial carcinomas to the ovary, we analyzed 10
clinically determined matched synchronous cancers and 11
known metastatic endometrial carcinomas for mutations in the
PTEN/MMACI gene and alterations in the PTEN/MMACJ gene
locus.
Materials and MethodsTissue Samples. Paraffin-embedded archival histology
slides for 10 cases of matched primary synchronous endometrial
and ovarian tumors and 1 1 cases of matched primary endome-
trial and metastatic tumors were retrieved from the Department
of Pathology at the Abington Memorial Hospital, Philadelphia,
Pennsylvania, from 1992 to 1996. Cases were chosen based on
availabilities of adequate tumor and normal tissue. Cases were
reviewed by two skilled gynecological pathologists (I. T. Y. and
R. A.). Table 1 shows the histology and clinical features of 21
cases studied. The median age of the synchronous group was 55
years (range, 46-73), and the median age of the metastatic
group was 70 (range, 44-81). In the synchronous group (n =
10), there were 7 cases of pure endometrioid cancers, 2 cases of
adenoacanthomas, and 1 case of clear cell carcinoma. In the
metastatic group (n = 1 1), there were 7 cases of pure endo-
metrioid cancers, 3 cases of papillary serous cancers, and 1 case
of adenosquamous carcinoma. The staging for the majority of
cases in the synchronous group was stage I based on the Fed-
#{233}rationInternationale des Gynaecologistes et Obstetristes clas-
sification (22), whereas the metastatic group were all stages
III-IV (Table 1).
Microdissection and DNA Isolation. Regions of pri-
mary endometrial and ovarian cancers and metastatic implants
(peritoneum, lymph node, and omentum) were identified. Pre-
cision microdissection was performed on targeted tumor regions
to obtain pure tumor populations without normal tissue contam-
ination. Approximately 500-1000 tumor cells were microdis-
sected for each case. For SSCP analysis, tumor tissues were
scraped using sterile blades to achieve populations >70% tumor
cells. Matched normal nodal tissue or normal stroma or myo-
metrium were used as a source of constitutional normal DNA.
DNA was extracted using proteinase K, as described previously
(23).
SScP Analysis. SSCP analysis was performed on
genomic DNA prepared from scraped paraffin slides. Two-
round PCR was performed using 13 primer sets covering nine
exons of the PTEN/MMACJ gene as described previously (15).
The products were diluted 1:5 in formamide dye, heat dena-
tured, snap chilled, and run on 0.5 X Mutation Detection En-
AAACAGAAC5A� A: AC IC CA GGGAG � � � � /#{149}� �G. GA A A C Pt3 � p�
Endometrial CA
AAAJGAhT�AA G � � �A �3fiW�A
Endometrial CA
A C � p�3 � � � AA��AA�A�AA
Ovarian CA Omentum
Fig. I SSCP analysis of representative synchronous and advanced metastatic endometrial cancers. N. normal stroma; E. endometrial carcinoma: 0.ovarian carcinoma; M, metastatic site. Mutations accounting for the shifted bands are given in Table 2. A, case 52. Identical SSCP shifts are shownin both the endometrial and ovarian cancers. B, case 54. A SSCP shift is shown only in the endometrial cancer and not in the ovarian cancer. C, caseM3. Identical SSCP shifts are shown in both the endometrial cancer and the omental metastasis.
2580 PTEN Gene in Synchronous Endometrial and Ovarian Cancers
Discussion
Clinical differentiation between synchronous endome-
trial and ovarian tumors versus metastatic primary tumors is
challenging. The final diagnosis determines the necessity and
the types of adjuvant treatment recommended. Molecular
analysis targeting critical tumor suppressor gene loci in-
volved in endometrial carcinogenesis may be useful in deter-
mining the likelihood of simultaneous distinct primary tu-
mors (25-28). With the recent identification of PTEN/
MMACJ, a putative tumor suppressor gene that is likely the
single most commonly mutated gene in endometrial carcino-
mas, we evaluated PTEN/MMAC1 mutations in 10 clinically
synchronous endometrial and ovarian tumors and 1 1 known
metastatic endometrial carcinomas to determine its clinical
utility as a novel genetic marker.
In our study, PTEN/MMACJ mutations occurred in 43% of
the endometnal cancers, which is similar to previous reports
( 13, 19). We hypothesized that PTEN/MMACJ mutations may
be a more common event or alternatively an unrecognized
germ-line predisposition in the clinically synchronous tumors
because the mean age of patients is less than those with primary
cancer alone (5, 6, 9). However, we found no statistical signif-
icance in the incidence of mutations within these two groups
(50% versus 36%; P 0.53). This analysis is limited, however,
by a small sample size.
Five cases of the clinically synchronous tumors had PTEN/
MMACJ mutations, and two of the five matched pairs had
common mutations in both the endometrial and the ovarian
tumors. Although PTEN/MMACJ mutations have been reported
in primary endometrioid ovarian carcinomas (21), it is highly
unlikely that the identical mutations occurred during embryo-
genesis within the mullerian tissues or occurred spontaneously.
