loss of heterozygosity and mutational analysis of the pten ... · north texas. dr. muller is an...

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.

on April 4, 2020. © 1998 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

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Ovarian histology

EndometrioidEndometrioidROVb clear cellLOVC endometrioid

EndometrioidEndometrioidEndometrioidEndometrioid

EndometrioidEndometrioidClear cell

Caseno. Age

Endometrialhistology

51 46 Endometrioid52 49 Endometrioid

53 62 Adenoacanthoma”

54 60 Endometrioid55 55 Endometrioid56 68 Endometrioid57 50 Endometrioid58 52 Endometrioid59 41 AdenoacanthomaSlO 73 Clear cell

Ml 49 EndometrioidM2 8! Pap serous”M3 70 EndometrioidM4 70 EndometrioidMS 44 AdenosquamouseM6 73 EndometrioidM7 74 Pap serousM8 80 EndometrioidM9 69 Pap serousMlO 79 EndometrioidMl I 66 Endometrioid

Ovarianstage/grade

IC/G2IA/Gl

LIC

IC/G3HB/G2

IHB/G1IA/Gl

IC/GlIIC/GlIIC

Endometrialstage/grade

IB/G2IA/Gl

IHA/G3

IB/G1

IB/G2

IB/Gl

IB/Gl

IAJG1

UA/Gl

IAfflC/G2HICIVB/G3fflC/G3fflC/G2IHC/G2IVBfflC/G3ifiCIHA/G2ifiC

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-



Clinical Cancer Research 2579

hancement gels (FMC Bioproducts, Rockland, ME) with and

without 10% glycerol at 5 W overnight and subjected to auto-

radiography for 24 h.

DNA Sequencing. Shifted SSCP bands were excised

from Mutation Detection Enhancement gels. The DNA was

eluted with distilled water and reamplified using the original

PCR primers. The reamplified PCR products were run on a 2%

agarose gel, excised, and purified by Geneclean (BlO 101, Inc.,

Vista, CA). Automated bidirectional sequencing was performed

on the purified DNA template using the ABI 377 Dye Termi-

nator cycle sequencer (Perkin-Elmer Corporation, Foster City,

CA). Sequences were analyzed and compared with the wild-type

PTEN/MMACJ sequence (GenBank Accession nos. AF000726

to AF000734; www.ncbi.nlm.nih.gov) with DNAStar software

(DNAStar, Inc., Madison, WI).

LOH Analysis. To evaluate LOH, a panel of four mic-

rosatellite markers were used, of which two are flanking

the PTEN/MMAC1 gene (D10S185, GDB 187826; D10S215,

GDB 188322), and two are intragenic to PTEN/MMACJ

(AFMO86wg9, GDB 1222085; D10S2491, GDB 6654876). The

primer sequences were obtained from the GDB (www.gdb.org).

A two-round multiplex PCR method was used to amplify the

microsatellite markers from microdissected cells as described

previously (24). Multiplex (four primer sets in each reaction)

PCR was done during the first amplification, followed by uni-

plex PCR for the individual markers. These results were con-

firmed in duplicate. The final product was separated on a 6%

denaturing polyacrylamide gel and subjected to autoradiogra-

phy. The sizes and relative intensities of alleles from the normal

stroma and invasive tumors were compared directly. Markers

that identified two distinguishable alleles of different sizes but

similar intensities in the lane having constitutional normal DNA

were termed as “informative” (i.e., heterozygous). Markers that

depicted a single allele in the normal DNA were termed as

“noninformative” (i.e., homozygous). Visual detection of com-

plete absence of either allele was required to designate LOH at

that marker due to the precision of the microdissection tech-

mque. Markers that gave repeatable shifted bands or expanded

bands as compared with the normal control DNA were termed

as MAs.

Statistical Analysis. The difference in the frequency of

PTEN/MMACJ mutations and MA between the clinically syn-

chronous tumors and the known metastatic tumors were ana-

lyzed by two-sided x2 or Fisher’s exact test. The association

between LOH at PTEN/MMACJ markers and PTEN/MMACJ

mutations were also analyzed by two-sided x2 or Fisher’s exact

test. P < 0.05 was considered significant.

