University of Groningen Hormonal and molecular aspects of ......between postmenopausal estrogen levels and endometrial cancer.1-4 Obesity leads to increased adrenal secretion of androgens

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Hormonal and molecular aspects of endometrioid endometrial cancerJongen, Vincentius Hubertus Willibrordus Maria

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Hormonal and molecular aspects of

endometrioid endometrial cancer

V.H.W.M. Jongen

Hormonal and molecular aspects of endometrioid endometrial cancer

Vincent Jongen

STELLING EN

Behorend bij het proefschrift

Hormonal and molecular aspects of endometrioid endometrial cancer

van

V.H.W.M. Jongen

Groningen

12 november 2008

1. Aromatase is de grote gemene deler in de carcinogenese van het endometrium­en mammacarcinoom (dit proefschrift).

2. De mate van ovariele stroma hyperplasie en de lokale productie van androgenen in het ovarium zijn aan elkaar gerelateerd in het endometrioid endometriumcarcinoom ( dit proefschrift ).

3. Gelijktijdige aanwezigheid van zowel androgenen als het enzym aromatase in het endometrium ondersteunt de hypothese dat lokale aromatase activiteit een rol speelt bij het ontstaan van endometrioid endometriumcarcinoom ( dit proefschrift).

4. Een zelfde relatie tussen aromatase en HER-2/neu bij endometrioid endometrium- en mammacarcinoom suggereert een vergelijkbare regulatie van aromatase expressie.

5. P53 positief endometrioid endometriumcarcinoom vertoont een biologisch gedrag dat lijkt op dat van non-endometrio'id endometriumcarcinoom.

6. Bepaling van de oestrogeen en progesteron receptor alpha is zinvol bij endometrioid endometriumcarcinoom ten behoeve van een meer op de individuele patient toegespitst behandelplan.

7. Voor de meeste vrouwen geldt dat hun eierstokken vrienden zijn, het is een bittere ervaring dat het opeens ook vijanden kunnen zijn.

8. Een ziekenhuis is een 24-uurs bedrijf en geen digitale papierwinkel.

9. Bezuiniging leidt niet alleen tot een afname van de thuiszorg maar tevens tot een toename hiervan (R. van Doom).

10. Een reeds gepromoveerde echtgenote is de beste prikkel om een proefschrift te voltooien, waarbij geldt: "twee promovendi op een kussen, daar komt niemand tussen".

11. In een goed huwelijk mag het best eens donderen. Als na elke donderdag maar weer een vrijdag komt (bron: onbekend).

C1..1,trJ!r. U

tvleGt',. he M Bi� Ii ,.lirck C

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Publication of this thesis was generously supported by Abbott Laboratorie� Bayer Schering Pharma, GlaxoSmithKline, Merck Serono, Novartis Oncology, Organon Nederland BV ( onderdeel Schering-Plough Corporation), and Sanofi Pasteur MSD, and is gratefully acknowledged.

Hormonal and molecular aspects of endometrioid endometrial cancer Thesis Rijksuniversiteit Groningen

Cover: immunohistochemical staining for aromatase expression in endometrioid endometrial cancer, using monoclonal aromatase 677 antibodies.

© V.H.W.M. Jongen, 2008

ISBN: 978-90-367-3543-8

Printed by: Drukkerij Bari et, Ruin en, The Netherlands

RIJKSUNIVERSITEITGRONINGEN

Hormonal and molecular aspects of endometrioid endometrial cancer

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

woensdag 12 november 2008 om 14.45 uur

door

Vincentius Hubertus Willibrordus Maria Jongen

geboren op 23 mei 1963 te Valkenswaard

Cl!nttale Medische

Bibliotheek Groningen

-�-

u M C G

Promotores: Prof. Dr. A.G.J. van der Zee Prof. Dr. M.J. Heineman Prof. Dr. H. Hollema Prof. Dr. H.W. Nijman

Beoordelingscommissie: Prof. Dr. J.G. Aalders Prof. Dr. J.H.H.Thijssen Prof. Dr. P.H.B. Willemse

Opgedragen aan onze lieve Renske

Contents

Chapter 1 General outline of the thesis and aims of the studies.

Chapter 2 General introduction.

The postmenopausal ovary as an androgen-producinggland;

hypothesis on the etiology of endometrial cancer.

9

Maturitas 2002;43: 77-85. 15

Chapter 3 Aromatase in the context of breast and endometrial cancer.

A review.

Minerva Endocrinologica 2006;31:47-60.

Chapter 4 Ovarian stromal hyperplasia and ovarian vein steroid levels

in relation to endometrioidendometrial cancer.

35

British Journal of Obstetrics and Gynaecology 2003; 110:690-5. 67

Chapter 5 Is aromatase P450 involved in pathogenesis of endometrioid

endometrial cancer?

International Journal of Gynecological Cancer 2005; 15:529-36. 87

Chapter 6 Aromatase, COX-2, HER-2/neu and P53 as prognostic factors in

endometrioid endometrial cancer.

Accepted for publication in the International Journal of

Gynecological Cancer 107

Chapter 7 Expression of estrogen and progesterone receptors alpha and beta in

a large cohort of patients with endometrioid endometrial cancer.

Submitted. 131

Chapter 8 Summary and future perspectives. 153

Chapter 9 Samenvatting en overwegingen voor de toekomst. 165

Authors and affiliations 176

Dankwoord 179

List of publications 184

Curriculum vitae 187

General outline of the thesis and aims of the studies

9

10

This thesis concerns the expression and prognostic value of various hormones

and molecular markers playing a role in endometrioid endometrial cancer.

Especially we were interested in the enzyme aromatase, its expression and

(prognostic) role in endometrioid endometrial cancer. Endometrial cancer is the

most common genital tract malignancy in women. Two major histological types

can be distinguished; endometrioid endometrial cancer, and nonendometrioid

endometrial cancer including high-grade malignancies like serous papillary and

clear cell carcinoma. The most common form, involving more than 75% of all

cases of endometrial cancer is endometrioid carcinoma. Generally it is believed

that continuous estrogenic hyperstimulation of the postmenopausal endometrium

gives rise to endometrial tissue hyperplasia followed by atypical hyperplasia.

Eventually endometrioid endometrial cancer may develop. These estrogens

mainly originate from peripheral conversion in body fat of androgens,

originating from the adrenal glands and ovaries. The enzyme complex

cytochrome P450 aromatase, main subject of this thesis, plays a key role in the

(peripheral) conversion of androgens into estrogens.

However, it is possible that various hormonal pathways exist contributing to the

early development or promotion of endometrioid endometrial cancer. In this

context it cannot be excluded that increased production of androgens by

postmenopausal ovaries leads to increased availability of aromatizable

androgens, which may contribute to estrogen synthesis in the endometrial tissue

as well. Following the local conversion of ovarian androgens into estrogens, a

reaction catalyzed as well by the cytochrome P450 aromatase, estrogens can

function as a local mitogenic factor contributing to the development of

endometrial cancer. We consider the local availability of androgens and the local

activity of aromatase in endometrial tissue relevant for this process.

11

Shortly, in this thesis we will demonstrate that androgens are present in the local

hormonal environment of the uterus, and that aromatase expression is present in

the endometrial tissues. If so, this means that the local ingredients are available

to facilitate another pathway contributing to the development or promotion of

endometrioid endometrial cancer. In this view it may be possible that different

hormonal pathways exist, possibly next to or promoting each other, ultimately

leading to the development of endometrioid endometrial cancer.

Our studies will examme the following hypothesis, which will be further

elaborated in the Introduction (Chapter 2):

"The local availability of androgens in the utero-ovarian circulation, possibly

related to the amount of ovarian stromal hyperplasia, and the local availability of

aromatase activity in the endometrial tissue, support the hypothesis that ovarian

androgens contribute to the development of endometrioid endometrial cancer

through aromatase driven local conversion into estrogens."

Are there similarities between estrogen dependent breast and endometrial cancer?

The presence of aromatase expression appeared to be a relevant factor in the

development of breast cancer, and nowadays treatment with aromatase inhibitors

has found an essential place in breast cancer treatment. Breast cancer aspects

will be discussed in relation to endometrioid endometrial cancer, since both

disorders are partly estrogen dependent ( Chapter]).

To verify our hypothesis the following questions were asked:

1. Is the degree of ovarian stromal hyperplasia related to the presence of

endometrioid endometrial cancer? In a retrospective study, the degree of ovarian

stromal hyperplasia of patients with and without endometrioid endometrial cancer

was compared ( Chapter 4).

12

2. Are steroid levels in the utero-ovarian circulation related to the degree of

ovarian stromal hyperplasia, and to the presence or absence of endometrioid

endometrial cancer? In a prospective study steroid levels in the utero-ovarian

circulation of patients with and without endometrioid endometrial cancer were

compared to the degree of ovarian stromal hyperplasia ( Chapter 4).

3. Are androgen levels in the utero-ovarian circulation in endometrioid

endometrial cancer related to aromatase activity in endometrial and body fat

tissues? A prospective study was performed, in which aromatase activities in

endometrial tissue were related to the utero-ovarian venous steroid levels, using

tritium water release assays ( Chapter 5).

4. What is the expression and prognostic value of aromatase in endometrioid

endometrial cancer? Two retrospective studies were performed in archival paraffin

embedded tumour tissue, using tissue microarray analysis. We determined the

expression and prognostic value of the molecular markers aromatase in both

cancer and stromal cells, next to the prognostic value of COX-2, HER-2/neu and

P53 in a large cohort of endometrioid endometrial cancer patients. In a second

study using the same cohort of endometrial cancer patients, we assessed the

prognostic value of the estrogen and progesterone receptors alpha and beta in

endometrioid endometrial cancer. Expression of receptors and molecular

markers were determined by immunostaining and related to classical clinico­

histopathological parameters, in addition to disease-free and overall survival.

( Chapter 6 and 7).

In Chapter 8 and 9 our findings will be summarized and discussed in relation to

each other.

13

14

2

The postmenopausal ovary as an androgen

producing gland; hypothesis on the etiology

of endometrial cancer

VHWM Jongen

AVSluijmer

MJHeineman

Maturitas 2002;43:77-85

15

Abstract

Postmenopausal estrogens originate from the peripheral conversion of androgens

which are produced by the adrenal glands and the ovaries. Estrogens are

considered to contribute to the neoplastic development of endometrium.

Hyperplasia of ovarian stroma is associated with an increased androgen production

by the ovaries and with the development of endometrial pathology. We

hypothesize that, in cases of endometrial pathology, an increased production of

aromatizable androgens by postmenopausal ovaries will lead to elevated

prehormone availability for estrogen formation in utero. Following the conversion

of ovarian androgens, a reaction catalyzed by the cytochrome P450 aromatase,

estrogens may function as a local mitogenic factor eventually leading to the

development of endometrial cancer. We consider the local availability of

androgens and the local activity of aromatase relevant for this process. If this

hypothesis proves to be right it may give rise to the introduction of aromatase

inhibitors in treatment strategies of hormone dependent endometrial malignancies.

16

Introduction

Some endometrial cancers are considered to be estrogen-dependent. After

menopause estrogen biosynthesis is mainly peripheral. In fat and other tissues

androgens from the adrenal glands and the ovaries are converted to oestrogens:

androstenedione and estrone are converted to testosterone and estradiol

respectively under the influence of 17B OH-dehydrogenase, and androstenedione

to estrone and testosterone to estradiol by the enzyme complex cytochrome P450

aromatase. Continuous hyperstimulation of the endometrium by ( endogenous and

exogenous) estrogens is supposed to be the causative mechanism eventually

leading to cancer or tumour progression. A positive correlation was reported

between postmenopausal estrogen levels and endometrial cancer.1-4 Obesity leads

to increased adrenal secretion of androgens and increased peripheral conversion

of androgens to estrogens in adipose cells.5•6 Possibly androgen production by the

postmenopausal ovary relates to estrogenic stimulation of endometrial tissues.1- 12

Apart from estrogens of peripheral origin local activation or failure of the

inhibition of aromatase activity in the endometrium may promote estrogenic

stimulation of the endometrium and eventually give rise to neoplastic development

by local conversion of androgens to estrogens.11•13•14 Indeed aromatase activity was

demonstrated in endometrial tumours, whereas its expression was not detected in

disease-free endometrium!1•15 Whether systemically delivered or locally produced,

elevated estrogen levels will promote the growth of steroid-responsive tissues:

speculatively these estrogens may result from an increased estrone conversion

from androstenedione in adipose tissues, or from an increased ovarian and adrenal

androgen secretion. In this article, we discuss the available evidence that a causal

relationship may exist between elevated ovarian androgen production after

menopause, the presence of ovarian stromal hyperplasia, aromatase activity and

the development of hormone-dependentendometrial cancer.

17

Postmenopausal ovarian function

During recent years the concept was developed of the postmenopausal ovary as a

viable androgen-producinggland.1-10,12,16-20 Apart from changes in morphology the

ovary also appears subject to changes in function, producing 40% respectively

20% of the total amount of testosterone and androstenedione in postmenopausal

years.7•2 1 Steroid measurements across the postmenopausal ovarian gland showed

that there exists a testosterone gradient across the postmenopausal ovary .10, 12, 19•22 In

order to study hormone production of the postmenopausal ovary techniques were

developed for the collection of utero-ovarian vein blood.23 As the amount of

ovarian stroma increased, the androgen content of the ovarian vein increased .17•18

Almost all studies in postmenopausal women comparing utero-ovarian vein and

peripheral vein levels of estrone, 17B-estradiol, androstenedione and testosterone,

demonstrate that the utero-ovarian androgen level is elevated when compared with

the peripheral serum concentration.8-1 3•17-19•2 1-25 Testosterone seems to be the more

important androgen secreted by the postmenopausal ovary. After oophorectomy

testosterone and androstenedione levels in postmenopausal women indeed

dropped significantly.21•25•26 Postmenopausal ovarian testosterone production

appears to be at least partly gonadotrophin (LH) dependent, since pituitary down­

regulation with a GnRH-agonist resulted in a significant decrease of testosterone

levels.25•27 Elevated levels of luteinizing hormone (LH) were demonstrated in

postmenopausal women with endometrial cancer, which may possibly contribute

to increased ovarian androgen production.28 Receptors for LH and to a lesser

extent follicle stimulating hormone (FSH) are found in ovarian stromal cells.29

Ovarian androgen secretion after menopause could be stimulated by human

chorionic gonadotropin and LH. 30•31 Using an aromatase-specific monoclonal

antibody, the distribution of the aromatase cytochrome P450 enzyme was studied

in human ovaries. It was found that in premenopause ovarian follicles but not

18

stromal cells stained for the aromatase, whereas aromatase immunostaining was

present in three of seven postmenopausal ovaries. Rather conflicting results were

found recently in a small but interesting study in postmenopausal women with

complete adrenal insufficienci2 In the absence of adrenal steroids after

menopause, no circulating androgens were found, suggesting the climacteric ovary

not being a source of androgens at all. No hormonal changes were noted after

administration of human chorionic gonadotropin. Also no FSH and LH receptors

or aromatase P450 enzyme could be demonstrated in the postmenopausal ovaries.

A better understanding of postmenopausal ovarian endocrine function will help to

reveal more of the etiology of endometrial carcinoma.

After menopause the ovaries decrease in size, ovarian follicles disappear and the

ovary consists mainly of stromal cells. Histological distinction can be made

between postmenopausal ovaries with minimal stroma ( atrophic ovaries) and

ovaries with an increased volume of stroma (stromal hypeiplasia). It is suggested

that the extent of stromal hyperplasia correlates well with postmenopausal ovarian

steroid production, which would mean that any evaluation of the hormonal pattern

in postmenopausal women should also take into account the stromal characteristics

of the ovary.10•17• 18•33 Marked stromal hyperplasia cannot be considered a

premalignant condition as it may occur as well in benign as in malignant

conditions. We report a significant increase of ovarian stromal hyperplasia in case

of endometrioid endometrial cancer [submitted]. Contrary to our results Lucisano

found no correlation between the degree of ovarian stromal hyperplasia and

endometrioid endometrial cancer.17 Comparison with other studies is hampered by

the fact that the exact histological type of endometrial cancer most often is not

mentioned. Also widely varying percentages of endometrial cancer in these studies

prevent statements on odds ratio for developing endometrioid endometrial cancer

in the presence or absence of ovarian stromal hyperplasia.18•3 1

•34

•35

19

Endometrial cancer

Estrogens

Generally, two different clinicomorphologic forms of endometrial cancer have

been described: a low-grade malignancy related to unopposed estrogenic

stimulation, and a second, more virulent estrogen- independent form.36•37 This

second pathogenetic type, denominated nonendometrioid endometrial cancer,

includes poorly differentiated and high-grade malignancies like serous papillary

and clear cell carcinoma. Estrogen or progesterone receptors are rarely found in

these poorly differentiated, clear cell, and serous papillary uterine cancers, which

also do not respond to hormonal therapy !8•39 The former type is called endome­

trioid endometrial carcinoma, and arises mainly in women with obesity,

hyperlipidemia, hypertension or diabetes and signs of hyperestrogenism:

anovulatory uterine bleeding, nulliparity, early age at menarche and late onset of

menopause, and a background of hyperplasia of the stroma of ovaries or

endometrium. Obesity increases the risk, the postulated mechanism being incre­

ased conversion of androgens to estrogens in the adipose tissue. Sherman

demonstrated higher circulating estrogen levels in women with endometrioid

endometrial cancer than in women with serous carcinoma.40 Also, obesity was

associated with a decrease of sex hormone binding globulin production in the

liver and an increase of available free estradiol.16 The concept of continuous

estrogenic hyperstimulation of the postmenopausal endometrium was developed,

in which endometrioid endometrial cancer gradually develops from atypical

hyperplasia. In this view endometrial cancer is a disease of the proliferative effects

of estrogen on the endometrium, especially when unopposed by the differentiating

action of progesterone. Estrogenic stimulation drives cell proliferation and by the

increase of the number of cell divisions the risk of random DNA replication errors

(activation of oncogenes, inactivation of tumour-suppresion genes) increases.

Depending on the kind of genetic damage a malignant phenotype may arise with

20

progressive growth under the influence of estrogens. Thus, estrogens act as tumour

promotors but not as carcinogens. Initially anti-hormone therapy by e.g. progestins

is able to slow down the process of progression of cancer via different anti­

estrogenic mechanisms, including a reduction in estrogen receptors and an

increase in metabolic inactivation of estrogens, which suggests that interruption of

continuous estrogenic stimulation may disturb the process of neoplastic

deterioration, until a 'point of no return' is reached.5'36

'41 Progestins also counteract

the development of hyperplasia by inducing cellular differentiation.

Postmenopausal women have low estradiol levels and may be sensitive to even

small increases in estrogen levels, which may enhance mitotic rate and thus

increase the risk of malignant degeneration.16 Under continuous estrogenic

stimulation nearly one quarter of atypical hyperplasias progress to well­

differentiated endometrial carcinoma, indicating that atypical hyperplasia is a

precursor of endometrioid carcinoma.42 Endometrioid carcinoma is the most

common form of endometrial cancer, explaining for more than 75% of all cases.

These tumours, by definition, do not contain areas showing more than I 0% of

squamous, mucinous, or clear cell differentiation. Estrogen receptor generally

appears positive. Also, mixed endometrioid/serous carcinomas exist, which are

associated with endometrial hyperplasia in 46% of cases. Finally, 5-8% of serous

endometrial cancers are associated with endometrial hyperplasia.43 The

antiestrogen tamoxifen, used a.o. as an alternative endocrine treatment for

endometrial cancer, was associated with an increased risk of endometrial cancer

after menopause, so this strategy was left.44 It was suggested that tamoxifen

functions as an estrogen agonist in the estrogen-poor environment existing after

menopause, dependent on cumulative dose and duration of use.45

21

Androgens

Nagamani reported significantly higher ovarian venous levels of both

androstenedione and testosterone in thin women with endometrioid

adenocarcinoma of the endometrium compared to those in obese women with can­

cer.46 These findings could imply that an increase in prehormone availability from

the ovaries plays a role in the development of endometrioid cancer in thinner

women, who are also known to be more prone to nonendometrioid cancer. An

interrelation may exist between postmenopausal ovarian morphology, local

androgen production and hyperplastic and malignant diseases of the endometrium.

Remarkably the testosterone levels in the utero-ovarian vein blood samples from

women with endometrial carcinoma were significantly elevated as compared with

the utero-ovarian vein levels from women without endometrial carcinoma, without

significant increases in the peripheral levels of these androgens.47·48 Two large

epidemiologic studies report an elevation of plasma androstenedione level in case

of endometrial cancer, which also may reflect increased precursor availability for

peripheral conversion.2·4 In vitro the ovarian stroma of postmenopausal women

with endometrial neoplasm released significantly more androstenedione,

testosterone and dehydroepiandrosterone than that of women without cancer.49

Stromal hyperplasia in the postmenopausal ovary is related with an increase of the

ovarian vein levels of androstenedione and testosterone?· 18·33 Increased proouction

of aromatizable androgens by the ovarian stroma may lead to increased pre­

hormone availability for estrogen formation from peripheral conversion in fat

tissue. Alternatively, the demonstration of androgen receptors in the endometrium

by immuno-histochtmical techniques suggests a possible direct role of androgens

in causing endometrial cancer.50-52 However, it was suggested as well that intra­

tumoral aromatase is able to convert circulating androgens derived from adrenal

cortex and/ or ovary to estrogens, and thus give rise to a growth promoting effect

on endometrium. In a in vivo mouse model androgen excess had little growth-

22

promoting effect on a well-differentiated hormone-responsive human endometrial

cancer line.53 Possibly low androgen receptor expression influenced the results.

Aromatase

Aromatase activity

In postmenopausal women approximately 2% of circulating androstenedione is

converted to estrone, which can be further converted to the biologically more

potent estradiol by 17B OH-dehydrogenase. Aromatase is the key enzyme for

estrogen production. The conversion of androgens to estrone is catalyzed by

aromatase cytochrome P450 (p450arom, product of the CYP19 gene, which is

involved in steroidgenesis and steroid metabolism). Aromatase is responsible for

binding the C 19 steroid substrate and catalyzing the series of reactions leading to

formation of the phenolic A ring characteristic of estrogens. The enzyme

aromatase is found in ovarian granulosa cells, adipose tissue, placental

syncytiotrophoblast, adipose and skin fibroblasts, vascular endothelial and aortic

smooth muscle cells, bone and brain. Conversion of androstenedione to estrone

increases as a function of age, obesity and aromatase activity in extraglandular

(adipose) tissue.54 These peripheral tissues, especially adipose cells, are dependent

for substrate circulating androgens from primarily the adrenal cortex and to a

lesser extent from the ovaries.

Estrogens may thus originate in extragonadal sites where they act locally in a

paracrine or intracrine way and may only partly escape into the circulation

without significantly affecting circulating levels.55 This means that the total

amount of estrogens produced from these sites may be small, but the local tissue

levels may be high with consequently significant biological influences, as the

estrogenic effects of locally produced steroids add to and amplify the effects of

systemic estrogens. Taking into account the above, it is hypothesized that, apart

23

from extraglandular aromatase activity in adipose tissue, aromatase activity in the

endometrium may play a role in malignant degeneration.1 1 • 13 Earlier research by

Tseng et al on testosterone aromatization in the human endometrium showed

estrogen production in these tissues.56 Increased aromatization activity might result

in increased estrogen concentrations in neoplastic endometrium. Intratumoral

estrogens derived from in situ aromatization may function as an autocrine growth

and mitogenic factor, regardless of serum concentration of estrogens. Indeed

aromatase activity was found in endometrial malignancy, but not in normal

endometrial tissue. 1 1•56-62 As endometrial carcinoma tissue consists of different cell

types ( epithelial, stromal, malignant cells), localization of aromatization sites in

endometrial cancer is of importance. Aromatase immunoactivity was found

mainly in stromal cells of endometrioid endometrial carcinoma, but not in normal

endometrium or tissue with ( atypical) hypeiplasia. Also mRNA hybridization

signals of aromatase accumulated in stromal cells but not in neoplastic or

(atypical) hyperplastic cells (without stromal invasion). The distribution of

aromatase mRNA correlated well with the immunohistochemical localization of

aromatase. It was concluded that intratumoral aromatase in endometrial malignan­

cy is associated with stromal invasion and is expressed during the process of carci­

noma-stromal interaction. The presence of aromatase in stromal or interstitial cells

suggests that locally produced estrogens may act on carcinoma cells in a paracrine

way. Watanabe et al reported no correlation between aromatase expression or acti­

vity and steroid receptor status, clinical stage or histological grade. 15 Aromatase

activity was noted to increase proportionally with increasing concentrations of

insulin, suggesting that hyperinsulinemiamay predispose to endometrial neoplasia

by enhancing endogenous endometrial estrogen production.63 It has long been

recognized that conversion of androstenedione to estrone, and subsequently to

estradiol, increases with both age and body weight.64•65 These findings support the

alleged association between diabetes, obesity, aromatase activity and endometrial

cancer.

24

Gene expression

Regulation of aromatase ( over-)expression in endometrial cancer remains an

unsolved issue. Interaction between hormonal and genetic factors during the

development of precancerous lesions may eventually cause cancer, but it remains

still questionable which kinds of genetic predispositions favour malignant

degeneration The human CYP 19 gene contains a number of promotors that direct

tissue-specific aromatase expression. In adipose stromal cells this transcription is

regulated through the use of promotor 1.4 stimulated by glucocorticoids.66 In

endometrial cancer levels of aromatase transcripts were reported similar to those in

adipose tissue, whereas disease-free endometrium did not express aromatase.1 1•60

In small studies promotor II ( exon 1 d and 1 b) and 1.3 ( exon 1 c) seem to be

preferably used for aromatase expression in endometrial malignancy.1 1 • 1 4 Various

hormones, growth factors or cytokines may increase intracellular cAMP levels to

produce a signal transduction mechanism, responsible for stimulation of

transcription, in the end leading to local estrogen biosynthesis. Above observations

suggest that in endometrial cancer the normal mechanisms that inhibit aromatase

expression may be lost or, alternatively, that aromatase-stimulatingfactors or their

receptors become overexpressed. Women who may be at increased risk are those

who are not able to deal with endogenous or exogenous hyperestrogenism, due to

e.g. polymorphisms in estrogen metabolizing genes. Recently it was demonstrated

that CYP 19 polymorphism might be a genetic risk factor for the development of

endometrial cancer, as women carrying the A6 and/ or A 7 alleles of CYP 19 were

over-represented in patients with endometrial cancer. 67 Like CYP19 also CYPl 7

is involved in steroid production. Postmenopausal women who were homozygote

for the A2 allele of CYP 17 had higher estrogen and androgen levels.68

Mutation of the PTEN tumour suppression gene, which is found in 34-80% of

endometrioid endometrial malignancies and hyperplasia, has emerged as a primary

cause of cancer origin.69-7 1 A relationship between PTEN expression and steroid

25

hormone levels and thus hormonal endometrial risk factors is suspected: possibly

PTEN mutation enhances (pre-)malignant development as a promotor or initiator,

in this way genetically predisposing to the development of endometrial cancer.

PTEN immunostaining may be a marker for identification of premalignant lesions

that are likely to degenerate in a malignant fashion.69

Endometrial microsatellite instability, mutations m the ras oncogene and

replication error repair (RER) phenotype are found in 9 to 45% of endometrial

premalignancy and adenocarcinoma. Microsatellites are short repeating noncoding

units widely dispersed throughout the genome. The basis of microsatellite

instability is probably epigenetic silencing of DNA mismatch repair genes. It is

associated with neoplastic endometrium and may develop long before clinical

manifestation of malignant disease, but is rarely found in randomly sampled

histologicallynormal endometrial tissues.72•73

Possibly due to these genetic disorders or mutations clonal clusters are formed,

derived from common progenitor endometrial cells. In these proliferative

monoclonal expansions atypical endometrial hyperplasia may be histologically

diagnosed as a direct precursor of endometrial cancer.74 Whether aromatase

expression also plays a role in these hypothethical processes by promoting

protracted unopposed estrogen exposure remains questionable.