Therefore, these findings suggest that identical mutations in
both endometrial and ovarian tumors within these cases are
consistent with metastatic endometrial cancer to the ovary. The
clinical stage assigned to the endometrial cancer in case S 1 was
stage IB/G2, which would be predicted to have a 15-30%
metastatic risk and could explain the apparent IC ovarian cancer
as a metastasis (29). However, we were surprised to find the
same mutation in two clinically assigned stage IA/G 1 endome-
trial and ovarian cancers in case 52 that would predict a low
likelihood of metastasis in either primary organ site. Therefore,
our data suggest that the stage and grade alone cannot ade-
quately predict the synchronicity of these tumors.
synchronous endometrial and ovarian carcinomas, suggesting an
ovarian metastasis from the primary endometrial tumor rather
than independent synchronous tumors. Furthermore, we corre-
lated the LOH with PTEN/MMACJ mutations, supporting the
role of PTEN/MMACJ as a tumor suppressor gene in the patho-
genesis of endometrial carcinoma. These findings suggest that
the putative tumor suppressor gene PTEN/MMACJ may be a
viable genetic marker for differentiating clinically synchronous
endometrial and ovarian carcinoma from metastatic endometrial
carcinoma to the ovary.
AcknowledgmentsWe are grateful to Dr. John D. Minna for advice, support, and
review of the manuscript.
References1. Eisner, R. F., Nieberg, R. K., and Berek, J. S. Synchronous primary
neoplasms of the female reproductive tract. Gynecol. Oncol., 33: 335-
339, 1989.
2. Ayhan, A., Yalcin, 0. T., Tuncer, Z. S., Gurgan, T., and Kucukali, T.Synchronous primary malignancies of the female genital tract. Eur. J.Obstet. Gynecol. Reprod. Biol., 45: 63-66, 1992.
3. Czernobilsky, B., Silverman, B. B., and Mikuta, J. J. Endometrioid
carcinoma of the ovary. A clinicopathologic study of 75 cases. Cancer
(Phila.), 26: 1141-1152, 1970.
4. Kline, R. C., Wharton, J. T., Atkinson, E. N., Burke, T. W., Gersh-enson, D. M., and Edwards, C. L. Endometrioid carcinoma of the ovary:retrospective review of 145 cases. Gynecol. Oncol., 39: 337-346, 1990.
5. Pearl, M. L., Johnston, C. M., Frank, T. S., and Roberts, J. A.Synchronous dual primary ovarian and endometrial carcinomas. Int. J.Gynaecol. Obstet., 43: 305-312, 1993.
6. Falkenberry, S. S., Steinhoff, M. M., Gordinier, M., Rappoport, S.,Gajewski, W., and Granai, C. 0. Synchronous endometrioid tumors ofthe ovary and endometrium. A clinicopathologic study of 22 cases. J.Reprod. Med., 41: 713-718, 1996.
7. Garzetti, G. G., Ciavattini, A., Goteri, G., De Nictolis, M., Cignitti,M., Tranquilli, A. L., and Romanini, C. Endometrioid carcinoma of theovary. Retrospective study. Eur. J. Gynaecol. Oncol., 14: 51-55, 1993.
8. Choo, Y. C., and Naylor, B. Multiple primary neoplasms of the ovaryand uterus. hit. J. Gynaecol. Obstet., 20: 327-334, 1982.
9. Gitsch, G., Hanzal, E., Jensen, D., and Hacker, N. F. Endometrialcancer in premenopausal women 45 years and younger. Obstet. Gy-necol., 85: 504-508, 1995.
10. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S., Puc,J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S., Giovanella,B., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M., and Parsons, R.PTEN, a putative protein tyrosine phosphatase gene mutated in humanbrain, breast, and prostate cancer. Science (Washington DC), 275:
1943-1947, 1997.
11. Stock, P., Pershouse, M., Jasser, S., Yung, W., Lin, H., Ligon, A.,Langford, L., Baumgard, M., Hattier, T., Davis, T., Frye, C., Hu, R.,Swedlund, B., Teng, D., and Tavtigian, S. Identification of a candidatetumour suppressor gene, MMACJ, at chromosome l0q23.3 that is mu-tated in multiple advanced cancers. Nat. Genet., 15: 356-362, 1997.
12. Guldberg, P., thor Straten, P., Birck, A., Ahrenkiel, V., Kirkin,A. F., and Zeuthen, J. Disruption of the MMACJ/PTEN gene by deletionor mutation is a frequent event in malignant melanoma. Cancer Res., 57:
3660-3663, 1997.
13. Tashiro, H., Blazes, M., Wu, R., Cho, K., Bose, S., Wang, S., Li, J.,Parsons, R., and Ellenson, L. Mutations in PTEN are frequent in endo-
metrial carcinoma but rare in other common gynecological malignan-cies. Cancer Res., 57: 3935-3940, 1997.
14. Rhei, E., Kang, L., Bogomolniy, F., Federici, M. G., Borgen, P. I., andBoyd, J. Mutation analysis of the putative tumor suppressor gene PTEN/
MMACJ in primary breast carcinomas. Cancer Res., 57. 3657-3659, 1997.