ResultsSSCP and Sequence Analysis. The entire open reading

frame of PTEN/MMACJ was analyzed using 13 primer pairs

across nine exons from 10 matched cases of primary synchro-

nous endometrial and ovarian carcinomas with respective nor-

mal lymph nodes and 1 1 cases of known metastatic endometria!

carcinomas with matched metastatic and normal lymph node

tissues. SSCP analyses were performed to screen for potential

mutations on a total of 37 samples (21 endometrial, 10 ovarian,

and 6 metastatic lesions). Case S3 had two separate ovarian

primaries (endometrioid and clear cell); however, only the clear

cell ovarian primary was available for adequate evaluation. The

overall incidence of PTEN/MMACJ mutation in our series of

endometrial cancer is 43%. Of the 10 matched primary synchro-

nous endometrial and ovarian tumors, five cases (50%) of the

endometrial carcinomas demonstrated PTEN/MMACJ muta-

tions. Interestingly, two of these five cases (51 and 52) demon-

strated the identical mutations in the corresponding ovarian

tumor (Fig. 1A; Table 2), whereas the corresponding ovarian

tumors in the three remaining endometrial cancers with PTEN/

MMACJ mutations (53, 54, and 55) demonstrated the wild-type

PTEN/MMACJ band pattern by SSCP (Fig. 1B). Of the 11

known metastatic endometrial carcinomas, 4 cases (36%) of

either endometrial carcinomas or the metastatic sites were found

to have PTEN/MMACJ mutations (Table 2). Because of the

availability of metastatic tissues (many metastases were small

microscopic foci of tumor cells within nodal tissues), only six

matched metastatic cases were adequate for SSCP analysis

(Table 3). As expected, three metastatic cases (Ml, M2, and

M3) showed common PTEN/MMACJ mutations in both the

endometrial carcinomas and the matched metastatic tumor site

(Fig. 1 C). However, one metastatic endometrial cancer (M4)

revealed a PTEN/MMACJ mutation in endometrial cancer cells

within one of two paraaortic lymph nodes tested and not in the

primary endometrial tumor. In all cases that demonstrated iden-

tical PTEN/MMACJ mutations (51, 52, Ml, M2, and M3), no

aberrant SSCP bandshifts were found in the normal lympho-

cytes, therefore excluding the possibility of germ-line muta-

tions.

LOH Analysis. All 21 patient cases (21 endometrial

carcinomas, 10 ovarian carcinomas, and 1 1 metastases) were

analyzed for LOH within the PTEN/MMACJ region using four

microsatellite markers, two flanking the PTEN/MMACJ gene

(D10S185 and D10S215) and two intragenic (AFMO86wg9 and

D10S2491) to the PTEN/MMACJ gene (Table 3; Fig. 2). Case

Ml demonstrates loss of the upper allele in both the endometrial

and metastatic tumors consistent with its metastatic biology

suggested by the clinical stage and molecularly by the PTEN/

MMACJ mutational analysis (Fig. 2; Table 2). Case S3 has loss

of the upper allele only in the ovarian tumor (Fig. 2). Because

case 53 demonstrated PTEN/MMACJ mutation only in the en-

dometrium, LOH seen at this locus likely represents an inde-

pendent acquired event in the synchronous primary ovarian

tumor. Case M8 shows loss of the upper allele only in the

metastatic tissue not seen in the endometrial primary tumor,

suggesting further alteration in the PTEN/MMACI gene locus in

the metastatic clonal population of tumor cells (Fig. 2). Com-

parison between case 53 and M8 demonstrates the unreliability

of using LOH as a sole predictor of metastatic clonality. As

expected, MAs were common and were found in 38% (16 of 42)

of all tumor sites, and there were no statistical differences

between the frequency of MAs in the clinically synchronous

group (7 of 20; 35%) versus the known metastatic group (9 of

22; 41%; P = 0.49). Of the 42 tumor specimens evaluated, LOH

at any PTEN/MMACJ marker was found in 55% (23 of 42).

Furthermore, PTEN/MMACJ mutations were detected in 52%

(12 of 23) of tumor sites with chromosome 10q23 LOH versus

26% (5 of 19) in tumor sites without LOH (P = 0.09).



A CaseS2 B CaseS4 C Case M3

N E 0 N E 0 N E M

I I

LI�..I*_ Shifted bWIdSExon 7

13 bp deletion

�

Shifted band

Exon 8

AtoG

+�AA�JGAAZJa��A 2� A5�.k57G2 � IC WC

Shifted bandsExon 8

4 bp deletion

++AAA AAAG �CA/(X�AA�(�G

Normal Stroma Normal Stroma Normal Stroma

Endometrial CA

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.