Aromatase inhibition in endometrial cancer

Although it is tempting to assume a causal relationship between ovarian stromal

hyperplasia, elevated androgen concentrations, and endometrial cancer, further

studies on aromatase activity in endometrial tissue are warranted. Treatment of

histocultures of endometrioid endometrial cancer with aromatase inhibitors

suggested depletion of in situ tumor estrogen, which might result in decreased cell

26

proliferation of tumor cells.75 The decreased cell proliferation due to aromatase

inhibitors in vitro appeared to be not directly related to intratumoral aromatase and

estrogen receptor status. Apart from their antagonistic oestrogenic action, anti­

aromatase agents have no in vivo oestrogen agonistic properties. In contrast to

tamoxifen aromatase inhibitors do not induce disturbing adverse events like

endometrial hyper- or neoplasia.76 A better understanding of intratumoral

aromatase in estrogen dependent malignancies like endometrioid endometrial

carcinoma may give rise to the potential development of endocrine therapy by

aromatase inhibitors, as already occured in breast cancer.77 Already in 1984

objective responses to the aromatase inhibitor aminogluthethimide were obtained

in four out of 18 women with advanced endometrial cancer.78 In vivo aromatase

inhibitors were dose-dependently able to inhibit aromatase activity in human

uterine tumours.79 A small pilot study showed decrease of aromatase activity in

endometrial tumour tissue after administration of the aromatase inhibitor

letrozole.80 In a recent Phase II study the use of the oral nonsteroidal aromatase

inhibitor anastrazole was studied in endometrial cancer.8 1 In a group of 23 women

with advanced or recurrent endometrial malignancy, most of whom had poorly

differentiated tumours and negative estrogen receptors, only two partial

responses were noted. However, only 39% of enrolled patients were diagnosed

with endometrioid endometrial cancer, the type of malignancy where aromatase

inhibibition might be of therapeutical benefit. The possible benefit of aromatase

inhibitors in well-differentiated or hormone-receptor-positive cancer in the

adjuvant situation remains yet to be ascertained.

Acknowledgements

We thank JLH Evers, J Grond, H Hollema, FH De Jong, HJMM Mertens, JG

Santema and AGJ van der Zee for their stimulation and support.

27

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34

3

Aromatase in the context of breast and endometrial

cancer. A review

VHWM Jongen

H Hollema

AGJ van der Zee

MJ Heineman

Minerva Endocrinologica 2006;31 :47-60

35

Abstract

Generally, estrogens are considered to be involved in the neoplastic

transformation of endometrium. After the menopause these estrogens mainly

originate from conversion of adrenal androgens by aromatization in body fat.

However, in case of stromal hyperplasia of the ovaries, it can not be excluded

that production of aromatizable androgens by postmenopausal ovaries leads to

increased availability of androgen precursors for intratumoural estrogen

synthesis in the endometrial tissue as well. The local presence of androgens and

the local expression and activity of aromatase is considered important for this

steroidogenesis. In this review, we will discuss the available evidence that

androgens, produced in hyperplastic ovarian stroma or body fat tissues, play a

role in the development of endometrial cancer through conversion into

estrogens, a reaction catalyzed in the endometrium by the enzyme aromatase

cytochrome P450. As the presence of aromatase appeared to be a

pathophysiological factor in the formation of breast cancer, the latter will be

evaluated in relation to the development of endometrioid endometrial cancer as

well, since both disorders appear partly estrogen dependent. As treatment with

aromatase inhibitors appeared feasible in breast cancer, current knowledge of

comparable treatment modalities in hormone dependent endometrial cancer will

be reviewed.

36

Aromatase

The enzyme complex aromatase plays an indispensable role in the production of

estrogens. Step by step cholesterol is converted to estrogen by steroidal

hydroxylation (Figure 1 ). Finally estradiol is biosynthesized from androgens by

aromatase. The enzyme complex aromatase consists of the cytochrome P450

aromatase enzyme, a haeme group, and an NADPH-reductase flavoprotein. 1

Cytochrome P450 is the product of the CYP19 gene. The enzyme aromatase is

localized in the endoplasmic reticulum of cells found in tissues as ovaries,

adipose tissue, placental syncytiotrophoblast, breast, bone and brain. Androgens

are important C 19 steroid precursor substrates for estrogen biosynthesis in these

tissues. Aromatase binds the androgen steroid substrate and catalyzes a series of

three consecutive reactions which will finally lead to estrogen production. After

two hydroxylation steps, the third aromatization step is defined by an oxidation

reaction in which cytochrome P-450 converts C19 androgens into C18

estrogens.2 In postmenopausal women approximately 2% of circulating

androstenedione is converted to estrone, which can be further processed to the

more potent estradiol.

From a clinicopathological point of view, estrogens are considered to be the

most important factor promoting cell growth of endometrioid endometrial

carcinoma. The principle precursors of estrogens are circulating androgens,

derived from primarily the adrenal cortex and to a lesser extent from the ovaries.

Before menopause the granulosa cells in the ovaries are the main source of

estrogens, after menopause the adipose tissue. The role of the postmenopausal

ovary in the production of hormonal precursors can not be neglected, as these

prehormones can be processed by a.o. the aromatase complex. After menopause,

the ovary still produces 40% respectively 20% of the total body quantity of

testosterone and androstenedione, which can be converted into estrogens. 3 With

37

increasing age, body weight and aromatase activity in extraglandular (adipose)

tissue more peripheral conversion of androgens to estrogens is observed. 4 The

contribution of aromatase activity to estrogen formation must be considered

important, as aromatization is the rate-limiting factor. So it seems logical that

endocrinological treatment of estrogen levels in hormone dependent tumours

focusses on inhibition and reduction of aromatization processes. In breast cancer

reduction of estrogenic stimulation became the main pharmacological target,

leading to the discovery that treatment with aromatase inhibitors is quite

effective in depriving the tumour of estrogenic growth stimulation.

Cholesterol ----+ Dehydroepiandrosterone ----+

+

Androstenediole

Androstenedione ----+ (J ) Estrone

t (2) t (2)

Testosterone ----+ ( J l Estradiol

Fig 1 . Pathways of sex hormone metabolism and biosynthesis, influenced by a.o. ( 1 )

aromatase, and (2) l 7beta-hydroxysteroid dehydrogenase activity.

Breast cancer and hormone-dependent endometrial cancer; biological

similarities or not?

The incidence of breast and endometrial cancer is near 28% and 1 0% of all

cancers in women. 5 Invasive breast cancer and endometrioid endometrial

carcinoma are both preceded by premalignant disorders, characterized by

estrogen induced hyperproliferation.6'7 Both breast and endometrioid

endometrial cancer are partly considered as estrogen sensitive and in both

bioconversion of androgens to estrogens plays a role in tumour development.

Aromatase expression and activity are of importance in this process . Although

endogenous hormones are not an easily modifiable risk factor, the hormone

38

dependent evolution m both disorders and the biological alterations and

differences ask for comparison, as prognostic factors and targets for

endocrinological therapy may be shared.

Breast cancer

Cumulative doses of unapposed estrogens over time increase the risk of

developing breast cancer, so a lower age at menarche, a higher age at menopause

and a higher body mass index have been associated with an increasing risk of

developing breast cancer.6 Prolonged lactation diminishes the number of

menstrual cycles and subsequently breast cancer risk decreases, whereas alcohol

consumption may increase breast cancer rbisk by increasing plasma estrogen

levels. Postmenopausal obesity causes increased conversion of androstenedione

to estrone in adipose tissue, and is an accepted risk factor for breast cancer.

Two distinctive clinicopathological pathways have been described in the

histological pathophysiology of breast cancer.8 Ductal carcinoma, accounting for

85% of invasive breast cancers, develops from terminal duct units, which give

rise to hyperplastic unfolded lobules, followed by atypical ductal hyperplasia,

ductal carcinoma in situ and beyond. A second pathway involves the so-called

lobular neoplasia, where atypical lobular hyperplasia evolves to lobular

carcinoma in situ. Finally invasive breast cancer establishes.

Nearly all premalignant lesions are estrogen receptor positive. The percentage of

estrogen receptor positive cells in breast cancer tissue differs.9 In one-third of

breast cancers, growth can be stimulated by estrogens, while tumour regression

has been observed after estrogen deprivation. 10 The most important prognostic

factor for response to hormonal treatment is the presence of estrogen or

progesterone receptors in the malignant tissues; in case of receptor positivity 60

to 70% of women respond to hormonal therapy, so estrogen receptor status is

39

considered as a prognostic biomarker predicting hormonal therapy sensitivity .

Breast cancers that have no or low estrogen receptor density do not appear to

require estrogen for their continued growth. These tumours are unlikely to

respond to therapy with aromatase inhibitors, designed to reduce intratumoural

estrogen levels. About 50 to 80% of breast cancers appear estrogen receptor

positive, but breast cancers with a more malignant histological profile are mostly

estrogen receptor negative, so it can not be excluded that breast cancer can

become estrogen receptor negative and estrogen independent as the tumour

progresses. Progesterone receptors are probably induced by estrogens through

the estrogen receptor and were observed in 40-45% of breast cancers. 1 1

Breast cancer is mainly diagnosed after menopause. Due to a decrease of

postmenopausal ovarian estrogen production plasma estrogen levels drop

significantly after menopause. However, as the majority of breast cancers

depend on estrogens for their growth and appear estrogen receptor positive,

growth of breast cancers is expected to depend more on local estrogen levels

than on circulating plasma estrogen levels. No significant differences in

intratumoural estrogen levels exist between pre- and postmenopausal women

with breast cancer. 1 2 Estrogens are produced locally within the breast tumour

after conversion of androgens due to the activity of the aromatase enzyme

complex, which enables the tumour to maintain high estrogen levels

independently of menopausal state. 1 •1 3 Estrogen biosynthesis in the tumour or

surrounding tissues is thought to be of importance. About 63 to 72% of breast

tumours appear to show measurable aromatase activity, but aromatase activity

between tumours can vary tremendously.1 4 Remarkably tumour aromatase

activity was found to be an independent endocrinological factor, not correlated

with estrogen or progesterone receptor status. 1 5 However, the exact localisation

of aromatase activity within the breast is still not certain. Controversial results

are reported concerning localisation of aromatase activity in tumour or adipose

40

stromal cells.5•1 6 Aromatase activity was detected in higher amounts in the

tumour-bearing part of the breast, but in breast fat tissues as well. 1 7 Possibly the

fat surrounding tumour cells is important, since cancer cells are often

surrounded by adipose stromal cells and adipocytes. To some degree

intratumoural aromatase activity was found to have predictive value as a

prognostic factor, but no consistent relationship was found between aromatase

activity in breast tumours and known prognostic factors like age, menopausal

status, tumour size, nodal status, estrogen and progesterone receptor

concentrations, tumour differentiation grade and survival. 1 8-2 1

Estrogen deprivation as a therapeutic target can be achieved by blocking the

hormone receptor with a selective estrogen receptor modulator (SERM) like

tamoxifen, which is first-line therapy for advanced breast cancer in

postmenopausal women and is most effective in estrogen receptor positive

disease. Alternatively, before menopause estrogen concentrations can be

lowered by ovariectomy or by the use of GnRH-analogues, but aromatase

inhibitors can be used as well, aiming at the cytochrome P450 enzyme complex

aromatase. Aromatase inhibitors are effective as second line treatment after

developing resistance to tamoxifen, as adjuvant treatment or as first line therapy

in metastatic disease. A major advantage of recently developed aromatase

inhibitors seem their improved efficacy and tolerability, when compared to the

earlier aromatase inhibitors. In the near future an even more prominent and first

line role for aromatase inhibitors is expected in the treatment of breast cancer.

The exact mechanism by which the use of aromatase inhibitors leads to tumour

reduction is still unknown, possibly apoptosis plays a major role. Most probably

a combination of reduced cell proliferation, increasing coagulative and extensive

hyelinisation of the cancer and stromal cells in correlation with host reactions

such as tumour infiltration by lymphocytes and/or macrophages are of

importance. 1 9 Until now, it remains difficult to determine aromatase positivity,

41

as conclusive results are hampered by the lack of specificity of the aromatase

antibodies used, which off course is a major problem and applies for endometrial

cancer as well, as will be discussed later on.

Endometrial cancer, estrogens and androgens

Analogous to breast cancer, carcinogenesis of endometrioid endometrial cancer

has been related to excessive life-time exposure to estrogens. Hyperlipidemia,

hypertension, diabetes and obesity increase life-time risk, probably due to incre­

ased conversion of androgens to estrogens in the adipose tissue. Ongoing

menopausal estrogen replacement therapy, sequential oral contraceptives,

chronic anovulation, anovulatory uterine bleeding, nulliparity, early age at

menarche and late onset of menopause increase the risk of developing

endometrioid endometrial cancer. This gives rise to the concept of continuous

estrogenic hyperstimulation of the postmenopausal endometrium, in which

endometrioid endometrial cancer gradually evolves from simple and atypical

hyperplasia. Diminished risks were observed in association with use of

combination-type oral contraceptives, and in case menopausal hormone

replacement therapy was combined with progestins, as progesterone response is

essential to control normal cell growth. With each pregnancy ( or lactation) the

life-time risk decreases, which is explained by the fact that no mitotic activity is

seen due to the continuously high placental production of progestagens.

Although both breast and endometrial cancer may develop under the influence

of estrogens, a major difference is evident: due to the endometrial capacity of

cyclic changes before menopause, progestins exert a protective action in

endometrial tissue. Conflicting data are present on the issue whether endogenous

progestins increase breast cancer risk, or not. 22•23 In the last two decades the

prevailing opinion has been that progesterone contributes to the development of

breast cancer, since exogenous progestins increase the breast cancer risk more

42

than estrogen alone when added to estrogen in hormone replacement therapy for

menopausal complaints.

On morphologic grounds endometrial cancer can be divided in two different

classes: endometrioid endometrial carcinoma related to unopposed estrogenic

stimulation, and a second estrogen independent form including poorly

differentiated and high-grade malignancies like serous papillary and clear cell

carcinoma, and mixed forms with endometrioid components. 24 Estrogen or

progesterone receptors are rarely present in these poorly differentiated cancers,

which also hardly respond to classic hormonal therapy. More than 75% of all

cases of endometrial cancers consist of estrogen and progesterone receptor

positive endometrioid endometrial cancers. As endometrioid endometrial cancer

has a relatively good prognosis, estrogen and progesterone receptor positivity

have been used as prognostic biomarkers. Especially progesterone receptor

positivity was associated with a more favourable outcome and disease-free

survival. 25 Endometrioid endometrial cancer gradually evolves from simple

hyperplasia to atypical hyperplasia and fullblown cancer. Under continuous

estrogenic stimulation nearly one quarter of atypical hyperplasias progress to

well-differentiated endometrial carcinoma. It is assumed that the increased

mitotic activity of the endometrium caused by estrogenic stimulation increases

the probability of the accumulation of random mutations, finally leading to

malignant transformation by activation of oncogenes, defective mismatch repair

genes and inactivation of tumour-suppressor genes. Continuous

hyperstimulation of the endometrium by ( endogenous and exogenous) estrogens

is supposed to be the causative mechanism eventually leading to endometrioid

endometrial cancer or its progression. However, androgens may play a role as

well. Several causative pathways may co-exist next to each other, or even

amplify each other, leading to the gradual development of endometrial cancer.

Since the ovary still produces considerable amounts of androgens after

43

menopause, these androgens may not only be processed to estrogens in

peripheral adipose tissues, but may also lead to elevated prehormone availability

for estrogen formation in utero. The hypothesis that ovarian androgens play a

role in endometrial carcinogenesis is supported by the fact that androgen levels

in the utero-ovarian veins from women with endometrial cancer were

significantly elevated as compared to women without malignant disease. 3•26-3 1

One may argue that ovarian androgens stimulate the development of endometrial

cancer through local aromatization into estrogens. In that case local aromatase

activity may give rise to high local estrogen concentrations which could result

in the stimulation of endometrial growth. Thus, in addition to extraglandular

aromatase activity through conversion of androgens into estrogens in adipose

tissue, aromatase activity in the endometrium itself may play a role in malignant

transformation. 32-34 The local availability of androgens and the presence of

aromatase activity within endometrial tissue, are the two essential conditions

supporting the hypothesis that estrogens may function as a mitogenic factor after

local conversion of ( ovarian) androgens. Thus, in this view some women may

develop endometrioid endometrial cancer through continuous hyperstimulation

of the endometrial tissue by estrogens systematically delivered from peripheral

conversion in body fat, while in other women malignant transformation is

enhanced by local conversion of ovarian androgens into estrogens, catalyzed by

(perhaps overexpression of) endometrial stromal aromatase cytochrome P450, or

both happen simultaneously. Possibly genetic risk factors like CYP19

polymorphisms increase the risk of developing endometrial cancer, as the normal

mechanisms that inhibit aromatase expression may be lost or aromatase­

stimulating factors or their receptors become overexpressed, causing

hyperestrogenism:5 The human CYP 19 gene contains a number of promotors that

direct tissue-specific aromatase expression. Especially women carrying the A6

and/or A7 alleles of CYP19 were over-represented in patients with endometrial

cancer.36 Remarkably, evidence was found that a correlation exists between CYP

44

19 gene expression and the endometrial cancer types until now generally

considered as hormone independent.36-38

Ovarian stromal hyperplasia and androgens

A relation may exist between the production of ovarian androgens and the degree

of ovarian stromal hyperplasia.32•39 Using the criteria developed by Boss, defining

stromal hyperplasia of the ovary as nodular or diffuse proliferation of ovarian

stroma, higher degrees of ovarian stromal hyperplasia were found in endometrioid

endometrial cancer.40 Also, with increasing degrees of ovarian stromal hyperplasia

levels of ovarian vein androgens were higher.32•4 1

-43 So a causal relationship in the

origin of hormone dependent endometrial pathology may exist between ovarian

stromal hyperplasia, ovarian vein androgen levels and endometrioid endometrial

carcinoma, with the aromatase complex as the main linking enzyme.

Aromatase and endometrial cancer

The question arises what evidence is available relating aromatase directly to

endometrial cancer? Aromatase activity in endometrial cancer was first

demonstrated by Tseng.44 Aromatase activity was found not only in endometrial

cancer, but also in endometriosis and adenomyosis. 1 7•33

•45

-53 Aromatase

immunoreactivity was demonstrated m stromal cells of endometrioid

endometrial carcinoma. 45•46 It was demonstrated that intratumoral aromatase in

endometrial malignancy is associated with stromal invasion and is expressed

during the process of carcinoma-stroma interaction. Varying levels of aromatase

expression responsible for local estrogen synthesis in endometrial cancer may

contribute to malignant degeneration. Possibly, aromatase expression inhibiting

mechanisms may be lost during the process of neoplastic transformation or

aromatase-stimulating factors are activated. No correlation was found between

aromatase expression or activity and steroid status, clinical grade or histological

45

stage, although aromatase expression was more often reported in low grade, than

in high grade stromal endometrial sarcomas, and higher expression of aromatase

was more frequently associated with advanced disease or more poorly

differentiated endometrial cancer.33•48

•54 One study reported aromatase expression

in 65% of endometrial cancers. A correlation was found between aromatase and

cyclooxygenase-2 expression, but not with estrogen or progesteron receptors,

indicating that indeed intratumoural production of estrogens in endometrial

cancer may be an important mechanism in carcinogenesis. 55

The presence of aromatase in stromal or interstitial cells rather than in the

epithelial carcinoma cells suggests that locally produced estrogens may act on

carcinoma cells in a paracrine way .48 Thus, intratumoural estrogens originating

from in situ aromatization may function as an extra local carcinogenic factor in

an already existing malignancy. Since invasive carcinoma may induce aromatase

activity in stromal cells, local estrogen production in an existing endometrial

cancer could play a role in its progression. Even a small increase in the quantity

of local estrogen synthesis may be of significance for carcinogenesis or tumour

progression, as in the microenvironment of the tumour tissue estrogen receptors

are available within the malignant cells.47 It may be that the presence of

aromatase within the endometrial tissues creates a growth advantage

independently of circulating estrogen levels. If so, aromatase positivity may be

associated with a negative prognosis for survival chances. As mentioned before,

indeed evidence was found that a correlation exists between CYP 19 gene

expression (and aromatase expression) and the so-called hormone independent

endometrial cancer types, which are generally considered to have a worse

prognosis than well differentiated endometrioid endometrial cancer.36-38 Increased

aromatase expression in breast and endometrial cancer, leiomyomas and

endometriosis do not give rise to elevated estrogen levels in the peripheral

blood. The relatively high aromatase activity in leiomyoma-derived smooth

46

muscle cells and (adipose) fibroblasts surrounding breast tumours suggest the

presence of significant quantities of locally produced estrogens in these tissues. 1 7

In human endometrial cancer cell lines a high aromatase activity correlated with

a high DNA synthesis responsiveness to steroid hormones, both estradiol and

testosterone. 46•56 Aromatase immunoreactivity was found in 67% of stromal cells

of endometrioid endometrial carcinoma, but not in hyperplastic or normal

endometrium. 48 Because marked aromatase expression was detected at the sites

of stromal invasion a role for aromatase in invasion was suggested.

Anti-estrogens and arom.atase inhibitors in breast and endometrial cancer

Effects of anti-estrogens like tamoxifen and aromatase inhibitors are illustrative,

as both may influence breast and endometrial cancer tissue in a different way.

Aromatase inhibitors suppress endogenous estrogen production without directly

interacting with estrogen receptors, and have a different mode of action than the

anti-estrogen, tamoxifen, which blocks estrogen receptors but leaves

endogenous hormone levels unchanged. Tamoxifen is a selective estrogen

receptor modulator with tissue-specific effects on breast and endometrium.

Tamoxifen competes with estradiol for binding to the estrogen receptor and

changes the receptor in such a way that downstream gene transcription is

inhibited. Possibly, intratumoural estrogens increase estrogen receptor gene

expression. It has been observed that breast tumours can become estrogen

receptor negative and tamoxifen resistant in the course of disease progression

and treatment. 1 6 Also in atypical hyperplasia of the endometrium and

endometrial cancer estrogen and progesterone receptor density decrease with

increasing histological grade. 57•58 In the endometrium, tamoxifen effects depend

on estradiol concentration: only in postmenopausal women estrogen-agonistic

properties predominate and the risk of developing endometrial cancer

increases. 59 As aromatase seems to play a significant role both in the

47

/

development of breast cancer and endometrial cancer, treatment targets may be

shared. The ATAC trial is a large phase III trial comparing primary adjuvant

tamoxifen with the aromatase inhibitor anastrozole for early breast cancer in

postmenopausal women.60 In the anastrozole group significantly less cases of

endometrial cancer or endometrial abnormalities occured, as compared to the

rate for the general population, suggesting that anastrozole offers a protective

effect on the endometrium.6 1•62 Also, median endometrial thickness appeared to

increase in the tamoxifen group, whereas it remained unchanged in the

anastrozole group, which could mean a protective role for aromatase inhibitors. 63

One may add that in breast cancer patients increased endometrial thickness,

related to treatment with tamoxifen after menopause, reversed following

treatment with aromatase inhibitors. 64 These findings are consistent with the

lack of correlation between aromatase activity and expression of estrogen and

progesteron receptors in breast and endometrial cancer. 1 6• 1 8• 1 9•48•65

In conclusion, pathophysiologic and therapeutic similarities and differences exist

between breast and endometrioid endometrial cancer. Both partly find their

origin in excessive exposure to unapposed estrogens and develop from

premalignant hyperproliferative disorders. Progestins may increase breast cancer

risk, but protect the endometrial tissue. Anti-estrogens like tamoxifen have

tissue-specific effects with estrogen-agonistic properties on the postmenopausal

endometrium, whereas tamoxifen blocks estrogen receptors in breast cancer.

Intratumoural estrogen production under the influence of the enzyme complex

aromatase is an important factor for the promotion of breast cancer growth, and

may similarly be of importance in the pathogenesis of endometrial

cancer. 1 8•32•33•39 The presence of aromatase within the endometrium may appear

to form a negative prognostic factor independently of hormone receptor

positivity with future implications for the further development and introduction

48

of aromatase inhibitors in treatment strategies of advanced or recurrent

endometrial cancer.

Aromatase inhibitors in advanced or recurrent endometrial carcinoma

Late stage endometrial carcinoma

Of the female genital tract malignancies endometrial cancer is the most frequent,

with a lifetime risk of 1.2% for women. Annual incidence rates between 10 to

130 per 100.000 women are found.66'67 More than seventy-five percent of all

cases of endometrial carcinoma involves endometrioid endometrial carcinoma. 68

The mean age of women with endometrioid carcinoma is 59 years. The FIGO­

stage is the surgical-pathological indicator of progression of the disease at the

time of diagnosis, and stage and age determine treatment. Staging includes

assessment of pelvic and paraaortic lymph nodes, adnexa, and peritoneal fluid

cytology, and requires a.o. histological evaluation of the tumour grade and depth

of invasion in the myometrium, but staging fails to assess individual risks of

local recurrence or distant metastases in the future. Most patients (7 5 to 80%) of

women present with a stage I or II malignancy with a total mortality of 10 to

25%. Creasman reports a 5-year survival of 9 1 % for stage Ia, 88% for stage lb

and 8 1 % for stage le. In stage II surival after 5 years drops to 67-77%, and to

52-60% in stage III. In case of distant metastases only 5% of affected women is

alive after 5 years. As most patients are diagnosed in early stages overall 5-year

survival was reported to be 73%.69 In a population-based Norwegian study, the

5- and 10-year survival for all FI GO-stages was 78% and 67%, respectively.70

For stage III patients these percentages were 39% and 34%, and for stage IV

patients these rates were only 27% and 23%. As patients often present with early

symptoms, such as vaginal bleeding, more than 80% of cases are diagnosed in

the early stages with coherent better prognosis. At diagnosis, 3% of women

present with a clinical stage IV, and about 4% have stage IV disease. Despite

49

treatment with surgery and radiotherapy, 50% of stage III tumours recur. Distant

metastases and local recurrence are major problems. Spread to the lungs occurs

in 36% of women. One must realize that these survival precentages may be

influenced by the fact that most patients present with endometrioid endometrial

cancer with a more favourable prognosis in the early, often curable stages.

In general, all patients undergo a hysterectomy and bilateral salpingo­

oophorectomy when feasible, supplemented by surgical staging and pelvic

radiotherapy is administered to patients with poor prognostic factors that

increase risk of recurrence. However, local recurrence was noted in 4% of

patients even if adequate radiotherapy was administered. 71 Adjuvant hormonal or

cytotoxic chemotherapy and palliative radiotherapy hardly contribute to cure

rates, and significant side effects are noted. One may conclude, that in case

surgery in combination with radiotherapy fails to prevent recurrence, or if the

patient presents with a stage III or IV disease, prognosis remains poor in spite of

adjuvant therapy. Introduction of additional hormonal treatment possibilities

would be welcomed.