15. Forgacs, E., Biesterveld, E., Sekido, Y., Fong, K., Muneer, S.,Wistuba, I., Milchgrub, S., Brezinschek, R., Virmani, A., Gazdar, A.,
and Minna, J. Mutation analysis of the PTEN/MMACI gene in lung
cancer. Oncogene, 17: 1557-1565, 1998.
16. Arch, E. M., Goodman, B. K., Van Wesep, R. A., Liaw, D.,Clarke, K., Parsons, R., McKusick, V. A., and Geraghty, M. T.
Deletion of PTEN in a patient with Bannayan-Riley-Ruvalcaba syn-drome suggests allelism with Cowden disease. Am. J. Med. Genet.,71: 489-493, 1997.
T., Wallin, G., Parsons, R., Longy, M., Larsson, C., and Eng, C. Somaticdeletions and mutations in the Cowden disease gene, PTEN, in sporadicthyroid Tumors. Cancer Res., 57: 4710-47 13, 1997.
18. Marsh, D. J., Dahia, P. L., Zheng, Z., Liaw, D., Parsons, R., Gorlin,R. J., and Eng, C. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat. Genet., 16: 333-334, 1997.
19. Risinger, J., Hayes, A., Berchuck, A., and Barrett, J. PTEN/MMACJmutations in endometrial cancers. Cancer Res., 57: 4736-4738, 1997.
20. Sakurada, A., Suzuki, A., Sato, M., Yamakawa, H., Orikasa, K.,Uyeno, S., Ono, T., Ohuchi, N., Fujimura, S., and Hon. A. Infrequentgenetic alterations of the PTEN/MMACJ gene in Japanese patients withprimary cancers of the breast, lung, pancreas, kidney, and ovary. Jpn. J.
Cancer Res., 88: 1025-1028, 1997.
21. Obata, K., Morland, S. J., Watson, R. H., Hitchcock, A., Chenevix-
Trench, G., Thomas, E. J., and Campbell, I. G. Frequent PTEN/MMACmutations in endometrioid but not serous or mucinous epithelial ovariantumors. Cancer Res., 58: 2095-2097, 1998.
22. Creasman, W. T. Announcement, FIGO stages, revisions. Gynecol.Oncol., 35: 125-127, 1989.
23. Hung, J., Kishimoto, Y., Sugio, K., Virmani, A., Mclntire, D. D.,
Minna, J. D., and Gazdar, A. F. Allele-specific chromosome 3p dde-tions occur at an early stage in the pathogenesis of lung carcinoma.J. Am. Med. Assoc., 273: 1908, 1995.
24. Wistuba, I. I., Behrens, C., Milchgrub, S., Virmani, A. K., Jagirdar,J., Thomas, B., loachim, H. L., Litzky, L. A., Brambilla, E. M., Minna,J. D., and Gazdar, A. F. Comparison of molecular changes in lungcancers in HIV-positive and HIV-indeterminate subjects. J. Am. Med.Assoc., 279: 1554-1559, 1998.
25. Symonds, D. A., Johnson, D. P., and Wheeless, C. R., Jr. Feulgen
cytometry in simultaneous endometrial and ovarian carcinoma. Cancer
(Phila.),61: 2511-2516, 1988.
26. Fujita, M., Enomoto, T., Wada, H., lnoue, M., Okudaira, Y., andShroyer, K. R. Application of clonal analysis. Differential diagnosis forsynchronous primary ovarian and endometrial cancers and metastatic
cancer. Am. J. Clin. Pathol., 105: 350-359, 1996.
27. Emmert-Buck, M. R., Chuaqui, R., Zhuang, Z., Nogales, F., Liotta,
L. A., and Memo, M. J. Molecular analysis of synchronous uterine andovarian endometrioid tumors. Int. J. Gynecol. Pathol., 16: 143-148, 1997.
28. Shenson, D. L., Gallion, H. H., Powell, D. E., and Pierem, M. Loss of
heterozygosity and genomic instability in synchronous endometrioid to-
mors of the ovary and endometrium. Cancer (Phila.), 76: 650-657, 1995.
29. Morrow, C. P., Bundy, B. N., Kurman, R. J., Creasman, W. T.,Heller, P., Homesley, H. D., and Graham, J. E. Relationship betweensurgical-pathological risk factors and outcome in clinical stage I and IIcarcinoma of the endometrium: a Gynecologic Oncology Group study.Gynecol. Oncol., 40: 55-65, 199!.
30. Kurman, R. J., Zaino, R. J., and Norris, H. J. Blaustein’s Pathologyof the Female Genital Tract, Ed. 4, pp. 439-486. New York: Springer-Verlag, 1994.
1998;4:2577-2583. Clin Cancer Res W M Lin, E Forgacs, D P Warshal, et al. carcinomas.PTEN/MMAC1 gene in synchronous endometrial and ovarian Loss of heterozygosity and mutational analysis of the