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.



Syn vs. Met” Sample Tumor site Mutation Exon Codon Predicted effect

Synchronous 51SiSI

EndometriumOvaryOvary

202 del TA202 del TAG425A

33S

6868

142

Stop at codon 72Stop at codon 72Arg to Gln

Synchronous S2S2

EndometriumOvary

792 del 13 bp792 del 13 bp

77

264264

Stop at codon 296

Stop at codon 296Synchronous S3 Endometrium G646A 7 216 Va! to MetSynchronous S4 Endometrium A992G 8 331 Asp to Gly

Synchronous SS Endometrium GS9A I 20 Gly to GluMetastasis to paraaortic node Ml

MlMl

EndometriumParaaortic nodeParaaortic node

800 del A800 del AC680T

777

267267227

Stop at codon 275Stop at codon 275Ser to Phe

Metastasis to iliac node M2M2M2

EndometriumIliac nodeEndometrium

97 del A97 del A228 del AT

224

343476

Stop at codon 53Stop at codon 53Tyr to Stop

Metastasis to omentum M3M3M4

EndometriumOmentumParaaortic node

955 del 4 bp955 del 4 bpG4SOT

88S

3193 19ISO

Thr to StopThr to StopGlu to AspMetastasis to paraaortic node

a Syn, synchronous tumors; Met, metastatic tumors.

Table 3 LOH analysis of PTEN/MMACI region

Case Tumor site” D10S185” AFMO86wg9” D10S2491” DJOS2JS” PTEN mutationc

SI ENDO EJ 0 0 +

SI OV EJ 0 0 +

S2 ENDO 0 0 +

S2 OV 0 0 +

S3 ENDO i::� 0 .4-

S3 OV 0 -

S4 ENDO i::� 0 +

S4 OV E:J 0 EJ -

S5 ENDO - 0 +

S5 OV EJ 0 E1 0 -S6 ENDO -S6 OV v� -Si ENDO 0 -

Si OV 0 r� -

S8 ENDO 0 .

S8 OV0 .

S9 ENDO E:J 0 t::i -

S9 OV - -

Sb ENDO 0 0 -

Sb OV 0 -

Ml ENDO - 0 +

MI MET - 0 EJ +

M2 ENDO c:� 0 E�J +

M2 MET EJ 0 EJ +

M3 ENDO 0 - +

M3 MET 0 +

M4 ENDO - 0 0 .

M4 MET - 0 +

MS ENDO 0 0 -

MS MET �?A

M6 ENDO 0 0 �l -

M6 MET 0 - ND

M7 ENDO EJ 0 EJ -

M7 MET EJ 0 �I ND

MS ENDO c:� 0 .

MS MET - 0 - -

M9 ENDO 0 -

M9 MET EJ 0 0 ND

Ml0 ENDO 0 -

MI0 MET 0 ND

MI! ENDO EJ 7/� c::� .

Mu MET ND

a ENDO, endometriurn; OV, ovary; MET, metastatic site.b 0, heterozygous; �, loss of heterozygosity; �, microsatellite alteration; 0, uninformative; blank, no amplification.

C ND, not done.


Table 2 PTEN/MMACJ mutations in synchronous and metastatic endometrial tumors



NEM NEO

‘4

Case

Marker D1OS18S D1OS18S

Ml S3

D1OS18S

M8


NEM

Fig. 2 LOH on chromosome

______ l0q23 within the PTENIMMACJgene locus. Representative exam-ples of chromosome l0q23 LOH

for marker DJOSJ8S are shownfor case Ml, M8 (advanced met-

astatic), and S3 (clinically syn-

chronous) cancers. N, normal

stroma; E, endometria! carcino-

ma; 0, ovarian carcinoma; M,metastatic site.

As a control population, our metastatic group also demon-

strated a similar incidence of PTEN/MMACJ mutation in endo-

metrial carcinoma. Three of the four matched tumors showed

common PTEN/MMACJ mutations in both the endometrial and

the metastatic lesions with the exception of patient M4, where

the PTEN/MMACJ mutation was reliably found in only one of

two paraaortic lymph nodes evaluated but not in the primary

endometrial tumor. This suggests that a late mutational event

occurred in a clonal metastatic focus in this case.