Palliative honnonal treatment

For recurrent or advanced hormone dependent cancer treatment is aimed at

slowing down tumour progression as long as possible, or to reduce symptoms of

local metastases. Endocrinological treatment aims at disease stabilisation and

preservation of quality of life, in the knowledge that disease progression is

unavoidable in the end. Particularly for women with well differentiated and/or

progesteron receptor positive endometrial cancers high-dose progestins like

megestrol acetate have been used with limited success. 72 Objective temporary

reponse rates of 10 to 25% were reported in advanced or recurrent disease. 73-75 In

spite of this relative success, one may also conclude that the therapy proved

ineffective in a high percentage of patients. Unfortunately, poor differentiation

50

and progression of endometrial cancers are associated with the loss of

progesterone receptor expression, which partly explaines the limited success of

progestin therapy in these patients. 76 Progesterone receptor positive tumours

show a six times higher response to hormonal treatment, when compared to

receptor negative tumours.25 The anti-estrogen tamoxifen, used a.o. as an

endocrine treatment for advanced endometrial cancer with objective responses

between O and 53%, was associated with an increased risk of endometrial

cancer, so this strategy was abandoned. 59•73 Growth inhibition of advanced

endometrial cancer can theoretically be achieved by treating patients with GnRH

analogs, but disappointing response rates between O and 35% were found.75•77

The potential of aromatase inhibitors

At present two types of aromatase inhibitors are prescribed to patients. Type I

steroidal aromatase inhibitors act as substrate analogues by binding to the

androgen substrate binding site, causing an increase of inactivated aromatase

enzymes. 1 8•78 The non-steroidal type II inhibitors bind to the haem group of the

aromatase enzyme complex and prevent NADPH utilisation, which obstructs

hydroxylation, and induces a decrease of estrogens. The first and most well

known inhibitor was the type II inhibitor aminoglutethimide.

Aminoglutethimide had several disadvantages like side-effects and a limited

specificity. New type I and II aromatase inhibitors were developed with more

binding affinity towards aromatase. These inhibitors like arimidex, vorozole and

letrozole appeared very powerfull in reducing estrogen levels. 18•79-83 Also, as

these new aromatase inhibitors showed less side-effects this was a major

improvement in the treatment of breast cancer. 18 Nowadays, these second

generation aromatase inhibitors are frequently used in the treatment of estrogen

dependent diseases such as breast cancer and also even in endometriosis. 84

Aromatase is overexpressed in endometriosis and reduction of aromatase

5 1

activity in endometriosis most likely diminishes estradiol production with

subsequent improvement of clinical symptoms. 85

The adjuvant use of aromatase inhibitors may offer new treatment approaches in

advanced or recurrent endometrial cancer with poor prognosis. The potent and

specific characteristics of the latest aromatase inhibitors could improve response

rates by suppressing estrogen levels in endometrial cancer with minimal side­

effects. Theoretically, aromatase blockers may inhibit both peripheral and

endometrial stromal aromatization, as the aromatase enzyme converting

androgens into estrogens appears present in all tissues. There exists some

controversy about the effects of aromatase inhibitors on breast cancer growth, as

some authors report local effects in and around the tumour, while others claim

suppression of total body aromatase activity, shown by a reduction of serum

estrogen levels. 1 8 The same controversy may exist in endometrial cancer, as both

mechanisms may apply for endometrial cancer as well. Aromatase inhibitors

may suppress estrogen levels more than by e.g. surgical adrenalectomy or

hypophysectomy alone. 86 Although the exact pathophysiology is unknown, most

likely tumour cell apoptosis is increased due to estrogen deprivation. Aromatase

inhibitors do not influence the estrogen receptor status of tumour cells in human

endometrial carcinomas, contrary to the estrogen agonistic properties of

tamoxifen. 1 9 Even endometrial thickness is reduced in patients treated with

aromatase inhibitors, concluding that aromatase inhibitors do not induce

endometrial hyperplasia as a side-effect in contrast to tamoxifen.60-63

•87 In vitro

it was shown that aromatase inhibitors reduce proliferation and increase

apoptosis m endometrial cancer. 1 9•88 Sasano detected aromatase

immunoreactivity in stromal cells of six out of 14 cases of endometrial

carcinoma. 1 9 The addition of testosterone as a substrate for aromatase, resulted

in an increase of aromatase activity in two cases. Addition of an aromatase

inhibitor, NKSO 1, blocked the [3H]Thymidine incorporation stimulated by

testosterone in four out of 15 endometrioid carcinomas. Some studies reported

52

objective responses and a decrease of aromatase activity after administration of

aromatase inhibitors to women with advanced endometrial cancer. Already in

1 984 objective responses to the aromatase inhibitor aminoglutethimide were

found in four out of 1 8 women with metastatic endometrial carcinoma. 89 A small

study showed decrease of aromatase activity after administration of the

aromatase inhibitor letrozole.90 One study reported very limited response rates to

aromatase inhibition in a phase II trial in patients with advanced or recurrent

endometrial cancer. Only two partial responses were found in 23 women with

advanced or recurrent endometrial malignancy, of whom most cancers were

poorly differentiated or presented an agressive histology ( clear cell carcinoma or

papillary serous carcinoma). Only 39% of these patients were diagnosed with

(hormone-dependent) endometrioid endometrial cancer, and women with poorly

differentiated tumours did not respond.9 1 One may argue that patient selection

and short duration of treatment (four weeks) influenced results negatively. In

breast cancer the tumours with no or low estrogen receptors do not require

estrogen for their continued growth.92 These tumours are unlikely to respond to

therapy designed to block estrogen synthesis. Women with estrogen receptor

negative breast tumours are not eligible for endocrine treatment. This may

possibly apply for some endometrial cancer as well. Poorly differentiated and

agressive tumours like serous papillary and clear cell carcinoma rarely show

estrogen or progesterone receptors and hardly respond to endocrinological

therapy like progestins.46 Unfortunately most women to be considered for

treatment with adjuvant therapy are those with advanced or recurrent aggressive

tumours, who simultaneously are not expected to respond to treatment with

endocrinological drugs, as they are most often hormone receptor negative. On

the other hand, no persistent correlation was found between steroid receptor

status and aromatase expression or activity, in accordance with the absence of

correlation between aromatase activity and steroid receptor expression in breast

carcinoma.48•65

•93 So it remains doubtfull whether estrogen or progesterone

53

receptor positivity should be used as a suitable criterion justifying treatment

with aromatase inhibitors. This would mean that aromatase inhibitors possibly

can be used also in estrogen and progesterone negative tumours with a suspected

poor prognosis. As a correlation was found between CYP 19 gene expression and

hormone independent endometrial cancer, aromatase positivity may be an

important marker relating to tumour growth due to hormonal stimulation from

locallly produced estrogens, with prognostic and therapeutic consequences

independently of estrogen receptor status.36-38 In a recent phase II trial the

aromatase inhibitor letrozole was administered for 12 weeks to patients with

recurrent or advanced endometrial cancer, of whom 86% appeared positive for

estrogen or progesteron receptor.94 In 28 women overall response was 34%. One

complete and two partial responses were noted, whereas 1 1 patients had a stable

disease for a median duration of 6. 7 months, suggesting that a proportion of the

population could benefit. A small report describes two women with endometrial

stromal sarcoma. Both patients developed pulmonar metastases, respectively

three and six years after surgery. Both patients achieved a complete response

after treatment with the aromatase inhibitor aminoglutethimide.95 Successful

response on treatment with letrozole was reported in advanced low-grade

endometrial stromal sarcoma, corresponding with the observation that aromatase

expression was more often reported in low grade, than in high grade stromal

endometrial sarcomas, and that higher expression of aromatase was more

frequently associated with advanced disease and poorer differentiation.33•54

•96

Two premenopausal obese women with well-differentiated endometrial cancer

were successfully treated with progesterone and anastrozole, with the purpose of

maintaining fertility.97 Recently Berstein et al found a decrease in aromatase

activity in endometrial cancer tissue after two weeks of treatment with letrozole

and exemestane. 37

54

Possibly the classification of endometrial cancers in two distinctive classes on

morphologic features, endometrioid endometrial carcinoma related to

unopposed estrogenic stimulation, and a second estrogen independent form

including poorly differentiated and high-grade malignancies like serous

papillary and clear cell carcinoma, should be subdivided in a new model

according to new endocrinological criteria; aromatase positive and aromatase

negative endometrial cancers may exist among both classes of endometrial

cancer, irrespective of body weight, or estrogenic or hormone receptor status,

but related more to genetic and molecular predispositions like CYP 19

polymorphisms. Several pathways on molecular levels may overlap the origin

and evolution of endometrial carcinogenesis and influence each other.

In conclusion, only limited evidence is available concerning the use of

aromatase inhibitors in advanced endometrial cancer. But what about the use of

aromatase inhibitors in the early treatment of endometrial cancer? The AT AC

trial is a large phase III trial comparing primary adjuvant tamoxifen with the

aromatase inhibitor anastrozole for early breast cancer in postmenopausal

women. 60•98 In the anastrozole group less cases of endometrial abnormalities or

cancer occured during two years treatment with anastrozole compared with

tamoxifen, suggesting that anastrozole offers some protective effect on the

endometrial tissue. 61 Also, median endometrial thickness appeared to increase

in the tamoxifen group, whereas it remained unchanged in the anastrozole

group, suggesting a protective role for aromatase inhibitors. 63 Another study

found that after treatment with aromatase inhibitors endometrial abnormalities

due to treatment with tamoxifen diminished in breast canbcer patients. 64 Thus,

one may consider to use aromatase inhibitors in the treatment of complex

atypical endometrial hyperplasia in order to prevent progression of atypia.

However, conservative treatment of atypical hyperplasia can only be achieved

under adequate hysteroscopic monitoring, and one should realize that almost

55

20% of atypical hyperplasias show a carcinoma m their hysterectomy

specimens. 74

As in breast cancer a positive but inconsistent correlation was found between

intratumoural aromatase activity and response to treatment with aromatase

inhibitors, reliable methods must be developed for detection of aromatase

positivity in the individual patient, in order to select those patients who may

benefit from aromatase inhibition. 99- 1 0 1 Taking into account the need for

treatment of patients with advanced or recurrent endometrial disease who are

often elderly and who present with some comorbidity, and the present lack of

long-lasting responses in the adjuvant treatment of endometrial cancer, the

usefulness of treatment with aromatase inhibitors in advanced endometrial

cancer ought to be evaluated. If so, a simple and quantifiable test for

measurement of aromatase activity in formalin-fixed or paraffin-embedded

tissue would be needed for prediction of possible responses to aromatase

inhibitors.54•99 In breast cancer expression of intratumoural aromatase was found

to show to have prognostic value concerning the chances of successful treatment

with aromatase inhibitors.100,101 Intratumoural aromatase expression was related

to poor survival in endometrioid endometrial cancer, but confirmation from

other studies is still indicated. 1 02 A reliable simple test for measurement of

aromatase activity in archival materials or formalin-fixed and paraffin-embedded

tissue would be welcomed for prediction of sensitivity to treatment with aromatase

inhibitors. Conflicting results were found in previous studies using

immunohistochemistry, and the most important problem appeared the fact that

specificity of antibodies could not be characterized well enough.99 New

monoclonal antibodies for immunohistochemistry, such as the 677 clones, seem

quite useful for detection of aromatase activity not only in breast cancer, but also

in endometrial carcinoma.99•103 The extent of aromatase positivity in these

tumours may offer new endocrinological treatment modalities in the

56

management of endometrial carcinoma and new insights m individual

prognostic factors and chances of disease-free survival.

57

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66

4

Ovarian stromal hyperplasia and ovarian vein steroid

levels in relation to endometrioid endometrial cancer

VHWM Jongen

H Hollema

AGJ van der Zee

JG Santema

MJHeineman

British Journal of Obstetrics and Gynaecology2003; 1 10:690-95

67

Abstract

In a retrospective and prospective study, respectively, the relationship was studied

between the presence of endometrioid endometrial cancer, the degree of ovarian

stromal hyperplasia and ovarian steroid production in postmenopausal women . . In

1 12 women with endometrioid endometrial cancer, 47 women with a benign

gynaecological condition and ten women with non-endometrioid endometrial

cancer, the degree of ovarian stromal hyperplasia was scored retrospectively on a

semi-quantitative scale (atrophy, slight, marked). All women were postmenopausal

and had undergone a hysterectomy with bilateral salpingo-oophorectomy.

Prospectively also blood sampling from the ovarian veins was performed in 60

women. Steroid levels ( estrone, estradiol, androstenedione, testosterone) were

determined and related to the degree of ovarian stromal hyperplasia and the

presence (n=52) or absence (n=8) of endometrioid endometrial cancer. In the

retrospective study the degree of ovarian stromal hyperplasia was higher in the

presence of endometrioid endometrial cancer (p=0.000). The prospective study

showed that an increasing degree of ovarian stromal hyperplasia was related to

higher ovarian levels of both testosterone and androstenedione (p<0.05 and

p<0.005 respectively), but not to estrone or estradiol. Mean ovarian vein levels of

both testosterone and androstenedione were higher in patients with endometrial

cancer than in patients with benign conditions, without reaching statistical

significance. In conclusion, higher degrees of ovarian stromal hyperplasia were

found in endometrioid endometrial cancer and with increasing degrees of ovarian

stromal hyperplasia levels of ovarian vein androgens were higher. A causal

relationship in the origin of hormone-dependentendometrial pathology may exist

between ovarian stromal hyperplasia, ovarian vein androgen levels and

endometrioid endometrial carcinoma.

68

Introduction

Endometrioid endometrial cancer is generally regarded as an estrogen-related

malignancy gradually developing from endometrial atypical hyperplasia towards

endometrial cancer. Estrogens have been demonstrated to act as promoting factors,

especially in the relative absence of progestagens.1 •2 Obese women are at risk for

developing endometrioid endometrial carcinoma due to the increased capacity in

adipose tissue to convert androstenedione to estrone and testosterone to estradiol

under the influence of the enzyme complex cytochrome P450 aromatase. The

postmenopausal ovary is a viable androgen-producing gland, producing 40% and

20% of the total body amount of testosteron and androstenedione, respectively; It

has been suggested that ovarian androgens, apart from being precursors for

peripheral conversion to estrogens in adipose tissue, could also play an indirect

growth promoting role in the development of endometrial pathology, as the

enzyme aromatase cytochrome P450 may be able to convert circulating androgens

to estrogens in the endometrium itself.4•7 The clinical significance of ovarian

stromal hyperplasia in the postmenopausal ovary is controversial. In smaller

studies it has been suggested that the extent of ovarian stromal hypeiplasia

correlates with postmenopausal ovarian steroid production. 8· 1 1 Thus, a causal

relationship between ovarian stromal hypeiplasia, ovarian steroid production and

the presence of endometrioid endometrial cancer may exist and the aim of the

present study was to explore this relationship.

69

Methods

Retrospective study

A retrospective study was performed in all 323 postmenopausal women, who

underwent a hysterectomy with bilateral sapingo-oophorectomy at the University

Hospital of Groningen between 1990 and 2001. The clinical data were extracted

from patients records (Table 1). Excluded were 126 women diagnosed with

ovarian metastases, cervical or ovarian cancer. Also not included were 24 women,

whose histological samples appeared absent or could not reliably be ascertained.

Women with atypical endometrial hyperplasia were excluded as a limited number

of four cases were found. A subdivision was made between 112 women with

endometrioid endometrial cancer, 47 women with benign disorders and a mixed

group of ten women with non-estrogen related endometrial cancer, like clearcell

carcinoma, serous papillary cancer, mixed mullerian tumour or uterine sarcoma.

The morphological characteristics of the ovaries of the included patients were

classified according to the degree of stromal hyperplasia using the parameters

originally described by Boss et al: an ovary with a cortex of less than 1 millimeter

(mm) was determined to be atrophic (class I), a cortex wider than 1 mm was

defined as slight stromal hyperplasia ( class II), an ovarian cortex wider than 1 mm

with apparent cellular cortical stroma present in the medulla was called moderate

or marked stromal hyperplasia ( class III).8-1 0

,1 3 The ovaries were judged without

being familiair with the patients' diagnosis. A representative part of the samples

(3 0%) was judged in almost full agreement by three investigators (VJ, HH, JS) to

rule out inter-observer variability. In the few cases of discordant histological

diagnoses full consensus was obtained after reassessment. The histological ovarian

tissue samples of the included patients were scored according to the degree of

ovarian stromal hyperplasia and related to the presence or absence of endometrial

cancer.

70

Table 1. Clinical data of 112 women with endometrioid endometrial cancer, 10

women with non-endometrioid endometrial cancer and 47 women with a benign

gynaecological disorder.

Age (years)

Year after menopause

Body weight (kg)

Hip-waist ratio (n=57)

Endometrioid

endometrial cancer (n= l 12)

66.2 ( 10.2)

14.7 ( 10.9)

8 1 .6 ( 19 .3)

1 .08 (0. 14)

Benign disorder Non-endometrioid

endometrial cancer (n=47) (n= l O)

63 .8 ( 10.3) 64.5 (9 .9)

12.7 ( 1 1 .6) 13.3 ( 12.7)

74.8 ( 12.7) 77.0 ( 12.6)

1.09 (0. 14)

Data represent mean values, standard deviation is shown in parentheses.

No significant differences were noted between subgoups.

Prospective study

In addition, a prospective study was performed between 1998 and 2001. Patients

suspected of endometrial cancer or a benign condition, who were scheduled to

undergo an abdominal hysterectomy with bilateral salpingo-oophorectomy in the

University Hospital of Groningen or the Medical Centre of Leeuwarden, were

invited to participate. Twenty of 94 eligible women were not included in the study

due to logistical problems. Fourteen women refused to participate in the study or

were not mentally fit enough to consider participation. All remaining 60 women

were included and fulfilled the same eligibility criteria as the women in the

retrospective part of the study. They were also part of the 169 women in the

retrospective study. None of the patients had received hormone replacement

therapy during the preceding 6 months and all had been postmenopausal for at

7 1

least one year. Approval of the Hospital Ethics committee was obtained. Tissue

ovarian samples obtained during surgery were fixed in formalin and repmentative

transverse sections were processed by routine methods including staining by

haematoxylin and eosin. Morphological characteristics of the ovaries of the

included patients were classified as described in the retrospective study. When

informed consent was received, peripheral blood samples and intra-operative

samples of blood from the utero-ovarian veins were collected as described

previously.12 When blood samples were collected, the samples were centrifuged

after clotting. The acquired serum was stored at -20° C until processed. Using non­

commercial radio-immunoassays (RIA) serum levels of estrone, estradiol,

androstenedione and testosterone were measured. 1 4- 1 6 For the estron assay intra­

and interassay coefficients of variation were less than 1 1 % and 15%, and for the

estradiol assay less than 5% and 14%, respectively. Intra- and interassay

coefficients of variation for the androstenedione assay were less than 7 .3% and

21 %, and for the testosterone assay less than 7 . 1 % and 19%. As sampling was

performed from left and right utero-ovarian veins, the average hormone levels

were calculated taking both sides together. Left and right vein levels are typically

similar.12

Hip-waist ratio and body weight were measured in 57 women. The hip-waist ratio

was defined as the hip circumference at the point of maximum protuberance of the

buttocks divided by the cirumference at the umbilical level, measured with a steel

tape while the subject was standing erect.

72

Statistics

For comparison of results in both studies data analysis was performed using the

SPSS 10.0 software package (SPSS Inc., Chicago, IL). Data are presented with

mean values and standard deviation or median and inter-quartile ranges, whenever

appropriate. Statistical comparison was made between the degree of ovarian

hyperplasia, the presence or absence of malignancy, and serum and ovarian vein

levels of estrogens and androgens. Differences in scores for hyperplasia were

evaluated by the Test of Yates and Cochran. Atrophy, slight and marked

hyperplasia of the ovaries were scored as 1,2 and 3. The Mann Whitney Test and

Kruskal Wallis Test were used for comparison of continuous variables. These non­

parametric tests were chosen, as the values were not expected to be normally

distributed. Chi-square analysis was used for comparison of frequencies. Linear

multiple regression analysis was performed for calculating statistical correlation

between the presence of endometrial cancer and ovarian vein steroid levels. P

values lower than 0.05 were considered to reflect statistical significance.

Results

Clinical data of patients involved in the study are shown in Table 1 and 2. The

whole retrospective study group consisted of 169 women; 1 12 women had

endometrioid endometrial cancer, 4 7 women had a benign gynaecological

condition (Table 2) and ten women had non-endometrioid endometrial cancer.

The three different patient categories were comparable with respect to age, age

after menopause and body weight. In the prospective study blood sampling was

performed in 60 postmenopausal women, of whom 52 had endometrioid

endometrial cancer and eight had a benign condition.

73

Table 2. Clinical or pathological diagnosis of 4 7 women with benign

gynaecological conditions.

Clinical or pathological diagnosis

Uterine leiomyoma Adenomyosis Reactive changes Abdominal hysterectomywith normal findings Genital prolapse with normal findings Benign glandulocystic endometrial polyp G landulocysticendometrial hyperplasia Complex hyperplasia(no atypia)

Number ofwomen (n=47)

12 1 3

12 4

5 5 5

Table 3. Degree of ovarian stromal hyperplasia in relation to endometrioid

endometrial cancer (n= 1 12), benign gynaecological disorders (n=47) and

nonendometrioidendometrial cancer (n= l 0).

Stromal atrophy Slight stromal Moderate/ marked Mean Hyperplasia Hyperplasia Score

(score= l ) (score=2) (score=3) Endometrioid endometrial 10 (8.9 %) 5 1 (45 .5%) 5 1 (45 .5%) 2.4 carcinoma(n= l 12Y

Benign disorder 14 (29.8%) 25 (53.2%) 8 ( 17 .0%) 1.9 (n=47)1

Non-endometrioid endometrial 4 (40.0%) 5 (50%) 1 ( 10.0%) carcinoma ( n= 10)

95% C.I 0.24-0.74; p=0.000

74

Ovarian stromal hyperplasia and steroid levels

In the retrospective study analysis of the association between endometrioid

endometrial cancer and degree of ovarian stromal hyperplasia is demonstrated in

Table 3. In 47 women with benign conditions a 17.0% prevalence of marked

ovarian stromal hyperplasia was observed, as compared to an incidence of 45 .5%

in 112 women diagnosed with endometrioid endometrial cancer (p=0.000). In the

group of ten women with non-endometrioid endometrial cancer only one case of

marked ovarian stromal hyperplasia was diagnosed.

In the prospective part of the research, an increasing degree of ovarian stromal

hyperplasia was related to higher ovarian levels of both testosterone and

androstenedione (Table 4; p<0.05 and p<0.005 respectively). When benign

disorders were excluded and mean ovarian vein levels were related to the degree

of ovarian stromal hyperplasia, no other relations were found. Median ovarian vein

levels of both testosterone and androstenedione were higher in patients with

endometrioid endometrial cancer as compared with patients with a benign

condition, without reaching statistical significance (Table 5). When comparing

women with endometrial cancer and atrophic stromal ovarian hyperplasia to

women with endometrial cancer and marked stromal ovarian hyperplasia, higher

ovarian levels of both testostosterone and androstenedione were found in the

second group (p<0.05), suggesting that the levels of androgens were related more

to the degree of ovarian stromal hyperplasia than to the presence of endometrial

cancer. By performing backward linear multiple regression analysis we confirmed

that the presence or absence of endometrial cancer did not influence serum values

of estrogens and androgens. The ovarian vein levels of estrone and estradiol were

not correlated to the degree of ovarian stromal hyperplasia. No statistical

difference was found between hormone levels of peripheral blood samples and

degree of ovarian stromal hyperplasia and the presence or absence of endometrial

cancer.

75

Table 4. Median ovarian vein and peripheral serum levels ( + inter-quartile

ranges) of Testosterone (T), Androstenedione (A), Estrone (El ) and Estradiol

(E2) in 60 postmenopausal women and the relationship with the degree of

ovarian stromal hyperplasia.

Ovarian stromal

atroEhY (n=5) T ovarian vein

(nmol/L) 3 . 1 (2. 1 :4.9) 1

T peripheral

(nmol/L) 1 .7 ( 1 .6:2.2)

A ovarian vein

(nmol/L) 3.7 (2.9 :4 . 1)2

A peripheral

(nmol/L) 3.0 (2.5:4.6)

E2 ovarian vein

(nmol/L) 0. 1 (0. 1 :0. 1)

E2 peripheral

(nmol/L) 0. 1 (0. 1 :0.2)

E 1 ovarian vein

(nmol/L) 0. 1 (0.0:0. 1)

E 1 peripheral

(nmol/L) 0.0 (0.0:0.0)

I p < 0.05

2 p < 0.005

Slight stromal

hype�lasia (n=30)

3.8 (2.9 :7.3) 1

2.0 ( 1 .8 :2.4)

6.3 (4 .8:9.3)2

3 .5 (2.5 :4 .7)

0. 1 (0. 1 :0.3)

0. 1 (0.0:0.2)

0. 1 (0.0:0.2)

0.0 (0.0:0. 1)

Marked stromal

Hype!:£lasia (n=25)

7.0 (3 .3 : 18.4) 1

2. 1 ( 1 .5 :2.6)

14 .0 (8.9 :29.3)2

3 .6 (2.4 :5 .3)

0. 1 (0.0:0. 1)

0. 1 (0.0:0.2)

0. 1 (0.0:0.2)

0. 1 (0.0:0. 1)

76

Table 5. Median ovarian vein and peripheral serum levels (+ inter-quartile

ranges) of Testosterone (T), Androstenedione (A), Estrone (E 1) and Estradiol

(E2) in 60 women with endometrioid endometrial cancer or a benign

gynaecological condition.

Endometrial Benign Cancer disorders p-value (n=52) (n=8)

T ovarian vein 5.0 (2.8: 10.0) 4.7 (3 .5 : 16.7) 0.5 (nmol/L)

T peripheral 2. 1 ( 1 .6:2.6) 1 .9 (1 .8:2. 1) 0.4 (nmol/L)

A ovarian vein 9 .0 (4.8 : 15 .4) 8. 1 (4.4 :8.5) 0.3 (nmol/L)

A peripheral 3 .4 (2.5 :4 .7) 3 .6 (2.5 :5. 1) 1.0 (nmol/L) E2 ovarian vein 0. 1 (0.0:0.2) 0. 1 (0. 1 :5.8) 0.4 (nmol/L)

E2 peripheral 0. 1 (0.0:0.2) 0. 1 (0.0:0.5) 0.4 (nmol/L)

E 1 ovarian vein 0. 1 (0.0:0.2) 0. 1 (0.0:0.2) 0.4 (nmol/L)

E l peripheral 0. 1 (0.0:0.2) 0.0 (0.0:0. 1) 0.4 (nmol/L)

N.S.

One woman with endometrioid adenocarcinoma was later excluded from the

prospective study as she was also diagnosed with a well differentiated

endometrioid adenocarcinoma in the left ovary, possibly due to metastatic disease,

whereas a second primary tumour could not be excluded. In her case a high

ovarian vein androstenedione level (average 85.1 nmol/L; left side 90.9 and right

side 79.3 nmol/L) and elevated testosterone level (average 18.4 nmol/L; left side

21.5 and right side 15.2 nmol/L) were found, possibly due to high androgen

77

production from local stromal reaction. Remarkably androgens from the ovary

without malignancy were elevated as well. In three women with endometrioid

endometrial cancer and marked stromal hyperplasia an extremely high mean

ovarian vein androstenedione level of 1 15 nmol/L and a mean testosterone level

of 1 10 nmol/L were found, but pathological reexamination showed no sign of

micrometastasis in these ovaries.

Bodyweight

Degree of ovarian stromal hyperplasia was related to body weight (p<0.005)

(Figure 1). In 57 women hip-waist ratio had been measured prospectively, but no

correlation was found between degree of hyperplasia, estrogen or androgen levels

and upper or lower body obesity. Comparison between degree of stromal

hyperplasia and age or age after menopause or histological grade of endometrioid

endometrial carcinoma did not appear statistically significant.

u � If) 0)

1 00

90

80

:C 70

>, u 0

.0 C (I] Q)

� 60

Fig 1 . Relation between body weight, ovarian

stromal hyperplasia and endometrial cancer

I I

atrophy slight

Degree of ovarian stromal hyperplasia

I 0 I I Benign disorder

□

I Endometrlal cancer

□ I

marked

78

Discussion

After menopause, simultaneously with the decreasing number of follicles the

ovarian cortex becomes thin and gyrated, creating an irregular surface.1 7•

1 8 Ovaries

of postmenopausal women are composed mainly of stromal cells. Although the

ovarian volume decreases after menopause, the postmenopausal ovary is generally

regarded as a gland producing significant amounts of androgens.3• 1 0• 1 1 •1 9•20 A

postmenopausal ovary may show cortical and stromal atrophy, but also stromal

hyperplasia can be seen. Stromal hyperplasia is characterized by nodular or diffuse

proliferation of ovarian stroma. Varying degrees may be present. Until the criteria

developed by Boss were introduced, some regarded the amount of stromal

collagen or the presence of cortical stroma as features of stromal hyperplasia,

whilst others made that classification only in case nodules of cortical stroma of

one mm. in diameter or more were present. Boss himself found no consistent

correlation between degree of stromal proliferation and ovarian size.1 3 Marked

stromal hyperplasia appeared sometimes evident in a normal sized ovary, as the

amount of stroma is only one factor determining the size of the ovary, concluding

that the volume perse is not a reliable indicator of the content of the ovary. Several

studies showed that the extent of ovarian stromal hyperplasia is associated to some

degree with the degree of postmenopausal ovarian steroid production.8-10 Any

evaluation of the hormonal patterns in postmenopausal women with regard to the

development of hormonal dependent endometrial cancer should also take into

account the stromal characteristics of the ovary, as ovarian morphology may

reflect to the endocrine status and endometrial disease.1 1

We compared the degree of stromal hyperplasia in relation to the presence or

absence of endometrial cancer and report a higher frequency of ovarian stromal

hyperplasia in case of endometrioid endometrial cancer. Our study supports the

hypothesis that a relationship exists between endometrioid endometrial cancer and

79

ovarian stromal proliferation. Contrary to our results, Lucisano et al found no

correlation between the degree of stromal hyperplasia and the presence of

endometrial cancer, but this lack of relation might be explained by the relative

small number of patients in their study.9 In case of endometrial carcinoma we

found a 45.5% incidence of marked stromal hyperplasia in ovaries, which is

higher than Lucisano, who reported an incidence of 19% in 21 women with

adenocarcinoma of the endometrium. Other studies report incidences of 1 1 %,

28% and 4 7% in patients with and without endometrial cancer mixed together, but

comparison is hampered by widely varying percentages of endometrial malig­

nancy in these studies.8• 1 3•2 1 Decades ago, without using the criteria developed by

Boss in 1965, marked ovarian hyperplasia was found in 84% of endometrial

malignancies, compared to only 44% in autopsy controls.1 3•2 1 Boss himself

mentions marked ovarian stromal hyperplasia in 8 1 % of 16 patients with

endometrial cancer.1 3 Except the study by Lucisano, all of the studies mentioned

failed to record the exact histological type of carcinoma in case of endometrial

pathology? The present study is the first one analysing only women with

endometrioid endometrium carcinoma separately. Endometrioid cancer is

supposed to be related to estrogenic stimulation and accounts for 75% of all cases

of endometrial cancer. Taking into account the assumption that increased androgen

production from ovarian hyperplasia may be related to endometrial pathology,

only hormone-dependent endometrioid endometrial malignancies should be

included in comparative studies. This conclusion is supported by our findings that

androgen levels from the ovarian veins were higher with an increasing degree of

ovarian stromal hyperplasia, and that the degree of ovarian stromal hyperplasia

was higher in the presence of endometrioid endometrial cancer. Analysis of

women with non-endometrioid endometrial cancer showed only 10% marked

stromal hyperplasia, suggesting that no increase of ovarian stromal hyperplasia is

found in hormone-independent uterine cancer. However, it should be noted that

this subgroup is rather small to draw firm conclusions.

80

Endometrioid endometrial cancer arises more often in women with obesity. In our

study mean body weight was higher in case of malignancy. Taking into account

the reported correlation between ovarian stromal hyperplasia and endometrioid

endometrial cancer, it is not surprising that in the present study a correlation was

found between ovarian stromal hyperplasia and body weight, although a causal

relationship cannot be presumed.

Whether increased androgen production by the hyperplastic ovary is a causative

mechanism leading to endometrial cancer remains to be ascertained. In 1 8

postmenopausal women Sluijmer et al demonstrated a correlation between the

degree of ovarian stromal hyperplasia and the production of androgens, both

androstenedione and testosterone.8 Other studies showed that ovaries with marked

stromal hypeiplasia secreted significant amounts of androgens, and that the degree

of hyperplasia correlated to some degree with the amount of androgen secreted.9'10

Androgen levels in the utero-ovarian vein blood samples from women with endo­

metrial cancer were significantly elevated as compared with the utero-ovarian vein

levels from women without endometrial carcinoma.22-24 Like androgens from the

adrenal glands also androgens produced by the ovaries can be converted to

estrogens by aromatase cytochrome P450. Increased production of aromatizable

androgens by the ovarian stroma may lead to increased precursor availability for

estrogen production. It is an intriguing question whether, apart from extraglandular

aromatase activity in adipose tissue, aromatase activity in the endometrium plays a

role in malignant degeneration.6'7 Aromatase (over-) expression in endometrium is

hypothesized to be a possible mechanism linking ovarian stromal hyperplasia and

increased steroid production to the development of uterine cancer. In endometrial

cancer indeed aromatase activity has been reported, whereas disease-free

endometrium did not express aromatase.25-29 These findings may support the

alleged correlation between steroid secretion from the ovarian stromal hyperplasia

8 1

and endometrial disease.30 In their excellent work from the forties of the last

century Smith, Woll and Hertig were the first to focuss on this relationship.2 1 •3 1

Many decades later it still remains questionable whether ovarian stromal

hyperplasia simply reveals a local stromal reaction in the presence of endometrial

cancer or reflects an ovarian predisposition favouring future malignant

degeneration due to its endocrine properties. Therefore future research should aim

at the relationship of ovarian stromal hyperplasia, ovarian steroid production and

aromatase activity in the endometrium. Under the influence of the increased

steroid levels from the hyperplastic ovary clonal cellular clusters may arise in the

endometrium with progressive growth resulting in an increasing risk of DNA

replication errors, especially in case of specific genetic susceptibility, such as e.g.

CYP 19 polymorphisms, that have been associated with increased steroid

concentrations and might be related to a.o. aromatase activity.32 Eventually

endometrial cancer may develop, with ovarian stromal hyperplasia as another

concomitant hormonal risk factor promoting endometrial carcinogenesis.

Acknowledgements

The authors are grateful to Mrs. KA Ten Hoar and J Koerts for laboratory

assistance and to Dr. WJ Sluiter for his contribution in statistical analysis.

82

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endometrialcancer. EndocrRel Cancer2000; 7: 227-42. 2. Akhmedkhanov A, Zeleniuch-Jacquotte A, Toniolo P. Role of exogenous and

endogenous hormones in endometrialcancer. Ann NY Acad Sci 2001; 943 ; 296-3 15.

3. Adashi EY. The climacteric ovary as a functional gonadotropin-driven androgen­

producing gland. Fertil Steril 1994; 62: 20-7 . 4 . Sasano H, Harada N. Intratumoral aromatase in human breast, endometrial and ovarian

malignancies.EndocrineRev 1998; 19 : 593-607. 5. WatanabeK, Sasano H, Harada N, et al. Aromatase in human endometrial carcinoma and

hyperplasia. Immunochemical, in situ hybridization, and biochemical studies. Am J

Pathol 1995; 146: 49 1-500. 6. Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, et al. Endocrine

disorders associated with inappropriately high aromatase expression. J Steroid Biochem

Molec Biol 1997; 61 : 133-9.

7. Bulun SE, Economos K, Miller D, Simpson ER. CYP19 (aromatase cytochrome P450)

gene expression in human malignant endometrial tumors. J Clin Endocrinol Metab 1994;

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8. Sluijmer A V,Heineman MJ, Koudstaal J, et al. Relationship between ovarian production of estrone, estradiol, testosteron, and androstenedione and the ovarian degree of stromal

hyperplasiain postmenopausalwomen. Menopause 1998; 5: 207- 10. 9. Lucisano A, Russo N, Acampora MG, et al. Ovarian and peripheral androgen and

estrogen levels in post-menopausal women: correlations with ovarian histology.

Maturitas 1986; 8: 57-65.

10. Ala-Fossi SL, Maenpaa J, Aine R, et al. Ovarian testosterone secretion during

perimenopause.Maturitas 1998; 29 : 239-45 .

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degeree of ovarian stromal hyperplasia in postmenopausal women. Menopause 1998; 5 : 205-6.

12. Heineman MJ, Sluijmer AV, Evers JLH. Utero-ovarian vein blood sampling in

postmenopausalwomen. Fertil Steril 1993; 60: 184-6.

13 . Boss Jh, Scully RE, Wagner KH, et al. Structural variations in the adult ovary: clinical

significance. Obstet Gynecol 1965; 25 : 747-64.

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14. Jurjens H, Pratt JJ, Woldring MG. Radioimmunoassay of plasma estradiol without extraction and chromatography.J Clin EndocrinolMetab 1975; 40: 19-25 .

15 . Pratt JJ, Wiegman T, Lappohn RE, Woldring MG. Estimation of plasma testostosteron without extraction and chromatography.Clin Chim Acta 1978; 84: 329-37 .

16. Pratt JJ. Steroid immunoassay in clinical chemistry. Thesis Groningen 198 1 . 17. Flaws JA, Rhodes JC, Langen berg P, et al. Ovarian volume and menopausal status.

Menopause2000; 7: 53-61 . 18 . Goswamy RK, Campbell S, Royston JP, et al. Ovarian size in postmenopausal women.

Br J Obstet Gynaecol 1988; 95 : 795-801 . 19. Sluijmer AV.The postmenopausal ovary. Functional and morphological aspects. Thesis

Maastricht 1999. ISBN 90-9012802-6. 20. Dennefors BL, Janson PO, Knutson F, et al. Steroid production and responsiveness to

gonadotropin in isolated stromal tissue of human post-menopausal ovaries. Am J Obstet Gynecol 1980; 136: 997- 1002.

21 . Woll E, Hertig AT, Smith GVS, et al. The ovary in endometrial carcinoma: with notes on the morphological history of the aging ovary. Am J Obstet Gynecol 1948; 56: 617-33.

22. Mertens HJMM, Heineman MJ, de Jong FH, et al . Postmenopausal ovarian function and endometrial cancer. Hum Reprod 14, Abstractbook, Uune 1999): 89-90.

23 . Heineman MJ, Sluijmer AV, Kampscoer PHNM, et al. Utero-ovarian vein blood smapling in postmenopausal women with and without endometrial cancer. Fertil Steril

1995; Suppl: S92 (Abstract O- 187).

24. Nagamani M, Stuart CA, Doherty MG. Increased steroid production by the ovarian stromal tissue of postmenopausal women with endometrial cancer. J Clin Endocrinol Metab 1992; 74: 172-6.

25. Bulun SE, MahendrooMS, Simpson ER. Polymerasechain reaction amplificationfails to

detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua.

J Clin EndocrinolMetab 1993 ; 76: 1458-63 . 26. Tseng L, Mazella J, Mann WJ, et al. Estrogen synthesis in normal and malignant humen

endometrium. J Clin EndocrinolMetab 1982; 55 : 1029-3 1. 27. Yamaki J, Yamamoto T, Okada H. Aromatization of androstenedione by normal and

neoplasticendometriumofthe uterus. J Steroid Biochem 1985 ; 22: 63-6. 28. Yamamoto T, Kitawaki J, Urabe M, et al. Estrogen productivity of endometrium and

endometrial cancer cells. J Steroid BiochemMol Biol 1993 ; 44: 463-8 .

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29. Sasano H, Kaga K, Sato S, et al. Aromatase P450 gene expression in endometrial

carcinoma. Br J Cancer 1996; 74 : 154 1-4 . 30. Longcope C. Editorial. The relationship of ovarian production of hormones and degree of

ovarian stromal hyperplasia in postmenopausalwomen. Menopause 1998; 5: 205-6. 3 1. Smith GVS. Carcinoma of the endometrium: a review with results of treatment trough

1935 . N Engl J Med 194 1 ; 225 : 608- 15 . 32. Berstein LM, Imyanitov EN, Suspitsin EN, Grigoriev MY, Sokolov EP, Togo A, et al.

CYP 19 gene polymorphism in endometrial cancer patients. J Cancer Res Clin Oncol

2001; 127: 135-8.

85

86

5

Is aromatase cytochrome P450 involved in the

pathogenesis of endometrioid endometrial cancer?

VHWM Jongen

JHH Thij ssen

H Hollema

GH Donker

JG Santema

AGJ van der Zee

MJ Heineman

International Journal of Gynecological Cancer 2005; 15:529-36

87

Abstract

Prospectively the relationship between androgen levels in the utero-ovarian

circulation, aromatase activity in endometrial and body fat tissue, and the presence

or absence of endometrioid endometrial cancer was studied in postmenopausal

women. In 43 women with endometrioid endometrial cancer and 8 women with a

benign gynaecological condition a hysterectomy with bilateral salpingo­

oophorectomy was performed. Using tritium water release assays, aromatase

activities in endometrial and body fat tissue were determined and related to the

steroid levels from the peripheral and the utero-ovarian venous circulation

( estradiol, androstenedione, testosterone) and to the presence or absence of

endometrial cancer. Significant aromatase activity was found in both benign and

malignant endometrial tissue samples. Aromatase activity in samples of

endometrial tissue and in samples of body fat did not correlate with steroid levels

in peripheral or utero-ovarian venous blood. Aromatase activity in samples of

benign or malignant endometrium did not differ. Remarkably, in four women with

mainly poorly differentiated endometrial cancer very high aromatase activity was

found in endometrial tissue. It is likely that multiple pathogenetic pathways exist

which eventually lead to the formation of endometrioid endometrial cancer. The

local availability of androgens and the finding that aromatase activity is present in

both endometrial cancer and benign endometrial tissue, support the hypothesis that

aromatase activity in the endometrium may play a role in malignant

transformation, by converting androgens into mitogenic estrogens in the

endometrial tissue.

88

Introduction

The enzyme complex aromatase consists of the cytochrome P450 aromatase

enzyme, its associated haeme group, and a NADPH-reductase flavoprotein. 1

Aromatase is responsible for the binding of the C 19 steroid substrate

( androstenedione and testosterone) and catalyzes the series of reactions which

will eventually lead to estrogen production. The conversion of androgens to

estrogens involves three subsequent steps. The third step leads to the

aromatization of the A-ring, giving the characteristic phenolic ring of estrogens.2

Aromatization inside the ovaries leads to the synthesis of estrogens during the

menstrual cycle, after menopause the so-called peripheral conversion is

responsible for the production of estrogens. The overall peripheral conversion of

androgens to estrogens increases as a function of age, obesity and aromatase

activity in extraglandular (adipose) tissue. 3 The contribution of aromatase to

estrogen formation must be considered extremely important, as aromatization is

the essential and rate-limiting step. The principle substrates of estrogens are

circulating androgens, derived primarily from the adrenal cortex and to a lesser

extent from the ovaries. Estrogens are considered to be the main promoting

factor of endometrioid endometrial carcinoma. It can not be excluded that

androgens may play a role as well. After menopause the ovary still produces

40% respectively 20% of the total amount of testosterone and

androstenedione.4-7 Androgens, produced by the ovaries, are not only converted

to estrogens in peripheral adipose tissues, but also give rise to elevated

prehormone availability for estrogen formation in utero. In several studies it has

been demonstrated that the androgen levels in the utero-ovarian veins from

women with endometrial cancer were significantly elevated as compared to

women without malignant disease. 7- 1 0 In a recent study these findings were not

confirmed. 1 1 Ovarian androgens may enhance the development of endometrial

cancer through local aromatization into estrogens. Local aromatase activity may

89

even give rise to very high local estrogen concentrations which could result in

the stimulation of endometrial growth. Thus, in addition to extraglandular

aromatase activity by converting androgens into estrogens in adipose tissue,

aromatase activity in the endometrium may play a role in malignant

transformation. 12 Aim of the present study is to explore the possible relationship

between androgen levels in the utero-ovarian circulation, aromatase activity in

the endometrial tissue and the presence of endometrioid endometrial cancer.

Patients and methods

A prospective study was performed between 1998 and 2001. Patients suspected of

endometrial cancer or a benign condition, who were scheduled to undergo an

abdominal hysterectomy with bilateral salpingo-oophorectomy in the University

Hospital of Groningen or the Medical Centre of Leeuwarden, were invited to

participate, and were included after informed consent. Women with ovarian

metastases, cervical or ovarian cancer were not included. None of the patients had

received hormone replacanent therapy during the preceding 6 months and all had

been postmenopausal for at least one year. Approval of the Hospital Ethics

committee was obtained. Patients were part of a study described in a previous

paper.1 1

During surgery samples of blood from the utero-ovarian and peripheral veins were

collected, as described previously.13 When blood samples were collected, the

samples were centrifuged after clotting. The acquired serum was stored at -20° C

until processed in order to determine steroid levels. During or directly after the

procedure tissue specimens were obtained from both the subcutaneous abdominal

fat, and the endo- and myometrium. In case of endometrial cancer a

representative piece of endometrial tissue was chosen, directly next to or on the

border of the suspected endometrial cancer tissue. The acquired specimens

90

( approximately one cm3) were frozen at -20° C as well, until aromatase assays

could be carried out.

Steroid measurements

Using non-commercial radio-immunoassays (RIA) serum levels of estrone,

estradiol, androstenedione and testosterone were measured. 14-1 6 Intra- and

interassay coefficients of variation were less than 11 % and 15% for the estrone

assay and for the estradiol assay less than 5% and 14%, respectively. Intra- and

interassay coefficients of variation for the androstenedione assay were less than

7 . 3 % and 21 %, and for the testosterone assay less than 7. 1 % and 19%. As

sampling was performed from both ovarian sides, the average hormone levels

were calculated taking left and right utero-ovarian vein together, as they are

typically similar.13

Aromatase assays

[ l �-3H]-androst-4-ene-3, l 7-dione (spec.act. 25 Ci/mmol) was provided by NEN

(Boston MA, USA); Exemestane by Pharmacia & Upjohn (Milan, Italy); NAD

by Sigma (St.Louis, Missouri ,USA); NADP and glucose-6- phosphate by Roche

(Mannheim,Germany); ATP and glucose-6- phosphate dehydrogenase by ICN

(Ohio,USA); charcoal by Merck (Darmstadt, Germany); Dextran T by

Amersham Pharmacia (Uppsala, Sweden); diethylether by Rathbum

(Walkerbum, Scotland); scintillation fluid Ultima Gold by Packard (Meriden

CT, USA); Coomassie brilliant blue reagent by Biorad (Hercules CA, USA) and

human serum albumin by Kabi Vitrum (Stockholm,Sweden). Buffer A: 0.01 M

phosphate buffer pH 7.4 containing Trasylol 500 KIE/ml (Bayer, Leverkusen,

Germany). Buffer B: buffer A + co-factors: NAD (10 mM), glucose-6-

phosphate dehydrogenase (2 U/ml) and 2 mM each of ATP,NADP and glucose-

6- phosphate. Charcoal: 2.5% Dextran T70, 5% charcoal in aqua dest. (w/v).

91

Aromatase activity assay

Measurement of aromatase enzyme activity was based on the tritium water

release assay as described previously. 1 7 In brief: tissue samples (0.4-2 gram)

were minced on ice and pulverized at - 1 96° C using a Micro-Dismembrator

(Braun, Melsungen, Germany) and stored at -80° C. Just prior to assay the

powder was suspended in 2-3 ml ice-cold buffer A, centrifuged at 4° C at 800 g

for 5 min, the fatty layer was removed and 1 ml of the supernatant was used.

Substrate 1 µCi [ 1 �-3H]-androstenedione ( 15 nM) was added in 1 ml of buffer

B. In order to obtain blanks for each of the individual samples identical parallel

incubations were done to which the aromatase inhibitor exemestane (final

concentration 10-5 M) had been added. Aspecific influences have been

minimised because all individual samples have been corrected for by the use of

an identical sample to which exemestane had been added. Incubations were done

1 6 hours at 3 7 °C with continous shaking and finished by placing the tubes on

ice. To remove tritiated steroids, supernatants were extracted 6 times with 4 ml

diethylether; subsequently 0.5 ml suspension of dextran coated charcoal was

added and incubated for 20 minutes on ice. Samples were centrifugated at 4800g

at 4°C for 30 minutes. Two ml of the supernatant was added to 1 0 ml Ultima

Gold scintillation fluid and assayed for 3H radioactivity in a W allac model 14 14

liquid scintillation counter.

Protein assay

Protein content of the supernatants was measured according to the method of

Bradford, using a two wavelenght OD-ratio modification to improve linearity.5

µl sample was added to 200 µl Coomassie brilliant blue reagent. The OD was

measured at 595 nm and 450 nm in a Biorad microplate reader. Human serum

albumin was used as a standard at concentrations ranging from 0-700 µg/ml.

The sensitivity of the aromatase assay is approximately 0. 3 fmol/mg. Results of

aromatase levels below 0. 3 fmol/mg were considered to be 0.3 fmol/mg.

92

Statistics

Data analysis was performed using SPSS I 0.0 software package (SPSS Inc.,

Chicago, IL). Data are presented with mean values and standard deviation or

median and inter-quartile ranges, whenever appropriate. Statistical comparison

was made between aromatase concentrations in endometrial tissue and body fat,

the presence or absence of malignancy, and ovarian vein levels of androgens and

estrogens. The Mann-Whitney-U Test was used for comparison of continuous

variables. Using Spearman rank-correlation, aromatase levels were compared to

utero-ovarian steroids. P values lower than 0.05 were considered to reflect

statistical significance.

Results

Inclusion

In the study 60 postmenopausal women were included, of whom 52 had

endometrioid endometrial cancer and eight had a benign condition. Atypical

hyperplasia of the endometrium (N=2) was regarded as a benign condition.

Clinical data of patients involved in the study are shown in Table I . Of 43 women

with endometrioid endometrial cancer 21 women had a grade I malignancy, 13

women a grade II maligancy, and 9 women were reported with a grade III

malignancy. Five women were reported with stage la endometrial cancer, 16

women with a stage l b, and 16 women with a stage l e malignancy.Stage 2a and

stage 2b were found in 2 respectively 3 women. Finally, one woman had stage 3

endometrial cancer. No correlation could be detected between histopathological

stage, grade and aromatase activity or steroid hormone levels. No patients had

diseases of liver, kidney or of the endocrine system, apart from one woman with a

(grade I) stage I a malignancy. She had suffered from hyperadrenocorticism, but

all steroid hormone levels were below median levels. Nine patients with

93

endometrial cancer were excluded later, because not enough endometrial or fat

tissue was available for determination of aromatase levels, so aromatase levels

could be compared in 43 patients with endometrial cancer. In Table 2 we describe

clinical or pathological diagnoses or reasons for hysterectomy with bilateral

salpingo-oophorectomy in 8 women with a benign condition. Unfortunately,

only 8 women with a benign condition could be included in the study. The size

of the control group was limited by the fact that an abdominal hysterectomy

with bilateral salpingo-oophorectomy is rarely performed in case no malignancy

is present.

Table 1 . Clinical data of 51 postmenopausal women undergoing an abdominal

hysterectomy with bilateral salpingo-oophorectomy.

Endometrioid Benign Endometrial cancer Disorder

(n=43) (n=8)

Age (years) 63 .9 (9.8) 62. 1 (8.6) Years after menopause 12.5 ( 10.9) 1 1 .9 (7.6) Body weight 78. 1 (14.8) 69. l ( 1 3 .6) Grade I Endometrioid endometrial cancer 21 Grade II Endometrioid endometrial cancer 1 3 Grade II Endometrioid endometrial cancer 9 Stage 1 malignancy 37 Stage 2 malignancy 5 Stage 3 malignancy 1

Data represent mean values, standard deviation is shown in parentheses

94

Table 2. Clinical or pathological diagnosis/ reason for hysterectomy with

bilateral salpingo-oophorectomy in 8 women with a benign condition.

Diagnosis or reason for hysterectomy n Recurrent postmenopausal bleeding leading to diagnostic

hysterectomy 3

Benign endometrial polyp 2

Atypical hyperplasia 1

Adenomyosis and atypical hyperplasia 1

Moderate cervical dysplasia after menopause 1

Table 3 . Median utero-ovarian vein and peripheral serum levels (+ inter­

quartile ranges) of Testosterone (T), Androstenedione (A), Estrone (E 1) and

Estradiol (E2) in 60 women with endometrioid endometrial cancer or a benign

gynaecological condition.

Benign Endometrioid p-value

disorders endometrial

(n=8) cancer (n=52) T ovarian vein 4.7 (3.5: 16.7) 5.0 (2.8 : 10.0) 0.5 T peripheral 1 .9 ( 1 .8 :2. 1) 2. 1 ( 1 .6:2.6) 0.4 A ovarian vein 8. 1 ( 4.4:8.5) 9.0 (4.8: 15.4) 0.3 A peripheral 3.6 (2.5:5. 1 ) 3.4 (2.5:4.7) 1 .0 E 1 ovarian vein 0. 1 (0.0:0.2) 0. 1 (0.0:0.2) 0.4 El peripheral 0. 1 (0.0:0.2) 0. 1 (0.0:0. 1 ) 0.4 E2 ovarian vein 0. 1 (0. 1 :5.8) 0 . 1 (0.0:0.2) 0.4 E2 peripheral 0. 1 (0.0:0.5) 0. 1 (0.0:0.2) 0.4 N.S.

95

Median utero-ovarian vein levels of both testosterone and androstenedione were

slightly higher in patients with endometrioid endometrial cancer as compared with

patients with a benign condition, without reaching statistical significance (Table

3 ). 1 1 When comparing activities of aromatase in endometrial and body fat tissues to

the presence or absence of endometrial cancer, no significant differences could be

noticed, although aromatase activities in benign disorders were generally higher.

Median activities of aromatase were higher in body fat tissues, both in the

presence and in the absence of endometrioid endometrial cancer (Table 4 ). Steroid

levels of androgens and estrogens from the peripherous and utero-ovarian veins

were compared to activities of aromatase in endometrial tissue and abdominal

subcutaneous fat (Table 5). No correlations could be found, although testosterone

levels in the utero-ovarian veins were related to the activity of aromatase in body

fat (p<0.05), but we do not have an explanation for that relation. In Figure 1

aromatase activities in endometrial tissue and body fat are shown in the presence

or absence of endometrioid endometrial cancer, respectively. It is shown that most

patients with endometrioid endometrial cancer had low aromatase activities in the

endometrial tissue samples; e.g. 20 times a low level of less than 0.3 fmol/mg

(limit of detection) was measured.

96

Table 4. Median aromatase activity in endometrial tissue and subcutaneous

body fat compared to the presence or absence of endometrioid endometrial

cancer.

Endometrioid Benign disorder Mann-Whitney-CT Test endometrial cancer ( p-value)

(n=43) (n=8)

Aromatase in endometrial 0.3 1 . 1 0.2 tissue (fmol/mg)

Aromatase in body fat 1 .6 2.7 1.0 (fmol/mg)

N.S.

Table 5. Comparison of aromatase activity in endometrial tissue and body fat to

utero-ovarian vein and peripheral levels of androstenedione (A), Testosterone

(T) and estradiol (E2).

Aromatase in endometrial tissue Aromatase in abdominal fat R p R p

A ovarian vein 0.019 0.895 0. 105 0.483

A peripheral -0.042 0.0773 -0. 138 0.355 T ovarian vein 0.050 0.0729 0.3 17 0.034* T peripheral 0.052 0.0721 -0.079 0.598

E2 ovarian vein 0. 106 0.503 0. 1 12 0.497 E2 peripheral 0.08 1 0.584 0. 197 0. 197

* Speannan-rank correlation; p<O. 05 Steroid hormone levels and aromatase

activity

97

Figure 1 . Aromatase activity (fmol/mg) in endometrial and body fat tissues of

patients with endometrioid endometrial cancer (n=43) and of patients with

benign disorders (n=8).