The ovarian tumor in case S 1, the paraaortic node in case Ml,

and the endometrium in case M2 each had two distinct PTEN/

MMACJ mutations, although we did not further study whether

these represented mutations in the same or alternate allele. In our

series in all cases of “double mutants,” at least one represented a

frameshift mutation introducing an early stop codon. These “double

mutations” have been reported in 16-38% of PTEN/MMACJ mu-

tations in endometrial carcinomas (13, 19). However, we also noted

that the ovarian tumor and endometrial tumor in case 51 shared an

additional loss within an intragenic marker. The requirement for

three genetic events to occur to inactivate the function of the

PTEN/MMACJ gene in the ovarian “metastasis” is unusual and

may represent the PTEN/MMACJ locus as a target for an alternate

mechanism of genomic instability. The presence of mutation in a

single metastasis in case M4 as well as the addition of a second

mutation in the endometrium in case M2 not seen in the metastasis

also lends support to this hypothesis. Because previous authors

have shown a high rate of PTEN/MMACJ mutations in the RER+

phenotype endometrioid endometrial cancers, it is possible that this

site is prone to errors in DNA mismatch repair (13). Although we

found a 38% incidence of MAs in our cases, this must not be

confused with the microsatellite instability represented by the

RER+ phenotype, because our cases were not classified for an

RER+ phenotype. MAs in a small focus of 500-1000 cells ob-

mined by precision microdissection has been described by other

authors (24), but its functional importance is unknown.

The majority of PTEN/MMACJ mutations (1 1 of 17; 65%)

lead to truncated proteins, suggesting a possible loss of phosphatase

activity and altered cell adhesion properties. Interestingly, all five

cases with identical mutation in the matched synchronous tumors

(Sl and 52) and the matched metastatic tumors (Ml, M2, and M3)

had bp deletions leading to predicted truncated proteins and thus

may be a predictor of the metastatic nature of the tumor. Loss of

heterozygosity analysis was also performed to determine the role of

PTEN/MMAC1 as a classic tumor suppressor gene in endometrial

cancer. The LOH analysis demonstrated a 55% LOH in at least one

PTEN/MMAC1 marker. Furthermore, the group with PTEN/

MMACJ LOH showed a relatively higher incidence of PTEN/

MMACJ mutations (52% versus 26%) with a trend toward signif-

icance (P = 0.09). These fmdings are all consistent with those of a

classic tumor suppressor gene and suggest that PTEN/MMACJ

may play a significant role in the pathogenesis of endometrial

adenocarcinoma.

In the LOH analysis, the patterns of loss of heterozygosity

or MA also provided information about the true synchronicity of

these tumors. Cases S3, 55, 58, 59, and 510 demonstrated

dissimilar LOH/MA patterns, suggesting different tumors.

Cases Sl, 52, 56, and S7 demonstrated similar LOH/MA pat-

terns, indicating the possibility of metastatic tumors. As ex-

pected, the known metastatic tumors demonstrated either similar

patterns of LOH/MA or progressive patterns of LOHIMA from

primary tumor to the metastatic site. Because of the quality of

paraffin-embedded DNA, several cases failed to amplify in

markers D10S2491 and D10S215. We believe that these are

largely due to the limitation of paraffin-embedded tissues, but

we recognize that this could also potentially represent homozy-

gous deletions within these markers.

Several epidemiological and clinicopathological studies

have shown that there are two pathogenetic forms of endome-

trial carcinoma. Type I (estrogen-related) neoplasm occurs in

younger, perimenopausal women and tends to be low grade,

whereas type II (non-estrogen-related) occurs in older postm-

enopausal women and is more virulent (30). The predominate

histological subtype demonstrating PTEN/MMACJ mutation as

described previously (13, 19) was the type I estrogen-dependent

endometrioid tumors, with the exception of few cases of adeno-

squamous carcinoma. The predominate histological subtype in

our series is also endometrioid, however, one case of advanced

endometrial adenoacanthoma (endometrioid cancer with squa-

mous differentiation) and one case of advanced metastatic pap-

illary serous (type II) endometrial carcinoma were also found to

have a PTEN/MMACJ mutation.

In summary, our analysis confirms the previously reported

PTEN/MMACJ mutation frequency in endometrial carcinomas,

which appears to be one of the most common mutational events

in the clinically synchronous endometrial cancers. We observed

common PTEN/MMACJ mutations in a subset of clinically




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.

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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

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