C ·a3

bO E

cE 0 (J}

C'd ... E

a

Aromatase levels endometrial and fatty tissues so ----------

.. ., •

1 0 ::-.,

.. y

'" .,, ... • .., I y ' •

� ' yly ""

t ... ... , ... 1 --- 'f' - ., • ...... - ., - 'flW • • • - ., • """" •

0.2 I I I I

I endometrium I I fatty tissue I

High aromatase activity

T malignant

• benign

Remarkably, whereas in all but four patients aromatase activities in the

endometrial cancer tissues were below 3 fmol/mg, activity of aromatase in those

four patients appeared extremely high ( 1 5.2, 17.5, 20. 1 and 2 1 .2 fmol/mg,

respectively) (Figure 1 ). No correlation could be found between aromatase

activities of the four patients with high aromatase concentrations in the

98

endometrial cancer tissue and utero-ovarian or peripheral steroid levels. None of

these four patients had a history of other malignancies like breast cancer.

Remarkably, three of these four women had poorly differentiated endometrioid

endometrial carcinoma, once with serous papillary components.

Discussion

Aromatase activity in endometrial cancer was first demonstrated by Tseng in the

early eighties. 18 In later years aromatase activity was found in endometrial

tumours, whereas its expression was not detected in disease-free endometrium.

Aromatase mRNA expression was demonstrable in several benign disease states

of endometrium, namely endometriosis and adenomyosis. 19-29 Very recently

evidence was presented showing aromatase activity in benign myometrial

disease.30 Watanabe et al found aromatase immunoactivity in 67% of stromal

cells of endometrioid endometrial carcinoma. 20 The presence of in situ

production of estrogens in the stromal cells of endometrial carcinoma was

proven by immunohistochemistry, in situ hybridisation and biochemical

methods. In stromal cells of endometrial cancer tissue, but not in neoplastic or

hyperplastic cells mRNA hybridization signals of aromatase accumulated and

the distribution of aromatase mRNA correlated well with the immunohistoche­

mical localization of aromatase. It was concluded that intratumoral aromatase in

endometrial malignancy is associated with stromal invasion and is expressed

during the process of carcinoma-stromal interaction. The presence of aromatase

in stromal or interstitial cells suggests that locally produced estrogens may act

on carcinoma cells in a paracrine way. Thus, intratumoral estrogens derived

from in situ aromatization may function as a local mitogenic factor. Contrary to

our results, Watanabe found no aromatase activity in normal or hyperplastic

endometrial tissue. Like in our study, no correlation was found between

aromatase expression, activity or mRNA transcript levels and

99

clinicopathological factors such

Immunohistochemically Sasano

as clinical or histological stage. 20•27

confirmed the presence of aromatase

immunoreactivity in the stromal cells of endometrioid endometrial carcinoma.22

Contrary to other reports, we conclude that considerable activities of the enzyme

complex aromatase are present not only in endometrial cancer, but also in

normal endometrial tissue.20,22

-29 We found even higher median activities of

aromatase in endometrial tissue of benign origin than in endometrial cancer

tissue (Table 4). Among the women with endometrial cancer, we identified four

women with very high aromatase activities (Figure 1) . Remarkably, three of

these patients had poorly differentiated endometrial cancer, once with serous

papillary components. It is well known that estrogen and progesteron receptors

are less prevalent in poorly differentiated tumours. The role of androgen

receptors and androgen sensitivity of the endometrium needs to be studied more

extensively. In a recent study Mertens et al found an increase of androgen

receptor content with increasing histological grade of endometrial cancer.8 We

report a correlation between testosterone levels from the utero-ovarian veins and

the activity of aromatase in body fat (Table 5; p<0.05), but we are unable to

explain this correlation. Although we can not report an increase of aromatase

activity in the endometrial cancer tissue, our observations still fit well within

the hypothesis that multiple pathogenetic pathways may exist eventually leading

to the formation of endometrioid endometrial cancer. The local availability of

androgens and the aromatase activity present within endometrial tissue, are the

two essential conditions supporting the hypothesis that estrogens may function

as a mitogenic factor after local conversion of ( ovarian) androgens. Thus, in this

view some women may develop endometrioid endometrial cancer through

continuous hyperstimulation of the endometrial tissue by estrogens

systematically delivered from peripheral conversion, while in other women

malignant transformation is enhanced by local conversion of ovarian androgens

into estrogens, catalyzed by ( overexpression of) endometrial stromal aromatase.

1 00

Indeed, data from endometrial carcinoma cell lines suggest that some but not all

endometrial carcinomas may possess a heterogenous aromatase-dependent

growth stimulating system.2 1•3 1

•32 Possibly the normal mechanisms that inhibit

aromatase expression may be lost or aromatase-stimulating factors become

overexpressed. 27 Bokhman described two different pathogenetic types of

endometrial carcinoma. 33 The first pathogenetic type arises in women with

obesity, hyperlipidemia and signs of hyperestrogenism. In these women mainly

highly and moderately differentiated tumours are found. In women with the

second type of endometrial cancer poorly differentiated tumours are found,

including high grade malignancies like serous papillary or clear cell carcinoma.

Women with this second more virulent estrogen-independent form tend to be

less obese. Our observations partly correspond with this hypothesis. In the four

patients with high aromatase activity of the endometrial tissue mainly poorly

differentiated endometrial carcinoma was found, once with serous papillary

components. Remarkably, these patients indeed had a lower body mass index

with a mean value of 25.4 kg/m2, compared to a mean value of 28.8 kg/m2 in the

remaining 39 patients with endometrial cancer. Possibly a further subdivision

should be made within the first pathogenetic type between obese women with

signs of hyperestrogenism, and less obese women with generally poorly

differentiated endometrioid endometrial carcinoma,who appear to be aromatase­

dependent.

Nowadays, the enzyme aromatase has become the most important target enzyme

in the endocrine treatment of advanced breast cancer. Generally, in about 70%

of breast tumours aromatase activity is found above the limit of detection. The

tumour-bearing quadrant of the breast was reported to have the highest

aromatase activity, suggesting that local estrogen production in the breast

increases the risk of tumour development, or that tumour tissue can stimulate

aromatase activity in the surrounding tissue. 34 It seems obvious to search for

10 1

similarities between breast and endometrial cancer in their pathogenesis and

endocrine treatment, as both are mainly hormone-dependant tumours. However,

as endometrial cancer in the early stages must be considered as an often curable

disease using surgery and radiotherapy, the potential use of aromatase inhibitors

may be of benefit mainly to only a minority of women with advanced disease.

Although in our study most women had detectable aromatase levels, we found

high aromatase levels in four of 43 women with endometrioid endometrial

cancer. Possibly these four women may be the ones with aromatase-dependent

endometrial cancer. It still remains unproven whether the adjuvant use of

aromatase inhibitors may improve prognosis of patients with endometrial

cancer, as in breast cancer. Theoretically, aromatase inhibition may influence

both peripheral and endometrial stromal aromatization. Some small studies

reported objective responses and a decrease of aromatase activity after

administration of aromatase inhibitors to women with advanced endometrial

cancer.35•36 One study reported very limited response to aromatase inhibition

therapy in patients with advanced or recurrent endometrial cancer. Only a

minority of these patients was diagnosed with (hormone-dependent)

endometrioid endometrial cancer, and women with poorly differentiated tumours

did not respond. 37

In conclusion, the local availability of androgens in the utero-ovarian

circulation, and the presence of aromatase activity in endometrium, support the

hypothesis that ovarian androgens may play a role in the development of

endometrioid endometrial cancer through conversion into estrogens.

Acknowledgements

The authors are grateful to Dr. WJ Sluiter for his contribution in statistical

analysis.

102

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106

6

Aromatase, COX-2, HER-2/neu and P53 as

prognostic factors in endometrioid endometrial

cancer

VHWM Jongen

JM Briet

RA de Jong

E Joppe

KA ten Hoor

HM Boezen

DB Evans

H Hollema

AGJ van der Zee

HW Nijman

Accepted for publication in the International Journal of Gynecological Cancer

107

Abstract

The prognostic value of aromatase, COX-2, HER-2/neu and p53 expression was

determined in endometrioid endometrial cancer. Tissue microarrays were

constructed comprising samples from 315 endometrioid endometrial cancer

patients. Expression of aromatase, COX-2, HER-2/neu and p53 was determined

by immunostaining and related to classical clinico-histopathological parameters,

in addition to recurrence of disease and survival. Median follow-up time for all

patients was 5.0 years. Patients were classified as FIGO stage I (59.0%), stage II

( 17. 1%), stage III ( 19.4%) and stage IV (4. 1%). Sixty-five patients (20.6%)

developed recurrent disease and 38 ( 12. 1 %) died because of endometrial cancer.

Aromatase, COX-2, HER-2/neu and p53 expression was observed in 133

(42.2%), 107 (34.0%), 17 (5.4%), and 21 (6.7%) tumour cases, respectively.

Aromatase expression in tumour cells was related to aromatase expression in

stromal cells (p<0.0001) and to HER-2/neu expression in tumour cells

(p=0.0 19). Aromatase expression in both tumour as well as stromal cells was

related to low stage of disease (p=0.02 and p=0.00 1, respectively), while

aromatase expression in stromal cells was also related to low tumour grade

(p=0.021). P53 expression was related to high stage and high grade (p=0.006

and p<0.0001, respectively). In multivariate analysis p53 overexpression was

independently related to death due to the disease (p=0.043, OR 3.0, 95% CI 1.0-

8.7). For COX-2, HER-2/neu and aromatase no relation with any other

histopathological parameter or survival was found. In conclusion, aromatase

expression in tumour and stromal cells and p53 expression in tumour cells are

related to tumour grade and stage of disease, while p53 is an independent

prognostic factor in endometrioid endometrial cancer. The possible interaction

of the studied parameters in the development of endometrial cancer is discussed.

108

Introduction

Over the past few years molecular markers such as aromatase, cyclooxygenase-2

(COX-2) and HER-2/neu have been identified as important cell biological

determinants, contributing to breast cancer development and simultaneously

becoming targets of new treatment modalities in breast cancer.1 •2 Studies in

breast cancer showed strong correlations between aromatase, COX-2 and HER-

2/neu enzyme systems, while interactions between these biomarkers were found

to play a role in the production of estrogens. 3•4 Comparable to breast cancer, also

in endometrial tissues aromatase activity may contribute to the development of

endometrioid endometrial cancer by local conversion of androgens into

estrogens, thereby inducing high local estrogen concentrations. 5•6 If indeed

activity of aromatase, COX-2 and HER-2/neu are involved in the development

of endometrioid endometrial cancer, a possible therapeutic role may be

considered for inhibitors of either aromatase, COX-2 or HER-2/neu in treatment

strategies of advanced or recurrent endometrial cancer.7 Likewise, strong

predictive markers could be used to further individualize treatment modalities,

next to classical clinico-histopathological factors.

Overexpression of p53 is another wellknown, cell biological prognostic factor

in endometrial cancer.8- 12 Data on a possible relation between p53 expression and

expression of HER-2/neu, aromatase or COX-2 are scarce and inconclusive in

endometrial cancer.13• 1 4 The aim of our study was to relate the expression of

aromatase, COX-2 and HER-2/neu to p53 expression, classical clinico­

histopathological characteristics and to disease-free and overall survival m a

large, well-documented series of endometrioid endometrial cancer patients.

109

Patients and Methods

Study population

All patients were consecutively treated at the department of Obstetrics and

Gynecology of the University Medical Center of Groningen, the Netherlands,

between 1984 and 2004. Data of 411 patients with endometrial cancer were

prospectively collected based on hospital records including surgical notes and

pathology reports. Corresponding histological material, suitable for tissue

microarray (TMA) construction, could be obtained from 367 out of 411 patients.

In the remaining 44 patients paraffin-embedded tumour tissue was not available

for TMA construction because of either lack of cancer tissue, or insufficient

tissue quality. Finally 315 out of 367 patients had an endometrioid tumour type

and were selected for for the present study. From 1988 pelvic lymphadenectomy

was performed in case of a grade 3 endometrioid endometrial tumour and/ or

cervical involvement. Para-aortic lymphadenectomy was done in case of suspect

para-aortic lymph nodes at surgery, or proven positive pelvic nodes. Extensive

lymph node dissection was omitted in case of severe co-morbidity. Clinico­

pathological characteristics and follow-up data were noted, using medical

records. Follow-up surveillance consisted of a clinical history, physical

examination and a yearly PAP smear of the vaginal vault with an increasing

interval until 10 years after treatment. Patient characteristics have been

described previously in a study from our group on lymph vascular space

involvement in endometrioid endometrial cancer. 1 5

Design and construction of the tissue microarray

The TMA was constructed, as described previously, using paraffin-embedded

tumour tissue of endometrial cancer patients. 16-19 In short, morphologically

representative areas of tumour were marked on hematoxylin- and eosin- stained

sections. Three 0.6 mm core biopsies of representative areas were taken from the

1 10

individual paraffin embedded tumour blocks. The core biopsies were then

assembled on a recipient paraffin block at pre-defined array locations, using a

precision instrument (Beecher Instruments, Silver Springs, Maryland).

Immunostaining

Four µm sections were cut and mounted on APES (3-aminopropyltriethoxylane;

Sigma-Aldrich, Diesenhofen, Germany) -coated glass slides. Sections were

dewaxed in xylene and rehydrated through graded concentrations of ethanol

( 100%, 90% and 70%) to distilled water. Antigen retrieval was performed when

needed by microwave method. For COX-2 antigen retrieval was performed for

15 minutes at 95 to 100° C in a microwave in lmM EDTA buffer (pH 8) and for

P53 1 OmM TRIS/EDT A (pH 9 .0) was used. Endogenous peroxidase activity was

blocked for 30 minutes with 0.3% H2O2. For aromatase endogenous avidin and

biotin activity was blocked for 15 minutes using a blocking kit (Vector

Laboratories, Burlingame, USA). Slides for aromatase staining were

preincubated with 1 % normal rabbit serum in Phosphate Buffered Saline (PBS)

pH 7.2 containing 1 % BSA. Slides for COX-2 and p53 were incubated with the

primary antibody for 60 minutes at room temperature and aromatase slides were

incubated overnight at 4 ° C. All dilutions were made in Phosphate Buffered

Saline (PBS) pH 7.2 containing 1 % BSA. For HER-2/neu a Ventana autostainer

was used (monoclonal anti rabbit 4B5 as primary antibody). As a primary

antibody for COX-2 we used anti mouse clone 33 (BD Biosciences) in a dilution

of 1 :200. For COX-2 rabbit anti mouse/HRP (DAKO) was used as secondary

antibody, followed by goat anti rabbit/HRP (DAKO), both 1 : 100 for 30 minutes

at room temperature. For p53 biomarker analysis anti mouse antibodies DO-7

(DAKO) were used as primary antibodies in a dilution of 1 :2000. Also for p53 a

goat anti-mouse-/rabbit secondary antibody conjugated with a peroxidase­

labelled polymer (Dako En Vision+ system) was used. For determination of

aromatase expression we used antibody 677 (Novartis), as primary antibodies in

1 1 1

a dilution of 1 : 1 00.20•21 Biotinylated rabbit anti mouse (DAKO) was used as a

secondary antibody ( 1 :300 for 30 minutes at room temperature) for aromatase

after which Streptavidine/HRP (DAKO) 1 :300 was applied for 30 minutes at

room temperature. For all slides the antigen-antibody reaction was visualized

with 3 ,3 '-diaminobenzidine (DAB substrate kit, Vector Laboratories,

Burlingame, U.S.A.) for 1 0 minutes, sections were then counterstained with

Mayer's haematoxylin and the slides were dehydrated in graded ethanol, dried

and cover slipped. Negative controls were obtained by omission of the primary

antibody. The TMA included breast and colon cancer as well as placental tissue

of known biomarker expression for use as positive control.

Figure 1 . Positive immunohistochemical staining for aromatase expression in

tumour cells.

1 12

Analysis of immunostaining on TMA

Immunostaining was semi-quantitatively scored based on the intensity of

nuclear or cytoplasmic staining. Intensity of immunostaining for receptor

expression was scored as negative (0), weak ( 1+), positive (2+) or strongly

positive (3+) staining. For aromatase both staining of stromal as well as tumour

cells was scored (Figure 1 ). In accordance to literature, weak to positive

cytoplasmic staining in stromal or tumour cells was considered as aromatase

expression positivie. 1 7•2° COX-2 was considered positive if more than 1 0% of the

tumour cells showed positive cytoplasmatic staining. 1 7 Immunostaining for DO-

7, an antibody which detects both wild-type and mutant p53 protein, was scored

according to the intensity of nuclear staining and to the percentage of positively

stained tumour cells. P53 was considered positive ( overexpression) in case a

substantial percentage of the nuclei (more than 50%) stained positive as

described by others.8 HER-2/neu expression was scored as recommended by the

manufacturer's scoring guidelines. HER-2/neu staining was considered positive

if there was moderate to strong complete membrane staining in more than 10%

of tumour cells, comparable to a recently published study by Morrison et al. 22

Immunostaining was scored by two independent observers (KH and VJ).

Observers had no prior knowledge of clinicopathological information. Cases

with a discrepancy in score between the two observers were re-examined with a

gynaecological pathologist (HH) until full agreement was reached. Minimally

two cores with representative tumour had to be present on the TMA for a sample

to be entered into analysis. The mean overall core loss was 6 .6% ( 6 .3-7 .6% ) .

Statistics

All analyses were performed using SPSS 14.0. Differences between groups were

tested using the X2 or the Fisher's exact test. Kaplan-Meier survival analysis and

Cox regression analysis were performed to study the role of aromatase, COX-2,

1 1 3

Table 1 . Histological and clinical characteristics of included patients with

endometrioid endometrial cancer (n=315).

Patient age, years Median 64.7 Range 32-89

Tumour Stage I 186 59.0 II 54 17. 1 III 6 1 19.4 IV 13 4. 1 Missing 1 0.3

Histological grade Grade 1 162 5 1.4 Grade 2 96 30.5 Grade 3 56 17.8 Undifferentiated 1 0.3

Myometrial invasion < 50% 185 58.7 > 50% 126 40.0 Missing 4 1.3

L VSI Negative 222 70.5 Positive 73 23 .2 Missing 20 6.3

Recurrence No recurrence 250 79.4 Local 26 8.3 Regional 8 2.5 Distant 3 1 9.8

Pelvic lymph nodes Negative 97 30.8 Positive 29 9.2 No sampling 189 60.0

Paraaortic lymph nodes Negative 45 14.3 Positive 12 3 .8 No sam lin 258 8 1.9

LVSI: Lymph Vascular Space Involvement

1 14

HER-2/neu, and p53 in relation to recurrence (cq disease free survival) and

death ( survival) as end points. Multiple logistic regression analyses were

performed with COX-2, HER-2/neu, aromatase and p53 as the dependent

variables and known risk factors lymph vascular space invasion, myometrial

invasion, grade and stage of disease as independent variables entered

simultaneously into the model. Patients were censored at date of last contact or

death in case of no relapse. P-values < 0.05 were considered statistically

significant (tested 2-sided).

Results

In Table 1 the clinico-pathological characteristics of 315 patients with

endometrioid endometrial cancer are shown. The median follow-up time for all

patients was 4.97 years (range 0.0-21.5). Follow-up data were completed until

September 2006. Median age at time of diagnosis was 64.7 years (range 32.0-

89.0). Patients were classified as FIGO stage I (59.0%), stage II ( 17.1 %), stage

III ( 19 .4 % ) and stage IV ( 4 . 1 % ) . Hysterectomy and bilateral salpingo­

oophorectomy was performed in 56.2% of the patients, and more extended

surgery in 41.9% of the patients. Adjuvant post-operative radiotherapy was

given to 168 patients (53.4%). In 65 patients (20.6%) recurrent disease was

diagnosed. Local relapse at the vaginal vault occurred 26 times, while regional

and distant metastases were diagnosed 8 and 31 times, respectively. The median

time to relapse was 1 . 7 years (range: 0.1-7 .6). During follow-up 8 1 out of 315

patients died (26.0%), 38 patients ( 12. 1 %) died from endometrial cancer.

1 15

Association of aromatase, COX-2, HER-2/neu, and P53 with clinico­

h istopathological factors.

Aromatase expression in tumour cells was observed in 133 (42.2%) tumours,

while 89 (28.3%) tumours showed aromatase expression in stromal cells. COX-

2, HER-2/neu and p53 expression was observed in 107 (34.0%), 17 (5.4%) and

2 1 (6.7%) patients, respectively. (Table 2). Expression of aromatase in tumour

cells was related to expression in stromal cells (p<0.0001 ). Aromatase

express10n in both tumour and stromal cells was related to HER-2/neu

expression (p=0.0 19 and p=0.02 1 respectively). Aromatase expression in tumour

and stromal cells was also related to low stage of disease (p=0.02 and p=0.00 1

respectively), while aromatase expression in stromal cells correlated with low

tumour grade (p=0.021 ) (Table 3). P53 expression was related to high stage of

disease and tumour grade (p=0.006 and p<0.000 1 , respectively) (Table 3 and 4).

In a multivariate (logistic regression) analysis absence of lymph vascular space

invasion was related to COX-2 expression (p=0.029, OR 0.46, 95% CI 0.2-0.9)

(Table 5). No relation was found between aromatase expression and disease free

or overall survival. In univariate analysis risk for death of disease was increased

in the presence of p53 (p=0.047) (Figure 2). P53 also had an independent

prognostic value in multivariate analysis with all prognostic factors for death

due to disease (p=0.043, OR 3.0, 95% CI 1 . 0-8.7) (Table 6). In a multivariate

analysis tumour grade was the strongest predictor for recurrence (p=0.003, OR

3. 6, 95% CI 1 . 5-8.3) (Table 7).

1 16

Table 2. Co-expression of aromatase, HER-2/neu, COX-2 and P53 in 315

patients with endometrioid endometrial cancer.

Aromatase Aromatase HER-2/ neu COX-2 cancer cells in stroma (n = 1 33) (n = 89) (n = 1 7) (n = 1 07)

Aromatase in cancer Positive X 83 (93.3%) 12 (70.6%) 87 (81 .3%) cells

Negative X 6 (6 .7%) 5 (29.4%) 20 ( 1 8.7%) Aromatase in stroma Positive 83 (62.4%) X 9 (52.9%) 24 (22.4%)

Negative 50 (37.6%) X 8 (47. 1 %) 83 (77.6%)

HER-2/ neu Positive 1 2 (9.0%) 9 ( 1 0. 1 %) X 1 1 ( 1 0.3%)

Negative 1 1 1 (91 .0%) 80 (89.9%) X 96 (89 .7%)

COX-2 Positive 43 (32.3%) 24 (27 .0%) 1 1 (64.7%) X

Negative 90 (67.7%) 65 (73.0%) 6 (35.3%) X

P53 Positive 9 (6.8%) 6 (6.7%) 4 (23.5%) 1 1 ( 1 0.3%)

Negative 1 24 (93.2%) 83 (93.3%) 1 3 (76 .5%) 96 (89.7%)

1 17

P53

(n=21 )

9 (42 .9%)

1 2 (57. 1 %)

6 (28.6%)

15 (71 .4%)

4 ( 1 9 .0%)

1 7 (81 .0%)

1 1 (52 .4%)

1 0 (47.6%)

X

X

Table 3 . Chi square analysis of receptor expression in relation to known

prognostic factors in patients with endometrioid endometrial cancer.

StaQe Grade Stage Stage Grade Grade 3 and I and I I I l l and IV 1 and 2 undifferentiated

Aromatase Neg in cancer n= 1 62 1 1 5 47 1 1 4 1 9 cells Pos

n= 1 33 1 09 23 1 26 36

p value 0.020 NS

Aromatase Neg in stroma n=206 146 60 1 61 45

Pos n=89 78 1 0 80 9

p value 0.001 0 .021 HER-2/neu Neg

n=274 2 10 64 220 54 Pos n= 1 7 1 2 5 1 5 2

p value NS NS

COX-2 Neg n= 1 87 140 47 1 46 41 Pos n=1 07 84 23 92 1 5

p value NS NS

P53 Neg n=274 2 1 3 60 229 45

Pos n=2 1 1 0 1 1 1 0 1 1

p value 0.006 <0.0001

P derived from 2 sided testing using Chi-square test Abbreviations Neg = negative

Pos = positive

LVSI LVSI Neaative

1 1 4

94

142

66

1 96

1 0

1 23

85

1 93

1 4

LVSI = Lymph Vascular Space Involvement

LVSI Positive

41

29

NS

53

1 7

NS

67

3

NS

55

1 6

0.006

66

6

NS

1 1 8

Table 4. The independent effect of stage of disease, tumour grade, myometrial

invasion and L VSI on the presence of p53 expression in endometrioid

endometrial cancer (logistic regression analysis).

95.0% Cl for OR EXP(B) P value

Lower Upper

Stage of 3 . 1 65 1 . 1 2 1 8.938 0.030 d isease

Tumour grade 4.797 1 .666 1 3.8 14 0.004

Myometrial invasion 0.732 0.250 2. 1 49 0.570

LVSI 0.51 7 0. 1 47 1 .8 1 5 0.303

LVSI = Lymph Vascular Space Involvement

Table 5. The independent effect of stage of disease, tumour grade, myometrial

invasion and LVSI on the presence of COX-2 expression in endometrioid

endometrial cancer (logistic regression analysis).

95.0% Cl for OR EXP(B) P value

Lower Upper

Stage of d isease 1 .201 0.633 2.279 0 .575

Tumour grade 0.787 0.387 1 .602 0.509

Myometrial invasion 0.822 0.473 1 .429 0.488

LVSI 0.461 0.231 0.923 0.029

LVSI = Lymph Vascular Space Involvement

1 1 9

Table 6. The risk of death due to endometrioid endometrial cancer according to

the presence of aromatase in cancer and stromal cells, HER-2/neu, COX-2, p53,

stage of disease, tumour grade, myometrial invasion and L VSI ( cox regression

analysis).

I -----OR�-

95.

�:�(�\

for

� Lower Upper

Aromatase in cancer cells

Aromatase in stroma

I HER-2/neu

COX-2

P53

Stage of disease

Tumour grade

Myometrial invasion

I LVSI

3 .702

0.584

0.660

0.474

3.01 0

1 .3 1 7

1 . 1 85

2. 1 1 4

1 .264

0.941 14 .562

0. 1 48

0. 1 02

0. 1 77

1 .037

0.583

0.435

0.866

0.5 1 9

2.300

4.278

1 .264

8.738

2.973

3.229

5. 1 56

3.080

LVSI = Lymph Vascular Space Involvement

P value

0.061

0.442

0.663

0 . 1 36

0.043

0.508

0.74 1

0 . 1 00

0.605

120

Table 7. The risk of relapse of endometrioid endometrial cancer according to the

presence of aromatase in cancer and stromal cells, HER-2/neu, COX-2, p53,

stage of disease, tumour grade, myometrial invasion and L VSI ( cox regression

analysis).

--95.0% Cl for

OR Exp(B) P value Lower Upper

Aromatase in 0.895 0.432 1 .856 0.766 cancer cells

Aromatase in 0.651 0.251 1 .688 0.377 stroma

HER-2/neu 1 .751 0.234 1 3.083 0 .585

COX-2 0.529 0.269 1 .037 0.064

P53 1 .086 0.359 3.282 0 .884

Stage of disease 0.854 0.487 1 .498 0.581

Tumour grade 3 .569 1 .535 8.299 0.003

Myometrial invasion 1 . 1 95 0.664 2. 1 50 0.553

LVSI 0.654 0.337 1 .272 0.21 1

LVSI=Lymph Vascular Space Involvement

12 1

Figure 2. Kaplan-Meier survival analysis concerning p53 expression and

cumulative disease-specific survival (p=0.047).

Survival Funct ions

0,0 2 ,0 4 P s p 8,0

follow-up in years

1 0,0

p53 e xp re ss ion -- negative

-- positive

122

Discussion

In this study in a large cohort of endometrial cancers aromatase expression was

frequently found both in tumour as well as in stromal cells. We found aromatase

in tumour and stromal cells to be related to low tumour grade and low stage of

disease. Also, aromatase expression in tumour cells was related to expression of

HER-2/neu. The aromatase enzyme is a product of the CYP 19 gene and is

capable of converting C 19 steroids (androgens), produced by adrenal glands

and ovaries, into C 18 steroids (estrogens). Conversion of circulating androgens

into estrogens may take place not only in peripheral adipose tissues, but in

endometrial tissue as well.6•7•17•23•24 In this way, aromatase activity may be

involved in the development of endometrial cancer.7•25-28 Our results suggest that

aromatase activity in tumour cells plays a role especially in lower stage and

grade of endometrial cancer, as the aromatase expression in tumour cells appears

to be lost with increasing stage and/ or dedifferentiation. As the presence or

absence of aromatase expression did not contribute to disease outcome, no

arguments could be found for adjuvant treatment with aromatase inhibitors in

the palliative setting. We found aromatase expression in cancer cells related to

aromatase expression in stromal cells. Possibly aromatase expression in the

nests of cancer cells is responsible for local conversion of androgens in

estrogens. Aromatase expression in the surrounding stroma may represent tissue

reaction, as aromatase expression was found far less frequently in stromal cells

than in tumour cells and expression in stromal cells was not related to expression

in cancer cells. In contrast, Watanabe localized aromatase expression in stromal

cells and suggested a paracrine growth mechanism through in situ production of

estrogens in stromal cells. 5 This different localization might be attributable to

differences in antibodies and techniques used. The aromatase antibody 677, that

was used for the first time in endometrial cancer in our study may be more

reliable, as its specificity was recently established in an international

123

collaborative effort, that had the specific purpose of intratumoural detection of

aromatase in archival or paraffin-embedded tissue.20•2 1

In the literature HER-2/neu overexpression has been correlated with non­

endometrioid endometrial cancer, advanced stage, high grade, myometrial

invasion and recurrent disease and was suggested to be used as a prognostic

indicator for hormone-independent growth.29-3 1 Interestingly, in our patient group

with only endometrioid endometrial cancer a correlation was found between

aromatase and HER-2/neu expression. Also in breast cancer HER-2/neu and

aromatase overexpression were reported to be related, with HER-2/neu as the

primary molecular determinant of increased aromatase activity and COX-2 as

the potential functional intermediate. 3•4 Increased prostaglandin synthesis from

COX-2 expression prompts formation of aromatase and may play a role in the

initial development of endometrial cancer.1•

1 3•32

-34 This may lead to the interesting

possibility that the regulation of aromatase expression m breast and

endometrioid endometrial cancer follows similar pathways, as both appear

examples of aromatase expressing estrogen dependent tumours.

We found COX-2 expression in 36% of endometrioid tumour cases and others

have shown positive immunostaining from 39 to 69% in endometrial

cancer.1 3•32

•35

-37 COX-2 did not appear to be a prognostic marker in our large

cohort study. In other studies, conflicting results were found on the relation

between COX-2 expression and survival and clinical variables such as stage and

histological type.4•

1 3•

1 7•32

•36 A strong relation in breast cancer was found between

COX-2 and CYP19 gene expression, of which the aromatase enzyme is the

product. 38•39 In contrast to a previous report, we did not find a correlation with

aromatase, although a tendency existed for aromatase expression in stromal cells

to be related to COX-2 expression (p=0.074). 1 7

P53 is a tumour-suppressor protein which causes cell cycle arrest in case of

DNA damage, to allow DNA repair or induction of apoptosis in case of

substantial damage. In malignancies p53 is often overexpressed due to mutations

124

and thereby uncapable of performing DNA repair. In our study in endometrioid

endometrial cancer p53 overexpression was independently related to a high

stage of disease, grade and to poor prognosis. Previous studies found p53

overexpression especially associated with non-endometrioid endometrial

cancer.29•40 Although in our study p53 over-expression was a relatively rare

observation (7. 1%), it appeared to be a strong prognostic marker.

We conclude that the local presence of aromatase in early stage and low grade

endometrioid endometrial cancer supports the hypothesis that aromatase may

contribute to the promotion of endometrial cancer. As aromatase expression in

both tumour and stromal cells was related to HER-2/neu expression, the

regulation of aromatase expression in breast and endometrioid endometrial

cancer may follow similar pathways. Also, in endometrioid endometrial cancer,

p53 is an independent prognostic marker for disease-specific survival.

Acknowledgements

We acknowledge the support of Novartis Pharma AG, Basel, Switzerland, for

their help in disposing the 677 aromatase antibodies.

HW Nijman is supported by the Dutch Cancer Society (Grant 2002-2768).

125

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129

130

7

Expression of estrogen and progesterone receptors

alpha and beta in a large cohort of patients with

endometrioid endometrial cancer

VHWM Jongen

JM Briet

RA de Jong

KA ten Hoor

HM Boezen

AGJ van der Zee

HW Nijman

H Hollema

Submitted

1 3 1

Abstract

The prognostic impact of estrogen receptor (ER) and progesterone receptor (PR)

alpha and beta was determined in endometrioid endometrial cancer. Tissue

microarrays were constructed from 315 endometrioid endometrial cancer

patients. Receptor expression was assessed by immunostaining, and their semi­

quantitatively determined expression levels were correlated to classical clinico­

histopathological parameters in addition to disease free and disease specific

survival. Patients were classified as FIGO stage I (59.0%), stage II (17.1%),

stage III (19.4%) and stage IV (4.1 %). Sixty-five patients (20.6%) developed

recurrent disease and 38 (12.1 %) died due to endometrial cancer. In univariate

analysis, expression of ER alpha was related to early stage endometrial cancer

(p=0.020), while expression of ER alpha, PR alpha and PR beta was associated

with lower grade tumours (p<0.0001, p<0.001 and p=0.001 respectively). A

ratio of ER alpha / ER beta < l was related to a shorter disease free survival

(p=0.027), while the ratio of PR alpha / PR beta <l both was associated with a

shorter disease free survival as well as a shorter overall survival (p=0.044 and

p=0.005, respectively). In early stage disease, using multivariate analysis,

absence of ER alpha was independently related to death of disease (p=0.017, OR

7.28, 95% CI 1.42-37.25), while absence of PR alpha (p=0.015, OR 4.2, 95% CI

1.32-13.33) appeared to be an independent prognostic factor for relapse of

disease. We conclude that in early stage endometrioid endometrial cancer

absence of PR alpha is an independent prognostic factor for disease-free

survival, while patients with ER alpha positive tumours have a better overall

survival.

1 32

Introduction

Endometrial cancer is the most frequently found genital tract malignancy in

women, and consists of two major histological types, endometrioid endometrial

cancer, and nonendometrioid endometrial cancer including high-risk malignancies

such as serous papillary and clear cell carcinoma. Endometrioid carcinoma is the

most common form, accountable for more than 7 5% of all cases of endometrial

cancer.1 Surgical treatment of endometrial cancer consists of total abdominal

hysterectomy with bilateral salpingo-oophorectomy next to pelvic and para­

aortic lymphadenectomy in high risk patients. Patients with stage I or II

endometrioid endometrial cancer and poor prognostic factors such as deep

myometrial invasion and / or poor differentiation grade are treated additionally

with pelvic radiotherapy, which reduces risk of local recurrence without

prolonging overall survival.2•3 Recently, it was suggested that patients with high­

risk early stage endometrial cancer might benefit from adjuvant chemotherapy.4

The question remains how to identify patients, who might benefit from adjuvant

treatment modalities. While some studies report an independent prognostic value

for estrogen receptor (ER), and progesterone receptor (PR) expression in

endometrioid endometrial cancer, others could not confirm this.5-13 Also, clinical

data in relation to the prevalence of steroid receptor isoforms ER and PR alpha

and beta are scarce. 7-9,11 The present study aimed to assess the prognostic value

of ER alpha and beta, and PR alpha and beta in a large cohort of endometrioid

endometrial cancer patients. We were particularly interested in the potency of

steroidmarkers for prediction of prognosis, as this may lead to a more patient

specific risk profile and treatment. 6•14

133

Patients and Methods

Study population

All patients were consecutively treated at the department of Obstetrics and

Gynaecology of the University Medical Center of Groningen, the Netherlands,

between 1 984 and 2004. Data of 41 1 patients with endometrial cancer were

prospectively collected based on hospital records including surgical notes and

pathology reports. Corresponding histological material, suitable for tissue

microarray (TMA) construction, could be obtained from 367 out of 4 1 1 patients.

In the remaining 44 patients paraffin-embedded tumour tissue was not available

for TMA construction because of either lack of cancer tissue, or insufficient

tissue quality. Finally 3 15 out of 367 patients had an endometrioid tumour type

and were selected for the present study. Clinico-pathologic and follow-up data of

the patients have been described previously. 1 5 From 1 988 pelvic

lymphadenectomy was performed in case of a grade 3 endometrioid endometrial

tumour or cervical involvement. Para-aortic lymphadenectomy was done in case

of suspect para-aortic lymph nodes at surgery, or proven positive pelvic nodes.

Extensive lymph node dissection was omitted in case of severe co-morbidity.

Clinico-pathological characteristics and follow-up data were noted, using

medical records. Follow-up surveillance consisted of a clinical history, physical

examination and a yearly PAP smear of the vaginal vault with an increasing

interval until 10 years after treatment.

Design and construction of the tissue microarray

The TMA was constructed, as described previously, using paraffin-embedded

tumour tissue of endometrial cancer patients. 16-1 9 Three representative cores of

0.6 mm diameter were taken from the tumour tissue block and placed in a

recipient paraffin block on pre-defined array locations, using a precision

instrument (Beecher Instruments, Silver Spring, Maryland). For each TMA

1 34

block control cores with known biomarker expression were incorporated in the

arrays, together with endometrial cancer tissue originating from the same donor

blocks for use as internal positive control for intra-run variability. Controls

included tissues of breast cancer and benign endometrium, gut, and placenta.

Immunostaining

Four µm sections were cut from the array blocks and mounted on APES (3-

aminopropyltriethoxylane; Sigma-Aldrich, Diesenhofen, Germany) -coated

glass slides. Sections were dewaxed in xylene and rehydrated through graded

concentrations of ethanol (100%, 90% and 70%) to distilled water. Antigen

retrieval was performed by either autoclave or microwave method when needed.

Antigen retrieval by autoclave treatment 3 times 5 minutes at 1 l 5°C was used

for PR beta in Blocking reagent (Boehringer Mannheim) (2% block + 0.2% SDS

in maleic acid, pH 6.0) and for ER alpha and ER beta in 1 OmM citrate buffer

(pH 6.0). For PR alpha antigen retrieval was performed for 15 minutes at 95 to

100°C in a microwave 1 mM EDT A buffer (pH 8). Endogenous peroxidase

activity was blocked by incubating the slides for 30 minutes with 0.3% H2O2.

ER alpha slides were preincubated with 1 :5 diluted normal goat serum for 15

minutes at room temperature. For ER beta endogenous avidin and biotin activity

was blocked for 15 minutes using a blocking kit (Vector Laboratories,

Burlingame, USA). Slides were then incubated with the primary antibody for 60

minutes at room temperature. For ER alpha 0.05M TRI S Buffered Saline (TBS)

pH 7.2 containing 1 % BSA was used for all dilutions. For ER beta the primary

antibody was diluted in 0.05M TRIS/HCL/1 % BSA, all other dilutions were

made in PBS pH 7 .2 containing 1 % BSA. The primary antibodies and dilutions

used are mentioned in Table 1. For ER alpha goat anti mouse IgG 1/HRP 1 :40

and for PR alpha and PR beta rabbit anti mouse/HRP (DAKO, Glostrup,

Denmark) 1: 100 was used as secondary antibody, followed by goat anti

rabbit/HRP (DAKO), 1: 100 for 30 minutes at room temperature. Biotinylated

135

rabbit anti mouse (DAKO) was used as a secondary antibody (1 :300 for 30

minutes at room temperature) for ER beta after which Streptavidine/HRP

(DAKO), 1 :300 was applied for 30 minutes at room temperature. For all slides

the antigen-antibody reaction was visualised with 3,3 '-diaminobenzidine (DAB

substrate kit, Vector Laboratories) and sections were counterstained with

Mayer's haematoxylin. The TMA included breast and colon cancer as well as

placental tissue of known biomarker expression for use as positive control.

Negative controls were obtained by omission of the primary antibody.

Table 1 . Primary antibodies and dilutions, used for receptor analysis.

Antigen Antibody Clone Company Dilution

Estrogen receptor alpha Monoclonal mouse 6F l 1 Serotec 1 :20

Estrogen receptor beta Monoclonal mouse ppg5/1 0 Serotec 1 :20

Progesterone receptor Monoclonal mouse HPRa7 Neomarkers 1 :50

alpha

Progesterone receptor Monoclonal mouse HPRa2 Neomarkers 1 :50

beta

Analysis of immunostaining on TMA

The receptor immunoreactivity for ER and PR was semi-quantitatively scored

according to the intensity and percentage of tumour cells showing positive

nuclear staining, as described previously.8•20 Intensity of immunostaining for

receptor expression was scored as negative (0), weak ( l+), positive (2+) or

strongly positive (3+ ). ER and PR staining was considered positive if nuclear

staining was present in more than 10% of the tumour cells (Figure 1 a and 1 b

respectively).

1 36

Figure la . Positive immunohistochemical staining for expression of

progesterone receptor alpha.

Figure 1 b . Positive immunohistochemical staining for expression of estrogen

receptor alpha.

137

lmmunostaining was scored by two independent observers (KH and VJ).

Observers had no prior knowledge of clinicopathological information. Cases

with a discrepancy in score between the two observers were re-examined with a

gynaecological pathologist (HH) until consensus was reached. Minimally two

cores with representative tumour had to be present on the TMA for a sample to

be entered into analysis.The mean overall core loss was 5.7% (4.8-6.3%).

Statistics

All analyses were performed using SPSS 14.0. Differences between groups were

tested using the X2 or the Fisher's exact test if appropriate. The ratio of ER alpha

and beta, and the ratio of PR alpha and beta were calculated using the semi­

quantitative numerical scores of the staining intensities, and classified as a ratio

> 1 or < 1, as described previously .8 Survival curves were constructed according

to Kaplan-Meier. Logistic regression analyses were performed with disease free

and disease specific survival as dependent variables, and ER alpha and PR alpha

as the independent variables, with adjustment for known risk factors as lymph

vascular space invasion, myometrial invasion, grade and stage of disease entered

simultaneously into the model. Cox regression analysis was used for time to

event analyses, with recurrence or death of disease as end point. Patients were

censored at date of last contact or death in case of no relapse. P-values < 0.05

were considered statistically significant (tested 2-sided).

Results

In Table 2 the clinico-pathological characteristics of 315 patients with

endometrioid endometrial cancer are shown. The median follow-up time was

4.97 years (range 0.0 - 21.5). Follow-up data were completed until September

2006. Median age at time of diagnosis was 64. 7 years (range 32.0-89.0).

Patients were classified as FIGO stage I (59.0%), stage II ( 17. 1 %), stage III

138

( 19.4%) and stage IV (4. 1%). Hysterectomy and bilateral salpingo­

oophorectomy was performed in 56.2% of the patients, and more extended

surgery in 4 1 .9% of the patients. Adjuvant post-operative radiotherapy was

given to 168 patients (53.3%). In 65 patients (20.6%) recurrent disease was

diagnosed. Local relapse at the vaginal vault occured in 26 patients, while in 8

and 31 patients regional and distant metastases were diagnosed, respectively.

The median time to relapse was 1 . 7 years (range: 0. 1 - 7 .6). During follow-up

82 out of 3 15 patients died (26.0%), 38 patients ( 12. 1 %) died from endometrial

cancer.

Association of ER alpha and beta, PR alpha and beta with h istopathological

factors

ER alpha and beta expression was observed in 185 (58.7%) and 23 1 (73.3%)

tumours respectively, in addition to PR alpha and beta expression in 1 16 (36.8%)

and 12 1 (38.4%) tumours respectively. Co-expression of ER and PR alpha and

beta in patients with endometrioid endometrial cancer is depicted in Table 3. In

87% of ER alpha positive tumours expression of ER beta was seen, while ER

alpha expression was found in 70% of ER beta positive tumours. Only half of

ER alpha and beta positive tumours expressed PR alpha and beta. Positive PR

alpha and beta expression were both combined with expression of ER alpha and

beta in 86% and 88%, respectively. PR alpha was seen together with PR beta

expression in 86%, while PR beta expression went together with PR alpha

expression in 83% of cases. In Table 4 receptor expression is shown in relation

to traditional prognostic factors such as stage, grade and lymphvascular space

invasion. ER alpha was found more often in early stage endometrial cancer

(univariate analysis p=0.020). Next, expression of ER alpha, PR alpha and PR

beta were related to low tumour grade (p<0.000 1 , p<0.001 and p=0.001

respectively). In the presence of lymph vascular space involvement the tumours

were more likely to have no ER alpha expression (p=0.0 19, OR 2.2, 95% CI

139

1 . 14-4.26) . High grade tumours were more likely to be negative for ER alpha

and PR alpha expression (p<0.005, OR 6.0, 95% CI 2.94- 12.31 and p<0.005,

OR 8.4, 95% CI 3.08-22.84 respectively) (Table 5 and 6).

Table 2 . Histological and clinical characteristics of patients with endometrioid

endometrial cancer (n=315).

Patient age, years Median 64.7 65.4 Range 32-89 35-86

Tumour Stage I 1 86 59.0 1 86 77.5 II 54 1 7. 1 54 22.5 III 6 1 1 9.4 IV 1 3 4. 1 Missing 1 0.3

Histological grade Grade 1 1 62 5 1 .4 1 39 57.9 Grade 2 96 30.5 7 1 29.6 Grade 3 56 1 7.8 29 12 . 1 Undifferentiated 1 0.3 1 0.4

Myometrial invasion < 50% 1 85 58.7 1 60 66.7 > 50% 126 40.0 78 32.5 Missing 4 1 .3 2 0.8

L VSI Negative 222 70.5 1 88 78.3 Positive 73 23.2 38 1 5 .8 Missing 20 6.3 14 5.8

Recurrence No recurrence 250 79.4 204 85.0 Local 26 8.3 20 8.3 Regional 8 2.5 6 2.5 Distant 3 1 9.8 1 0 4.2

Pelvic lymph nodes Negative 97 30.8 78 32.5 Positive 29 9.2 No sampling 1 89 60.0 1 62 67.5

Paraaortic lymph nodes Negative 45 14.3 27 1 1 .3 Positive 1 2 3.8 No sam ling 258 8 1 .9 2 1 3 88.8

LVSI: Lymph Vascular Space Involvement

140

Table 3. Co-expression of the receptors m patients with endometrioid endometrial cancer.

ER alpha ER beta PR alpha PR beta positive positive positive positive (n = 1 85) (n = 231 ) (n = 1 1 6) (n = 1 21 )

ER alpha X 1 6 1 (69.7%) 1 00 (86.2%) 1 04 (86 .0%)

ER beta 1 6 1 (87.0%) X 1 02 (87.9%) 1 06 (87.6%)

PR alpha 1 00 (54. 1 %) 1 02 (44.2%) X 1 00 (82.6%)

PR beta 1 04 (56.2%) 1 06 (45.9%) 1 00 (86.2%) X

Table 4. Chi square analysis of receptor expression in relation to known prognostic factors in patients with endometrioid endometrial cancer.

Neg

ER alpha n= l l 2 Pos

n= l 85 p value Neg

ER beta n=66 Pos

n=23 1 p value Neg

PR alpha n= 1 84 Pos

n= l 1 6 p value Neg

PR beta n= l 77 Pos

n= l 2 1 p value

Neg = negative Pos = positive

Stage

Stage I

and I I

76

148

56

1 69

1 3 8

90

136

90

Grade LVSI

Stage III Grade l Grade 3 and LVSI LVSI

and IV and 2 undifferentiated Negative Positive

35 69 42 64 42

36 1 7 1 14 144 30 0.020 <0.000 1 <0.0001

1 0 5 8 8 49 12

61 1 84 47 1 59 60 NS NS NS

45 1 32 5 1 126 52

25 1 1 1 5 85 20 NS <0.001 NS

4 1 1 33 44 12 1 49

30 1 09 1 2 89 23 NS 0.001 NS

LVSI = Lymph Vascular Space Involvement

141

Table 5. The independent effect of stage of disease, tumour grade, myometrial

invasion and LVSI on the presence of ER alpha expression in endometrioid

endometrial cancer (logistic regression analysis) .

95.0% C . l .for EXP(B)

OR Lower Upper P value Stage of disease 0.889 0.454 1 .743 0.733 Tumour Qrade 6.01 5 2.939 1 2 .309 0.000 Myometrial invasion 0 .943 0 .523 1 .698 0 .844

LVSI 2 .206 1 . 1 42 4.262 0 .01 9

LVSI = Lymph Vascular Space Involvement

Table 6. The independent effect of stage of disease, tumour grade, myometrial

invasion and L VSI on the presence of PR alpha expression in endometrioid

endometrial cancer (logistic regression analysis) .

95.0% C. l .for EXP(B)

OR Lower Upper P value Tumour grade 8.384 3.078 22.839 0.000 Stage of disease 0.700 0.357 1 .372 0.299 Myometrial invasion 0.867 0 .493 1 .526 0.621

LVSI 1 .297 0.654 2 .573 0.457

LVSI = Lymph Vascular Space Involvement

1 42

Figure 2a. Kaplan-Meier survival analysis concerning ER alpha expression and

cumulative disease-specific survival (p=0.0053) . .

1 , 0

0 ,8

.;!!: 0 ,6

::s tJ)

a 0 ,4

0 ,2

0 ,0

0 ,0 2 ] 4D 6] 8 D

Follow-up in years

1 0 ,0

ER a lfa exp ression ERA expression

NO ERA expression

143

Prognostic factors in endometrioid endometrial cancer

Univariate analysis using Kaplan-Meier survival analysis indicates that ER

alpha positive tumours had a better disease-specific survival and presence of PR

alpha reduces risk of recurrence (Figure 2a and 2b) (p=0.0053 and p=0.0335

respectively). Using cox regression analysis, high tumour grade (p=0.0 14, OR

2.4, 95% CI 1 . 19-4. 72) and absence of PR alpha expression (p=0.015, OR 4.2,

95% CI 1 .32- 13. 33) were independent prognostic factors for relapse of

endometrioid endometrial cancer (Table 7). Stage of disease was the strongest

independent predictor for survival (p=0.0 14, OR 2. 7, 95% CI 1 . 23-5.88) (Table

8).

Figure 2b. Kaplan-Meier survival analysis concerning PR alpha expression and

cumulative recurrence (p=0.0335).

1 ,0

0,8

C Q) t 0,6 :J

! E :J 0,4

0,2

0,0

0,0 1 ,0 2 , 0 3,0 4,0 5,0

timetill recurrencein years

PRalfa expession

NO PRA expesson

PRA

expesson

144

Table 7. The risk of relapse of endometrioid endometrial cancer according to the

presence of ER and PR alpha and beta, stage of disease, tumour grade,

myometrial invasion and L VSI ( cox regression analysis).

95.0% Cl for OR Exp(B) P value

Lower Upper

ER alpha 0.855 0.376 1 .943 0.708

ER beta 1 .076 0.499 2 .322 0.852

PR alpha 4. 1 98 1 .322 1 3 .333 0.01 5

PR beta 0.542 0. 1 82 1 .6 12 0.271 Stage of disease 0.859 0.483 1 .528 0.605 Tumour grade 2 .372 1 . 1 93 4.720 0.0 14 Myometrial invasion 1 .438 0.81 0 2 .553 0.2 1 5

LVSI 0 .91 4 0.489 1 .707 0.778

LVSI = Lymph Vascular Space Involvement

145

Table 8. The risk of death due to endometrioid endometrial cancer according to

the presence of ER and PR alpha and beta, stage of disease, tumour grade,

myometrial invasion and L VSI ( cox regression analysis).

95.0% Cl for OR Exp(B) P value

Lower Upper

ER alpha 2.079 0.670 6 .453 0.205

ER beta 1 .482 0.401 5.475 0.555

PR alpha 0.967 0. 1 75 5.362 0.970

PR beta 0.789 0. 1 87 3 .326 0.746 Stage of disease 2.685 1 .226 5.880 0.014 Tumour g rade 0.961 0.403 2.291 0.982 Myometrial i nvasion 2.009 0.851 4.747 0 . 1 1 2

LVS I 1 .093 0.470 2.544 0.836

LVSI = Lymph Vascular Space Involvement

Relevance of ER alpha and ER beta, PR alpha and PR beta in early stage

endometrial cancer

In patients with stage I and II (n=240) ER alpha expression was observed in 148

(6 1.7%) tumours, while ER beta expression was found in 169 cases (70.4%).

Both, PR alpha and beta expression was demonstrated in 90 patients (3 7 .5% ). In

patients with early stage disease local relapse was diagnosed in 20 patients, and

regional and distant metastases were diagnosed in 6 and 10 patients, respectively

(Table 2). In this early stage group, 14 patients (5.8%) died from endometrial

146

cancer. In a multivariate analysis absence of ER alpha was independently related

to death of disease (p=0.017, OR 7.28, 95% CI 1.42-37.25).

Prognostic value of ER alpha I beta and PR alpha I beta ratios

The ratio of ER alpha / ER beta <I was univariately related to a shorter disease

free survival, using Kaplan-Meier analysis (p=0.027). Patients with a ratio of

PR alpha / PR beta < I more often had a shorter disease free survival (univariate

analysis p=0.044) or died from endometrial cancer (p=0.005).

Discussion

We found ER alpha expression to be decreased in poorly differentiated tumours

and high stage of disease, and to be correlated with survival. Interestingly, in a

subgroup analysis for early stage of disease, absence of ER alpha appeared an

independent prognostic factor for death of disease. Our results suggest that

adjuvant or more extensive therapy may be considered in ER alpha negative

patients with early stage of disease, as these patients can be classified as being at

risk for death of disease. Recently adjuvant chemotherapy was suggested to

improve outcome in patients with high-risk early stage endometrial cancer.4

Immunohistological analysis of ER has been used previously to predict overall

prognosis of patients with endometrial cancer, but data on ER alpha and beta are

scarce. 7•8•2 1 •22 ER alpha consists of a single polypeptide chain of 595 amino

acids, and ER beta of 485 amino acids. ER beta has a similar binding affinity for

estrogens as ER alpha, but selective modes of estrogenic action in different types

of tissue may exist. 23 In contrast to our results, it has been suggested that ER beta

is important in the progession of myometrial invasion. 8•2 1 •24

Estrogen dependent carcinogenesis may depend on the ratios of the two ER

subtypes, as disruption of the balance between the isoforms ER alpha and beta is

likely related to different modes of estrogenic action. In literature ER alpha / ER

147

beta ratios decreased from normal to malignant endometrial tissue. 7•8•22•24 We

observed that the ratio of ER alpha / ER beta <1 appeared to be a predictor of a

shorter disease free survival, which was not found in a small study, using the

same semi-quantitative ratio.8 So status of both ER alpha and beta may be a

prognostic factor in endometrial cancer, as disturbances of the synchronized

expression of ER alpha and beta may play a role in the alteration of estrogen

action. As we did not demonstrate any correlation between ER beta and clinico­

histopathological characteristics, determination of the expression of ER beta

seems only useful for prognostic purposes if analysed together with ER alpha

expression as a ratio.25

Several studies reported a correlation between PR expression and disease-free

survival, but data on the relative expression of PR alpha and beta, as reported in

this study, and their involvement in the pathogenesis of endometrial cancer are

scarce.9•11•2 1 We found the absence of PR alpha expression to be a powerful

independent prognostic factor for relapse of disease in endometrioid endometrial

cancer. This is contradicted by a recent study reporting decrease of PR beta to

have independent prognostic value, but the power of that study may be biased by

heterogeneous tumour inclusion as only 73% of analysed tumour cases

concerned endometrioid adenocarcinoma. 25 The availability of PR is an essential

factor determining progesterone response. Progesterone decreases the risk of

developing estrogen-dependent endometrial cancer by controlling cell

proliferation, as it is the natural antagonist of estrogenic hormones. PR is

expressed by a single gene encoding two proteins (PR alpha and PR beta). PR

beta consists of 933 amino acids, and PR alpha is a truncated form of PR beta

lacking 164 amino acids from the N-terminus of PR beta.9 PR alpha was found

to inhibit PR beta function and ER activation, while PR beta is involved in

regulation of endometrial responsiveness. 1 1 It was hypothesized that decreased

PR levels observed in endometrial cancer in fact arise from the loss of one PR

148

isoform, PR alpha or PR beta, as express10n of a single PR isoform was

associated with higher grade of tumour.9•1 1

As we found both PR alpha and beta to be related to low grade of tumour, the

loss of PR alpha and beta possibly contributes to tumour progression. We found

the ratio of PR alpha / PR beta <1 to be related to relapse and death of disease.

These results need to be confirmed by others to determine the strength of the PR

alpha / PR beta ratio as a prognostic factor. So the cellular ratio of PR alpha / PR

beta possibly determines progesterone action and may be involved in the

regulation of endometrial reaction.

In conclusion, together with histological grade, the absence of PR alpha is an

independent prognostic factor for recurrence of disease, while patients with ER

alpha positive tumours have a better survival, especially in the early stages. The

data of our large study population support the general idea of the prognostic

significance of estrogen and progesterone receptors, in addition to the classical

clinico-histopathological factors. These findings suggest that the

immunohistochemical evaluation of steroid receptors contributes to predicting

the biological behaviour of the tumour, next to the classical parameters. This

could lead to a more patient specific risk profile and therefore improve patient

tailored treatment.

Acknowledgements

HW Nijman is supported by the Dutch Cancer Society (Grant 2002-2768).

149

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adjunctive external pelvic radiation therapy in intermediate risk endometrial

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cancer. Curr Opin Obstet Gynecol. 2007; 19(1) :42-7.

5. Gehrig PA, Van Le L, Olatidoye B, Geradts. Estrogen receptor status, determined by immunohistochemistry, as a predictor of the recurrence of stage I endometrial carcinoma. J Cancer 1999;86:2083-9.

6. Jeon YT, Part IA, Kim YB, Kim JW, Park NH, Kang SB. Steroid receptor expressions in endometrial cancer: clinical significance and epidemiological implication. Cancer Lett 2006;239: 198-204.

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8 . Mylonas I, Jeschke U, Shabani N, Kuhn C, Kriegel S, Kupka MS, et al. Normal and

malignant human endometrium express immunohistochemically estrogen receptor alpha (ER-alpha), estrogen receptor beta (ER-beta) and progestrone receptor (PR).

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Significance of progesterone receptor-A and -B expressions in endometrial

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Ajwani S, et al. Consequences of loss of progesterone receptor expression in

development of invasive endometrial cancer. Clin Cancer Res 2003;9 :4 190-9.

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1 1. Arnett-Mansfield RL, deFazio A, Wain GV, Jaworski RC, Byth K, Mote PA, Clarke CL. Relative expression of progesterone receptors A and B in endometrioid cancers of

the endometrium. Cancer Res 2001;61 :4576-82. 12. Hirai M, Hirano M, Oosaki T, Hayashi Y, Yoshihara T, Matsuzaki 0. Pognostic factors

relating to survival in uterine endometrioid carcinoma. Int J Gynaecol Obstet

1999;66: 155-62. 13 . Steiner E, Eicher 0, Sagemuller J, Schmidt M, Pilch H, Tanner B, et al. Multivariate

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18 1 patients : 10 years experience at the Department of Obstetrics and Gynecology of

the Mainz University. Int J Gynecologic Cancer 2003 ; 13 : 197-203 .

14. Oreskovic S, Babic D, Kalafatic D, Barisic D, Beketic-Oreskovic L. A significance of

irnmunohistochemical determination of steroid receptors, cell proliferation factor Ki-

67 and protein p53 in endometrial carcinoma. Gynecol Oncol 2004;93 :34-40.

15. Briet JM, Hollema H, Reesink N, Aalders JG, Maurits MJE, ten Hoor KA, et al.

Lymphvascular space involvment: an independent prognostic factor in endometrial cancer. Gyn Oncol 2005;96:799-804.

16. Hoos A, Cordon-Cardo C. Tissue microarray proofiling of cancer specimens and cell­lines: opportunities and limitations. Lab Invest 2001 ;8 1 : 133 1-8.

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Tissue microarray immunohistochemical expression of estrogen, progesterone, and

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22. Hu K, Zhong G, He F. Expression of estrogen receptors ER alpha and ER beta in endometrial hyperplasia and adenocarcinoma. In J Gynecol Cancer 2005 ; 15 :537-4 1 .

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152

Summary and future perspectives

153

154

Summary and future perspectives

As outlined in Chapter 1 the aims of the studies described in the first part of this

thesis was to explore whether endogenously ( ovary) produced androgens after

conversion to estrogens by aromatase activity of the endometrium may contribute

to endometrial carcinogenesis. This hypothesis was examined (i) by evaluation of

the relation between androgens present in the utero-ovarian circulation and the

degree of ovarian stromal hyperplasia, and (ii) by demonstration of aromatase

expression in endometrial tissue.

In the second part of the thesis, we assessed the expression and prognostic value of

(i) estrogen and progesterone receptor alpha and beta, next to (ii) aromatase, COX-

2, HER-2/neu and p53 in a large cohort of endometrioid endometrial cancer

patients. Using tissue microarray analysis, the expression of the hormonal and

molecular markers was related to classical histopathological factors and disease

free and overall survival.

In Chapter 2 an introduction is given on the hormonal interactions contributing to

the development of endometrioid endometrial carcinoma. After the menopause

estrogens mainly originate from peripheral conversion of androgens by

aromatization in body fat. Hyperplasia of ovarian stroma may be associated with

an increased androgen production by the ovaries. In case of stromal hyperplasia

of the ovaries, increased production of aromatizable androgens by

postmenopausal ovaries may contribute to increased availability of androgen

precursors for intratumoural estrogen synthesis in the endometrial tissue as well.

Following the aromatase driven conversion of ovarian androgens, estrogens may

contribute in situ to the development of endometrial cancer. The local availability

of androgens and the local activity of aromatase in endometrial tissue are

considered relevant for this process.

155

Chapter 3 reviews available evidence that androgens, produced in hyperplastic

ovarian stroma or body fat tissues, play a role in the development of endometrial

cancer through conversion into estrogens, a reaction catalyzed in the

endometrium by the enzyme aromatase cytochrome P450. As the presence of

aromatase appeared to be a pathophysiological factor in the development of

hormone dependent breast cancer, the latter is evaluated in endometrioid

endometrial cancer as well. As treatment with aromatase inhibitors appeared

feasible in breast cancer, current knowledge of comparable treatment modalities

in hormone dependent endometrial cancer is also reviewed. Treatment of

endometrial cancer with aromatase inhibitors appears to be useful to some

extent, but available evidence in the literature is limited, especially in advanced

and recurrent endometrial cancer, where new treatment modalities are urgently

needed.

In chapter 4 two separate studies report on the relationship between the presence

of endometrioid endometrial cancer, the degree of ovarian stromal hyperplasia and

ovarian steroid production in postmenopausal women. In 1 1 2 patients with

endometrioid endometrial cancer, 4 7 patients with a benign gynaecological

condition and 10 patients with non-endometrioidendometrial cancer, the degree of

ovarian stromal hyperplasia was scored retrospectively on a semi-quantitative

scale (atrophy, slight, marked) . All patients were postmenopausal and had

undergone a hysterectomy with bilateral salpingo-oophorectomy. Prospectively

also blood sampling from the ovarian veins was performed in 60 patients. Steroid

levels ( estrone, estradiol, androstenedione, testosterone) were determined and

related to the degree of ovarian stromal hyperplasia and the presence (n=52) or

absence (n=8) of endometrioid endometrial cancer. In the retrospective study the

degree of ovarian stromal hyperplasia was higher in the presence of endometrioid

endometrial cancer (p=0.000). Our prospective study showed that an increasing

1 56

degree of ovarian stromal hyperplasia was related to higher ovarian levels of both

testosterone and androstenedione (p<0.05 and p<0.005 respectively), but not to

estrone or estradiol. Mean ovarian vein levels of both testosterone and

androstenedione were higher in patients with endometrial cancer than in patients

with benign conditions, without reaching statistical significance. In conclusion,

higher degrees of ovarian stromal hyperplasia were found in endometrioid

endometrial cancer and with increasing degrees of ovarian stromal hyperplasia

levels of ovarian vein androgens were higher. A causal relationship in the origin

of hormone-dependent endometrial pathology may exist between ovarian stromal

hyperplasia, ovarian vein androgen levels and endometrioid endometrial

carcinoma.

In chapter 5 prospectively the relationship between androgen levels in the utero­

ovarian circulation, aromatase activity in endometrial and body fat tissue, and the

presence or absence of endometrioid endometrial cancer was assessed in a

prospective study. In 43 patients with endometrioid endometrial cancer and 8

patients with a benign gynaecological condition after menopause a hysterectomy

with bilateral salpingo-oophorectomywas performed. Using tritium water release

assays, aromatase activity in endometrial and body fat tissue was determined and

related to the steroid levels from the peripheral and the utero-ovarian venous

circulation ( estradiol, androstenedione, testosterone) and to the presence or

absence of endometrial cancer. Significant aromatase activity was found in both

benign and malignant endometrial tissue samples. Aromatase activity in samples

of endometrial tissue and in samples of body fat did not correlate with steroid

levels in peripheral or utero-ovarian venous blood. Aromatase activity in samples

of benign or malignant endometrium did not differ. Remarkably, in four patients

with mainly poorly differentiated endometrial cancer very high aromatase activity

was found in endometrial tissue. It is likely that multiple pathogenetic pathways

exist which eventually lead to the formation of endometrioid endometrial cancer.

157

The local availability of androgens and the finding that aromatase activity is

present in both endometrial cancer and benign endometrial tissue, support the

hypothesis that aromatase activity in the endometrium may play a role in

malignant transformation, by converting androgens into mitogenic estrogens in the

endometrial tissue.

In chapter 6 the prognostic value of aromatase, COX-2, HER-2/neu and p53

expression was determined in endometrioid endometrial cancer. Tissue

microarrays were constructed comprising samples from 315 endometrioid

endometrial cancer patients. Expression of aromatase, COX-2, HER-2/neu and

p53 was determined by immunostaining and related to classical clinico­

histopathological parameters, in addition to recurrence of disease and survival.

Median follow-up time for all patients was 5.0 years. Patients were classified as

FIGO stage I (59.0%), stage II ( 17. 1%), stage III ( 19.4%) and stage IV (4.1%).

Sixty-five patients (20.6%) developed recurrent disease and 38 ( 12. 1 %) died

because of endometrial cancer. Aromatase, COX-2, HER-2/neu and p53

expression was observed in 133 (42.2%), 107 (34.0%), 17 (5.4%), and 21

(6.7%) tumour cases, respectively. Aromatase expression in tumour cells was

related to aromatase expression in stromal cells (p<0.0001) and to HER-2/neu

expression in tumour cells (p=0.0 19). Aromatase expression in both tumour as

well as stromal cells was related to low stage of disease (p=0.02 and p=0.00 1,

respectively), while aromatase expression in stromal cells was also related to

low tumour grade (p=0.021). P53 expression was related to high stage and high

grade (p=0.006 and p<0.000 1, respectively). In multivariate analysis p53

overexpression was independently related to death due to the disease (p=0.043,

OR 3.0, 95% CI 1.0-8.7). Although rarely found, presence of p53 expression

leads to a worse prognosis of endometrioid endometrial cancer. From literature it

is known that p53 is more frequently found in non-endometrioid tumours. As

patients with non-endometrioid endometrial cancer generally have a worse

158

prognosis than patients with endometrioid tumours, it seems that p53 positive

endometrioid tumours have a biologic behaviour more resembling non­

endometrioid tumours. For COX-2, HER-2/neu and aromatase no relation with

any other histopathological parameter or survival was found. In conclusion,

aromatase expression in tumour and stromal cells and p53 expression in tumour

cells are related to tumour grade and stage of disease, while p53 is an

independent adverse prognostic factor in endometrioid endometrial cancer.

The frequent presence of aromatase in endometrioid endometrial cancer ( 42% of

tumour cases ! ) supports the hypothesis that aromatase activity in the endometrial

tissue may play a role in the development of endometrioid endometrial cancer.

With regard to the aims of the study, it must be noted that aromatase expression

was found predominantly in lower stage and lower grade of disease. Possibly its

expression therefore disappears with increasing stage and dedifferentiation. In

breast cancer a relationship was found between the COX-2, HER-2/neu and

aromatase enzyme systems, as interaction between these biomarkers was found

to play a role in the production of estrogens. In breast cancer HER-2/neu and

aromatase overexpression were related, with HER-2/neu as the primary

molecular determinant of increased aromatase activity. COX-2 is the functional

intermediate with prostaglandin E2, which regulates aromatase gene expression,

as its product. We found a correlation between HER-2/neu and aromatase, which

may suggest a similar correlation between these enzyme systems in endometrial

cancer. Our studies suggest a role for aromatase in the early stages and grades of

disease. It can not be excluded that, apart from local effects on the endometrial

tissue, a positive effect of the use of aromatase inhibitors on peripheral fat tissue

exists. Theoretically this estrogen decreasing effect in body fat will not

contribute much to a better prognosis of the higher stages and grades, as

estrogen receptor expression decreases in the higher stages and grades of disease

( chapter 7). So possibly some women develop endometrioid endometrial cancer

1 59

through continuous hyperstimulation of the endometrial tissue by estrogens

produced by peripheral conversion in body fat, while in other women malignant

transformation is enhanced by local conversion of ovarian androgens into

estrogens, catalysed by (perhaps overexpression of) endometrial stromal

aromatase cytochrome P450, or both phenomena may happen simultaneously.

In chapter 7 the prognostic impact of estrogen receptor (ER) and progesterone

receptor (PR) alpha and beta was determined in endometrioid endometrial

cancer. Tissue microarrays were constructed from 315 endometrioid endometrial

cancer patients. ER alpha and beta and PR alpha and beta were semi­

quantitatively assessed by immunostaining, and together with their alpha / beta

ratios related to classical clinico-histopathological parameters in addition to

disease free and disease specific survival. Patients were classified as FIGO stage

I (59.0%), stage II (17.1%), stage III (19.4%) and stage IV (4.1%). Sixty-five

patients (20.6%) developed recurrent disease and 38 (12.1 %) died due to

endometrial cancer. In univariate analysis, expression of ER alpha was related to

early stage endometrial cancer (p=0.020), while expression of ER alpha, PR

alpha and PR beta was associated with lower grade tumours (p<0.0001, p<0.001

and p=0.001 respectively). A ratio of ER alpha / ER beta <1 was related to a

shorter disease free survival (p=0.027), while the ratio of PR alpha / PR beta <1

both was associated with a shorter disease free survival as well as a shorter

overall survival (p=0.044 and p=0.005, respectively). These findings illustrate

that the relative distribution of these biomarkers is of importance in disease

progress and prognosis. In early stage disease absence of ER alpha was

independently related to death of disease (p=0.017, OR 7.28, 95% CI 1.42-

37.25), while absence of PR alpha (p=0.015, OR 4.2, 95% CI 1.32-13.33)

appeared to be an independent prognostic factor for relapse of disease. Although

aromatase expression ( chapter 6) was not part of the study described in chapter

7, we also assessed relations between expression of aromatase and ER and PR

160

subtypes. Although expression of aromatase and ER alpha were both related to

early stage endometrial cancer, no relations between the aromatase and ER or

PR expression could be found. We conclude that in early stage endometrioid

endometrial cancer absence of PR alpha is an independent prognostic factor for

disease-free survival, while patients with ER alpha positive tumours have a

better overall survival. The data of our large study population support the

significance of estrogen and progesterone receptors for prognostic purposes, in

addition to the classical clinico-histopathological factors, and this may lead to a

more patient specific risk profile and treatment.

Future perspectives

Future research should be aimed at the molecular basis of endometrial cancer

carcinogenesis, and the exact role of aromatase. Our studies can not be

considered as true evidence for a causative role for aromatase in endometrial

cancer carcinogenesis, as we only describe statistical associations and relations.

Further evidence for the role of aromatase needs to be derived from in vitro

studies with human endometrial tissue cells. In normal, hyperplastic and

malignant endometrial cells more extensively the influences of addition or

deprivation of androgens and aromatase should be explored. In endometrial

cancer cell lines the levels of aromatase activity in relation to the effects of

steroids on DNA synthesis deserve further in depth evaluation.

Although in the treatment of breast cancer a revolution took place, when

aromatase inhibitors became available for depriving the tumour of estrogenic

growth stimulation ( see chapter 3), it is not expected that this will happen in a

same manner with endometrial cancer. Contrary to breast cancer, prognosis of

endometrioid endometrial cancer generally remains good with effective surgical

and radiotherapeutical treatment possibilities. Most likely only small numbers of

patients may benefit from aromatase inhibitors. A palliative role for aromatase

161

inhibitors especially in stage IV patients or metastatic disease should be

considered. Unfortunately until now studies did not show therapy with

aromatase inhibitors to be very effective in endometrial cancer ( chapter 3).

Future studies on the use of aromatase inhibitors in recurrent or advanced

endometrial cancer should relate objective disease response to potential

predictive markers, as described in chapter 6 and 7, including aromatase. Until

now only one clinical phase II trial on the efficacy of aromatase inhibitors

reported disappointing disease response in endometrial cancers with positive

expression of ER, PR and HER-2/neu. 1 So the potential role of aromatase

inhibitors in steroid receptor positive endometrial cancer is not yet resolved, as

theoretically estrogen receptor positive tumours can be expected to respond to

therapy. As aromatase is known to convert androgens into estrogens, it is

unlikely that the enzyme plays a significant role in hormone independent cancer

development, so research on the possible role of aromatase expression in non­

endometrioid endometrial cancer does not seem useful. Indeed disappointing

response rates to aromatase inhibition were reported in a phase II trial in

patients with advanced or recurrent endometrial cancer. Only two partial

responses were found in 23 women with advanced or recurrent endometrial

malignancy, of whom most cancers were poorly differentiated or presented an

agressive histology ( clear cell carcinoma or papillary serous carcinoma). Only

39% of these patients were diagnosed with endometrioid endometrial cancer,

and women with poorly differentiated tumours did not respond. 2 One may argue

that patient selection and short duration of treatment influenced results

negatively. As aromatase may play a role in the origin of endometrial cancer,

future research should perhaps focus on the possible use of aromatase inhibitors

as prophylactic medical treatment of (atypical) endometrial hyperplasia, with the

aim of preventing a prophylactic hysterectomy eg. in young patients with

endometrial cancer or atypical hyperplasia who want to preserve fertility.

Another challenge may be found in evaluation of the endometrium in

1 62

postmenopausal breast cancer who are using aromatase inhibitors. Several

studies reported a potential protective effect of aromatase inhibitors on the

endometrium in patients previously treated with tamoxifen, but data are not

conclusive ( chapter 3). 3-5 As aromatase inhibitors are prescribed more frequently

in postmenopausal breast cancer patients according to new treatment protocols,

more research has to be done on the implications for the endometrial tissue.

Meanwhile the endometrial thickness of postmenopausal breast cancer patients,

who switched from treatment with tamoxifen to aromatase inhibitors needs to be

monitored carefully, until possible (late) effects of replacement of tamoxifen by

aromatase inhibitors are better defined.

References

1 . Ma BBY, Oza A, Eisenhauer E, Stanimir G, Carey M, Chapman W, et al. The activity

of letrozole in patients with advanced or recurrent endometrial cancer and correlation

with biological markers - a study of the National cancer Institute of Canada Clinical

Trials Group. Int J Gynecol Cancer 2004; 14 : 650-8.

2. Rose PG, Brunetto VL, VanLe L, Bell J, Walker JL, Lee RB. A phase II trial of

anastrozole in advanced recurrent or persistent endometrial carcinoma: a gynecologic

oncology group study. Gynecol Oncol 2000; 78 : 212-6.

3. Cohen I. Aromatase inhibitors and the endometrium. Maturitas (2008),

doi : 10. 1016/j.maturitas.2008.03 .001 .

4. Duffy SRG, Greenwood M. The endometrial data from the ATAC (Arimidex,

Tamoxifen, Alone or in Combination) trial indicates a protective effect of anastrozole

(arimidex) upon the endometrium. Breast Cancer Res Treat 2003; 83 : abs 134.

5. Duffy S, Jackson TL, Lansdown M, Philips K, Wells M, Pollard S, Clack G, Coibon M, Bianco AR. The ATAC ('Arimidex', Tamoxifen, Alone or in Combination)

adjuvant breast cancer trial; first results of the endometrial sub-protocol following 2

years of treatment. Hum Reprod 2006; 21(2): 545-53.

163

164

Samenvatting en overwegingen voor de toekomst

165

Samenvatting en overwegingen voor de toekom.st

Zoals uiteengezet in hoofdstuk 1 was bet doel van de in bet eerste deel van bet

proefschrift beschreven studies om te onderzoeken of door bet ovarium

geproduceerde androgenen bijdragen aan de carcinogenese van bet endometrioid

endomeriumcarcinoom, nadat deze androgenen tot oestrogenen zijn omgezet via

bet enzym aromatase. Hiertoe werd onderzocht of (i) in de utero-ovariele circulatie

aanwezige androgenen gerelateerd zijn aan de mate van ovariele stroma

hyperplasie, en of (ii) expressie van aromatase aangetoond kan worden in

endometriumweefsel. Daamaast onderzochten we in bet tweede deel van bet

proefschrift de expressie en prognostische waarde van (i) oestrogeen en

progesteron receptor alpha en beta, naast (ii) aromatase, COX-2, HER-2/neu en

p53 in een groot cohort van patienten met endometrioid endometriumcarcinoom.

Gebruikmakend van tissue microarray analyse werd de expressie van de

hormonale en moleculaire markers gerelateerd aan klassieke histopathologische

factoren en aan ziektevrije en totale overleving.

In hoofdstuk 2 wordt een introductie gegeven over de hormonale factoren en

interacties, die van belang zijn bij bet ontstaan van bet endometrioid

endometriumcarcinoom. Langdurige blootstelling van het postmenopauzale

endometrium aan oestrogenen is een van de factoren die van belang is bij bet

ontstaan van endometrioid endometriumcarcinoom. Toename van lichaamsvet en

toegenomen oestrogeenproductie in het vetweefsel bevordert de kans op

endometriumcarcinoom. In bet lichaamsvet worden androgenen door o.a. het

enzym aromatase omgezet in oestrogenen. Deze androgenen zijn oorspronkelijk

afkomstig van productie in de bijnierschors en postmenopauzale ovaria. Er lijkt

een relatie te bestaan tussen de mate van ovariele stroma hyperplasie

(schorsverdikking in de ovaria), productie van androgenen door de ovaria, en het

ontstaan van endometriumcarcinoom. Toegenomen productie van androgenen

1 66

door de ovaria zou via de utero-ovariele circulatie kunnen leiden tot een

toegenomen beschikbaarheid van androgenen in het endometrium. Indien het zo

blijkt te zijn dat in het endometrium het enzym aromatase aanwezig is, zou dit

kunnen leiden tot extra lokale omzetting van androgenen in oestrogenen, met

dientengevolge een toegenomen kans op het ontwikkelen van

endometriumcarcinoom. Wij beschouwen de aanwezigheid van androgenen en

aromatase activiteit in het endometrium als onmisbare voorwaarden voor het

mogelijk maken van dit proces. Wij tonen in het vervolg aan dat androgenen

aanwezig zijn in het hormonale milieu van het endometrium, in samenhang met

ovariele stroma hyperplasie, en dat aromatase inderdaad lokaal aanwezig is in

het endometrium. Dan zijn de noodzakelijke ingredienten aanwezig zijn om een

lokale route, leidend tot endometrioid endometriumcarcinoom, mogelijk te

maken. Dit is van belang voor de gedachtenvorming met betrekking tot het

ontstaan van endometriumcarcinoom. Ook heeft dit wellicht praktische

consequenties, daar een vergelijkbaar pathofysiologisch concept bij

mammacarcinoom aanwezig bleek, hetgeen leidde tot succesvolle behandeling

van hormoonafhankelijke borstkanker met aromataseremmers.

In hoofdstuk 3 wordt de stelling besproken dat androgenen, geproduceerd in

ovaria en lichaamsvet, bijdragen aan het ontstaan van endometrioid

endometriumcarcinoom doordat deze androgenen lokaal warden omgezet in

oestrogenen. Het enzym aromatase cytochrome P450 in het endometrium is

hiervoor verantwoordelijk. Daar aangetoond is dat het enzym aromatase van

belang is bij het ontstaan van mammacarcinoom, wordt in dit hoofstuk dieper

ingegaan op overeenkomsten en verschillen tussen mamma- en

endometriumcarcinoom. Omdat behandeling met aromataseremmers haalbaar en

zinvol bleek bij mammacarcinoom, is de "state of the art" van de

endocrinologische behandeling van endometriumcarcinoom geevalueerd. Tot op

zekere hoogte werden aromataseremmers met matig succes gebruikt bij de

167

behandeling van endometriumcarcinoom, echter het wetenschappelijke bewijs

hiervoor is beperkt.

In hoofstuk 4 worden twee studies beschreven. In een archiefstudie werd een

microscopische vergelijking gemaakt van ovariumweefsel van 169 patienten met

endometrioid endometriumcarcinoom, en benigne aandoeningen. De mate van

ovariele stroma hyperplasie bleek toegenomen m geval van

endometriumcarcinoom (p=0.000). In een tweede (prospectieve) studie werd de

relatie bestudeerd tussen hormoonproductie door de postmenopauzale ovaria en

endometrioid endometriumcarcinoom. Bij zestig postmenopauzale vrouwen

werden uterus en ovaria verwijderd wegens endometriumcarcinoom of een

benigne aandoening. Tijdens de procedure werd bloed afgenomen uit de utero­

ovariele circulatie. Spiegels van androgenen en oestrogenen uit het afgenomen

bloed werden gerelateerd aan de mate van ovariele stroma hyperplasie, en aan

de aanwezigheid (52 maal) of afwezigheid (8 maal) van endometriumcarcinoom.

Het bleek dat bij toename van de mate van ovariele stroma hyperplasie ook de

spiegels van testosteron en androsteendion toenamen (p<0.05 and p<0.005),

maar niet de spiegels van oestrogenen. Ook bleken de spiegels van de

androgenen hoger bij de vrouwen met endometriumcarcinoom, dan bij de

vrouwen met een benigne aandoening, zonder statistische significantie te

bereiken. Wij concludeerden <lat bij patienten met endometrioid

endometriumcarcinoom de mate van ovariele stroma hyperplasie hoger was.

Ook vonden wij bij toename van de mate van ovariele stroma hyperplasie

hogere spiegels van androgenen in de utero-ovariele circulatie. Deze

bevindingen suggereren een verband tussen ovariele stroma hyperplasie,

ovariele androgeenproductie en endometrioid endometriumcarcinoom.

In hoofstuk 5 wordt een prospectieve studie gepresenteerd, waarin de relatie

wordt onderzocht tussen de androgeen- en oestrogeenspiegels in de utero-

168

ovariele circulatie, en aromatase expressie m lichaamsvet en

endometriumweefsel bij patienten met en zonder endometriumcarcinoom. Bij 5 1

postmenopauzale vrouwen werd een abdominale hysterectomie met bilaterale

adnexectomie verricht. Gebruikmakend van een tritium water release assay werd

de aromatase expressie bepaald in endometriumweefsel, en in lichaamsvet.

Aromatase expressie werd gerelateerd aan androgeen- en oestrogeenspiegels in

de utero-ovariele circulatie, en aan de aan- of afwezigheid van

endometriumcarcinoom. Significante aromatase expressie bleek aanwezig in

zowel benigne als maligne endometriumweefsel. De mate van aromatase

expressie in endometriumweefsel en/of lichaamsvet correleerde niet met

steroidspiegels in de utero-ovariele circulatie. Ook bleek de mate van aromatase

expressie niet significant te verschillen tussen benigne of maligne

endometriumweefsel. Toch ondersteunt de lokale beschikbaarheid van

androgenen in de utero-ovariele circulatie, en de waarneming dat aromatase

expressie in het endometrium aanwezig is, de volgende hypothese; aromatase

activiteit lokaal in het endometrium speelt een rol bij het ontstaan van

endometrioid endometriumcarcinoom, doordat aromatase ter plekke in staat is

androgenen om te zetten in oestrogenen.

In hoofdstuk 6 wordt de prognostische waarde onderzocht van de expressie van

de moleculaire markers COX-2, HER-2/neu, p53 en aromatase bij het

endometrioid endometriumcarcinoom. Met behulp van tissue microarray analyse

werd een cohort van 315 chirurgisch behandelde patienten met endometrioid

endometriumcarcinoom onderzocht. Tissue microarray analyse is een nieuwe

technologie, die het mogelijk maakt tumorweefsel van vele patienten tegelijk te

beoordelen onder de microscoop. lmmunohistochemische kleuringen werden

semi-kwantitatief gescoord, gebaseerd op de intensiteit van nucleaire kleuring.

Ten aanzien van de aromatase antilichamen, moet warden opgemerkt dat in de

literatuur de specificiteit van de antilichamen in het verleden vaak niet goed kon

169

warden gekarakteriseerd, met onbetrouwbare resultaten als gevolg. Daarom

gebruikten wij monoclonale 677 aromatase antilichamen, die speciaal

ontwm-pen en gevalideerd zijn voor routinematige bepaling van aromatase

expressie in archief-materiaal. Onze studie is de eerste die deze antilichamen

gebruikt bij endometriumweefsel. Expressie van de moleculaire markers werd

gerelateerd aan klassieke klinische en histopathologische karakteristieken, naast

ziektevrije en algehele overleving. De mediane follow-up voor alle patienten

bedroeg 5.0 jaar. De patienten werden geklassificeerd als FIGO stadium I

(59.0%), stadium II ( 1 7. 1%), stadium III ( 19.4%) en stadium IV (4. 1%).

Vijfenzestig patienten ontwikkelden een recidief (20.6%) en 38 ( 12. 1 %) stierven

ten gevolge van endometriumcarcinoom tijdens de follow-up periode. Expressie

van aromatase, COX-2, HER-2/neu en p53 werd waargenomen bij 1 33 (42.2%),

107 (34.0%), 1 7 (5.4%), en 2 1 (6.7%) van de tumoren. Aromatase expressie in

tumorcellen bleek gerelateerd aan aromatase expressie in stromacellen

(p<0.0001), en aan HER-2/neu expressie (p=0.0 19). Expressie van aromatase in

tumor en stromacellen was gerelateerd aan een lager stadium

endometriumcarcinoom (p=0.02 en p=0.00 1 ), terwijl aromatase expressie in

stromacellen ook gecorreleerd bleek aan een lagere tumorgraad (p=0.021 ). Wij

concludeerden dat p53, hoewel slechts aanwezig bij 7% van de patienten, een

krachtige onafhankelijke prognostische factor bleek. Expressie van p53 bleek

gerelateerd aan hogere graad en stadium (p=0.006 en p<0.0001 ), met een

toegenomen kans op overlijden ten gevolge van de aandoening (p=0.043, OR

3.0, 95% CI 1 .0-8.7). Uit de literatuur is bekend dat p53 vaker voorkomt bij het

(prognostisch ongunstiger) non-endometrioid endometriumcarcinoom. Daar

p53 positieve tumoren een slechte prognose bleken te hebben bij endometrioid

endometriumcarcinoom, lijkt hun biologische gedrag meer op dat van non­

endometrioid endometriumcarcinoom. COX-2, HER-2/neu en aromatase bleken

geen prognostische betekenis te hebben. Wij concludeerden dat aromatase

expressie in tumor- en stromacellen, en expressie van p53 in tumorcellen

170

gerelateerd blijken aan de graad en het stadium van het endometrioid

endometriumcarcinoom, terwijl express1e van p53 een onathankelijke

prognostische voorspeller is.

De frequente aanwezigheid van aromatase expressie (42%!) ondersteunt de

hypothese dat aromatase activiteit een rol speelt bij het ontstaan van

endometrioid endometriumcarcinoom. Opvallend genoeg werd aromatase

expressie voomamelijk gevonden bij lager stadium en graad van het

endometriumcarcinoom. W ellicht verdwijnt de aromatase expessie bij progressie

van de maligniteit en toenemende dedifferentiatie. Bij mammacarcinoom werd

in de literatuur een relatie gevonden tussen COX-2, HER-2/neu en aromatase

expressie, daar interactie tussen deze biomarkers een rol speelt bij

oestrogeenproductie. Wij vonden ook een correlatie tussen HER-2/neu en

aromatase, hetgeen een vergelijkbare relatie zou kunnen suggereren bij

endometrioid endometriumcarcinoom. Aan de andere kant, moet er niet alleen

gekeken worden naar de effecten van aromataseremmers lokaal in het

endometrium, maar ook naar de effecten m het lichaamsvet. Bij

hormoonathankelijk mammacarcinoom worden aromataseremmers namelijk

ingezet juist mede vanwege hun effect in het perifere vetweefsel. Op

theoretische gronden zal dit oestrogeenverlagende effect van aromatase in

vetweefsel niet veel bijdragen aan een betere prognose, daar de expressie van

oestrogeenreceptoren m de hogere stadia en graden van het

endometriumcarcinoom juist afneemt.

In hoofdstuk 7 wordt de prognostische waarde onderzocht van de aanwezigheid

van de oestrogeen receptor (ER) en progesteron receptor (PR) alpha en beta, bij

het endometrioid endometriumcarcinoom. Endometriumweefsel van 315

chirurgisch behandelde patienten met endometrioid endometriumcarcinoom

werd onderzocht. Het doel van de studie was om de expressie van de genoemde

1 7 1

receptoren te relateren aan klinische karakteristieken, tumor recidief en

overlevingsduur. Expressie van ER alpha was gerelateerd aan de vroege stadia

endometriumcarcinoom (p=0.020), terwijl expressie van ER alpha (p<0.0001),

PR alpha (p<0.001) en PR beta (p=0.001) gerelateerd bleken aan lagere graad

endometriumcarcinoom. Afwezigheid van ER alpha was bij het vroege stadium

endometriumcarcinoom onafhankelijk gerelateerd aan sterftekans (p=0.017, OR

7.28, 95% CI 1.42-37.25). Dit suggereert dat adjuvante therapie overwogen zou

kunnen worden bij vroeg stadium ER alpha negatief endometriumcarcinoom.

Afwezigheid van PR alpha bleek een onafhankelijke voorspeller van ziekte

recidief (p=0.015, OR 4.2, 95% CI 1.32-13.33). Ook bleek de verhouding ER

alpha/ ER beta<l en PR alpha/ PR beta<l gerelateerd aan risico op ziekte

recidief en sterfte. Deze waamemingen illustreren dat de verhouding tussen deze

biomarkers van prognostische betekenis kan zijn. Hoewel aromatase geen deel

uitmaakte van de studie, beschreven in hoofdstuk 7, bleken er geen relaties te

bestaan tussen ER en PR expressie en aromatase. Wij concludeerden dat

expressie van ER en PR alpha en beta en hun ratio's nuttige parameters zijn om

inzicht te krijgen in de kans op ziektevrije periode en overleving bij

endometrioid endometriumcarcinoom.

Toekomstig onderzoek.

Verder onderzoek dient gericht te z1Jn op het verder ontsluieren van de

moleculaire basis van het ontstaan van endometriumcarcinoom, en de exacte rol

van aromatase hierbij, daar onze observaties vanwege het associatieve karakter

niet beschouwd kunnen worden als een bewijs voor een rol voor aromatase bij

de carcinogenese. Wellicht dient het bewijs voor de rol van aromatase bij het

ontstaan van endometriumcarcinoom gevonden te worden in in vitro studies: in

cellijnen met normaal, hyperplastisch en maligne endometrium dient de invloed

op DNA synthese bestudeerd te warden van toevoeging en deprivatie van

androgenen en aromatase. Hoewel zich bij de behandeling van

172

hormoonafhankelijk mammacarcmoom een ware revolutie voordeed met de

introductie van aromataseremmers, die de omzetting van androgenen in

oestrogenen succesvol bleken te remmen (hoofdstuk 3), is het niet

waarschijnlijk dat dit ook zal gebeuren bij het endometriumcarcinoom. De

prognose van het endometrioid endometriumcarcinoom is over het algemeen

goed te noemen, in tegenstelling tot mammacarcinoom, mits tijdig chirurgische

en/of radiotherapeutische behandeling wordt ingesteld. Een palliatieve rol voor

aromataseremmers bij gemetastaseerd of hoger stadium endometriumcarcinoom

kan wel overwogen warden. Daarom is het interessant onderzoek te doen naar

de hormonale interacties bij recidief endometriumcarcinoom, daar de patienten

met een recidief juist degenen zijn die zouden kunnen profiteren van een

endocrinologische behandeling met aromataseremmers. Objectieve respons van

de ziekte op behandeling in toekomstige studies dient dan gerelateerd te warden

aan mogelijke voorspellende markers, zoals besproken in hoofdstuk 6 en 7,

inclusief aromatase. Een fase II studie vermeldt teleurstellende resultaten bij

behandeling met aromataseremmers van patienten met gevorderd

endometriumcarcinoom en positieve expressie van ER, PR en HER-2/neu, maar

de aantallen patienten zijn klein. 1 De mogelijke rol van aromataseremmers bij

de behandeling van steroid receptor positief endometriumcarcinoom is dus nog

niet duidelijk, daar men theoretisch effect zou mogen verwachten van

behandeling met aromataseremmers bij ER positieve tumoren. Omdat aromatase

een rol speelt bij de omzetting van androgenen in oestrogenen lijkt onderzoek

naar de rol van aromatase expressie bij non-endometrioid

endometriumcarcinoom minder zinvol. Inderdaad werden in een fase II studie

teleurstellende resultaten gevonden van behandeling met aromataseremmers bij

patienten met gevorderd of gemetastaseerd endometriumcarcinoom. 2 Slechts 2

van de 23 patienten toonden gedeeltelijke verbetering. De meeste van de

pa ti en ten hadden slecht gedifferentieerd of non-endometrioid

endometriumcarcinoom. Men kan opmerken dat selectie van de patienten en

1 73

korte duur van de behandeling de resultaten wellicht negatief hebben beinvloed.

Daar aromatase een rol zou kunnen spelen bij het ontstaan van

endometriumcarcinoom zou toekomstig onderzoek zich kunnen richten op de

vraag of aromataseremmers profylactisch gebruikt zouden kunnen worden bij de

behandeling van ( atypische) endometriumhyperplasie, met het doel een

hysterectomie te voorkomen, met name bij jonge patienten die hun

vruchtbaarheid willen behouden. Een andere uitdaging kan gevonden worden in

evaluatie van het endometrium bij postmenopauzale patienten met

mammacarcinoom die worden behandeld met aromataseremmers. Verschillende

studies vinden namelijk een mogelijk beschermend effect van

aromataseremmers op het endometrium bij patienten die eerder werden

behandeld met tamoxifen. 3-5 De dikte van het endometrium dient bij deze

patienten zorgvuldig vervolgd te worden, totdat de (late) effecten van

vervanging van tamoxifen door aromataseremmers goed in kaart zijn gebracht.

References

1. Ma BBY, Oza A, Eisenhauer E, Stanimir G, Carey M, Chapman W, et al. The activity of letrozole in patients with advanced or recurrent endometrial cancer and correlation

with biological markers - a study of the National cancer Institute of Canada Clinical

Trials Group. Int J Gynecol Cancer 2004; 14 : 650-8. 2. Rose PG, Brunetto VL, VanLe L, Bell J, Walker JL, Lee RB. A phase II trial of

anastrozole in advanced recurrent or persistent endometrial carcinoma: a gynecologic

oncology group study. Gynecol Oncol 2000; 78: 2 12-6.

3. Cohen I. Aromatase inhibitors and the endometrium. Maturitas (2008),

doi: 10. 1016/j .maturitas.2008.03 .001.

4. Duffy SRO, Greenwood M. The endometrial data from the ATAC (Arimidex,

Tamoxifen, Alone or in Combination) trial indicates a protective effect of anastrozole

(arimidex) upon the endometrium. Breast Cancer Res Treat 2003 ; 83 : abs 134. 5. Duffy S, Jackson TL, Lansdown M, Philips K, Wells M, Pollard S, Clack G, Coibon

M, Bianco AR. The ATAC ('Arimidex', Tamoxifen, Alone or in Combination)

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adjuvant breast cancer trial; first results of the endometrial sub-protocol following 2

years of treatment. Hum Reprod 2006; 21(2): 545-53 .

175

Authors and affiliations

From the Department of Gynaecologic Oncology, University Medical Center

Groningen, University of Groningen, The Netherlands

JM Briet

KA ten Hoor

RA de Jong

E Joppe

HW Nijman

AGJ van der Zee

From the Department of Obstetrics and Gynaecology, University Medical Center

Groningen, University of Groningen, the Netherlands

( Present affiliation: Academic Medical Centre of the University of Amsterdam,

Amsterdam, The Netherlands)

MJ Heineman.

From the Department of Pathology and Laboratory Medicine, University

Medical Center Groningen, University of Groningen, The Netherlands

H Hollema

From the Department of Epidemiology and Statistics, University Medical Center

Groningen, University of Groningen, The Netherlands

M Boezen

176

From the Department of Endocrinology, University Medical Center Utrecht, The

Netherlands

GH Donker

JHH Thij ssen

From the Department of Obstetrics and Gynaecology of the Medisch Centrum

Leeuwarden, Leeuwarden, The Netherlands

JG Santema

From the Department of Obstetrics and Gynaecology of the Wilhelmina

Ziekenhuis Assen, Assen, The Netherlands

AVSluijmer

From Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG,

Oncology Research, K136-P65, CH-4002 Basel, Switzerland

DB Evans

177

178

Dankwoord

Promoveren doe je niet alleen, wordt vaak gezegd. Oat is zeker waar en dat heh

ik heel duidelijk ondervonden. Dit proefschrift was nooit tot een goed einde

gekomen zonder de prettige samenwerking met en stimulans van de

gynaecologische oncologie-groep in het UMCG. Met name dit hechte team hen

ik dank verschuldigd.

Hooggeleerde van der Zee. Beste Ate, als kritische stimulator van onderzoek en

"bijschaver" van artikels hen je een ongelooflijke kei. Je gaf me op bet juiste

moment de duw in de rug om de weg der tma' s in te slaan, zodat de tweede helft

van dit proefschrift geschreven kon worden. Oat duwtje had ik trouwens ook

nodig! Je gaf duidelijke en ongezouten kritiek op de stukken, maar het werd er

natuurlijk altijd beter van. Fijn dat je bet stokje van Maas Jan wilde ovememen,

naast je reeds zo voile agenda. Ik vind bet een groot voorrecht dat je mijn boekje

hebt willen begeleiden.

Hooggeleerde Heineman. Beste Maas Jan, je hebt een zeer grote bijdrage

geleverd aan bet tot stand komen van dit proefschrift. De wetenschappelijke

gedachtenvorming over bet onderwerp aromatase en de eerste stimulans om in

dit onderwerp te duiken komen van jou vandaan. Steeds weer wist je me

vriendelijk te motiveren door te gaan: "bet boekje moet wel af' ! De gesprekken

op je kamer heh ik altijd zeer gewaardeerd. Je bent een stimulerend voorbeeld.

Hooggeleerde Hollema. Beste Harry, geweldig hoe je je zeer grote vakkennis

weet te combineren met gevoel voor humor! De plezierige en laagdrempelige

manier waarop je me corrigeerde en nieuwe zaken bijbracht op bet mij

onbekende terrein der pathologie was van grote waarde ! Door een microscoop

179

ziet de wereld er inderdaad anders uit. Daamaast hielp jouw relativerende

houding als ik weer eens de moed liet zakken. Ik ben buitengewoon blij <lat ook

jij mijn promotor wilde zijn.

Hooggeleerde Nijman. Beste Hans, als endometriumonderzoeker werd jij toch

pas in de tweede helft van <lit geheel bij de studies betrokken. En direct bleek jij

"de man". Als directe begeleider op de werkvloer konden de TMA studies vlot

voortgang vinden, en gaf je in alle voortgangsgesprekken duidelijk en

stimulerend richting aan. Heel veel dank daarvoor !

De leescommissie, bestaande uit de hooggeleerden Prof. Dr. J.G. Aalders,

Prof. Dr. J.H.H.Thijssen en Prof. Dr. P.H.B. Willemse ben ik zeer erkentelijk

voor hun beoordeling en vanzelfsprekend ook voor hun goedkeuring van mijn

manuscript.

Hooggeleerde Aalders, mijn opleider in het UMCG en als gynaecologisch

oncoloog zelf gepromoveerd op endometriumcarcinoom. Hoewel woonachtig en

werkzaam in het buitenland, bleek u toch bereid zitting te willen nemen in de

beoordelingscommissie van <lit manuscript.

Hooggeleerde Thijssen. Ik ben zeer vereerd dat u als wandelende vraagbaak op

endocrinologisch gebied uw medewerking heeft gegeven aan een deel van de

"aromatase" studie. U vormde een brug tussen Groningen en Utrecht. Ik ben erg

verheugd dat u als deskundige op het gebied van aromatase ook als lid van de

beoordelingscommissie bij het manuscript betrokken bent.

Hooggeleerde Willemse. Als oncoloog draagt u bij aan een evenwichtige

samenstelling van de beoordelingscommissie, die behalve een gynaecologisch

oncoloog en een endocrinoloog natuurlijk ook een intemistisch oncoloog diende

1 80

te bevatten. Alle drie deze vakgebieden ZlJn van belang gezien het

multidisciplinaire karakter van de inhoud van het manuscript. Ik dank u

vriendelijk voor uw bereidwilligheid zitting te willen nemen in de

beoordelingscommissie en uw bijdrage hieraan.

Mw. Ten Hoor. Beste Klaske, zonder jouw onmisbare hulp en inspanning bij het

beoordelen van alle coupes en laboratoriumwerkzaamheden was het allemaal

niks geworden. Inmiddels had je in al die jaren al wel tien keer zelf kunnen

promoveren. De dinsdagmiddagen "scoren" van weefsels met een kopje thee

waren altijd erg gezellig. Klaske, je bent geweldig.

Justine, Renske, en Enja. Ook jullie waren fijne partners bij het beoordelen van

de tissue microarrays. Door jullie voelde ik me weer een "jonge onderzoeker".

Met name noem ik heel speciaal Justines onmisbare hulp en inzet bij de

statistiek ondanks de drukte van de opleiding, hetgeen een van de belangrijkste

schakels in de studie bleek en zeer werd gewaardeerd! Justine, buitengewoon

bedankt! Ik ben blij dat we elkaar op deze manier konden helpen. Ik ben ervan

overtuigd dat jullie vervolgopleiding ongetwijfeld succesvol zal zijn.

Mw Donker. Beste Truus, fijn dat je vanuit Utrecht zoveel hebt bijgedragen aan

de aromatase-bepalingen. Dank voor al je tijdsinvestering en belangstelling!

Zeergeleerde Boezen. Beste Marike, dank voor je waardevolle hulp, waardoor

de betrouwbaarheid van het geheel verder werd vergroot. Al werkende aan het

manuscript is mijn respect voor statistiek nog verder toegenomen!

Dear Dean, thanks for your help in disposing the 677 antibodies. Thanks to your

help and Novartis we were able to continue and complete our studies in

18 1

aromatase, using as reliable antibodies as possible. Thanks for this pleasant

collaboration! I hope our results will be of benefit for you as well.

Zeergeleerde Brouwer. Beste Willem, opleider van het eerste uur. Ook al hen je

niet direct bij dit boekje betrokken geweest, toch hoor je absoluut in dit

dankwoord thuis. Ik heh veel aan je te dank.en. Jij wist nog wel een tropenarts in

de bush van Tanzania, die belangstelling had voor de gynaecologie. Je haalde

me naar Leeuwarden en stimuleerde me steeds weer op de jou eigen wijze in de

zoektocht naar een opleidingsplaats. Je creatief enthousiasme zal me levenslang

bijblijven. Ik beschouw jou als de meester, en mij als je gezel. Ik zal je nooit

vergeten, en hen trots te kunnen zeggen "ik hen nog opgeleid door Brouwer".

Zeergeleerde Santema. Job, beste vriend, jij hebt letterlijk aan de wieg van dit

proefschrift gestaan. Wij tweeen weten wat met deze zinsnede bedoeld wordt.

Samen met Willem beschouw ik jou als mijn eerste opleider. Bedankt.

Maatschapsleden Leeuwarden en oncologisch gynaecologen te Groningen, en

collega arts-assistenten. Veel dank voor het indertijd steeds maar weer

includeren van patienten voor "dat onderzoek van Vincent". Angelique en Peet,

die geregeld vanuit Leeuwarden weefsel in een tank met vloeibare stikstof

meebrachten naar Groningen, bedankt. Elke keer was ik weer bevreesd dat de

vloeibare stikstof in de auto zou gaan lekken met dramatische gevolgen.

Gelukkkig ging het steeds weer goed!

Ook de vakgroep Pathologie te Leeuwarden en met name Dr Joris Grond dank

ik voor de vriendelijke bereidwilligheid steeds weer endometriumweefsel uit te

willen snijden en gebruik te mogen maken van de archieffaciliteiten.

Geke de Ruijter en Janny Abels wil ik hartelijk bedanken voor hun hulp in het

UMCG bij het plannen van data, versturen van manuscripten en nog veel meer.

182

Ook mijn beide zeer gewaardeerde paranimfen wil ik buitengewoon bedanken

voor hun hulp en inzet! Fijn dat jullie erbij waren!

Ik mag niet vergeten ook de patienten te bedanken, die bereid waren te

participeren in de prospectieve studies, beschreven in hoofdstuk 4 en 5, en bereid

bleken peroperatief weefsel en bloed af te staan ten bate van de wetenschap.

Maatschapsleden Meppel, beste George, Guus en Aster. Met je maatschapsleden

ben je inderdaad ook een beetje getrouwd, zoals velen reeds in hun boekje

opmerkten. Dank voor de goede samenwerking en harmonie de afgelopen jaren.

Laten we vooral doorgaan onze "afdeling" verder uit te bouwen tot een goede

gynaecologische kliniek.

Natuurlijk wil ik ook mijn ouders bedanken, die mij in staat stelden te komen

waar ik kwam, dankzij hun stimulatie vanaf mijn prille jeugd. Lieve mamma en

pappa, ook jullie mogen bier genoemd worden!

Zeergeleerde van Vuuren. Lieve, lieve Anke, dank voor alles. Ik kon natuurlijk

niet achterblijven na jouw promotie. Samen hebben we erg ftjne, maar ook

verdrietige tijden doorgemaakt. We beleefden avonturen in Tanzania en

Nederland. Onze band is in al die jaren alleen maar hechter geworden. Het is fijn

met jou en de kinderen op onze boerderij in Drenthe. Een reeds gepromoveerde

echtgenote blijkt toch de beste prikkel om een proefschrift te voltooien, waarbij

geldt: "twee promovendi op een kussen, daar komt niemand tussen". Dr. van

Vuuren, vanaf nu slapen er twee zeergeleerden op een kussen.

Lieve Marije, Karlijn, Hielke en Jolien, ik ben erg blij met jullie. Op zulke

kinderen kun je trots zijn! Nu kunnen jullie weer wat vaker op de computer.

183

List of publications

Jongen VHWM, van den Heuvel MM. An abdominal pregnancy. TropicalDoct<?r 1995 ; 25 : 124.

Jongen VHWM. Alternative external fixation for open fractures of the lower leg. TropicalDoctor 1995; 25 : 173-4.

Jongen VHWM. Ectopic pregnancy and culdo-abdomino-centesis.

Int J Gynecol Obstet 1996; 55 : 7 5-6.

Jongen VHWM, Koetsier DW. Pitfalls in the diagnosis of complete hydatidiform mole with

coexistent live fetus. Trop Med & Int Health 1997; 2: 289-90.

Jongen VHWM. Autotransfusion and ectopic pregnancy; an experience from Tanzania.

TropicalDoctor 1997; 27: 78-9.

Jongen VHWM, van Holten J, Ti ems J. Neonatal Borrelia Duttoni infection: a report of3 cases. TropicalDoctor 1997; 27 : 1 15-7.

Jongen VHWM, van RoosmalenJ. Complications of cervical cerclage in rural areas.

Int J Gynecol Obstet 1997; 57: 23-6.

Jongen VHWM, van Roosmalen J, Wetsteyn JCFM, et al. Tick-borne relapsing fever and

pregnancyoutcome in rural Tanzania.Acta Obstet Gynecol Scand 1997; 76: 834-8.

Jongen VHWM, Santema JG, Grond AJK, van VeelenH. Uterine amyloidosis in menopause.

Br J Obstet Gynaecol 1998; 105 : 362-4.

Jongen VHWM, Halfwerk MOC, Brouwer WK. Vaginal delivery after previous caesarean

section for failure of second stage oflabour. Br J Obstet Gynaecol 1998; 105 : 1079-8 1 .

Jongen VHWM. A case of dystocia in conjoined twins. Tropical Doctor 1999; 29 : 54-5 .

184

Renes WB, Jongen VHWM, van RoosmalenJ. De prijs van een cerclage.

Ned TijdschrObstet Gynaecol 1999; 1 12: 121-4.

Jongen VHWM, Brouwer WK. Comparison of the modified Pereyra procedure using permanent

suture material and Burch urethropexy; subjective results of a one to six years follow-up study.

Eur J Obstet Gynecol Reprod Biol 1999; 84: 7- 1 1 .

Jongen VHWM, Santema JG, Wallenburg HCS. Het primaire antifosfolipidesyndroom in de

zwangerschap.Ned TijdschrObstet Gynaecol2000; 1 13 : 79-82.

Jongen VHWM, Holm JP, Verschuuren EAM, van der Bij W. Vaginal delivery after lung

transplantation. Acta Obstet Gynecol Scand 2000; 79 : 1 132-3 .

Jongen VHWM, Collins J, Lubbers JA, van Selm GM. Unsuspected pregnancy at

hysterosalpingographyFertil Steril 2001; 76: 610- 1.

Jongen VHWM, Sluijmer AV, Heineman MJ. The postmenopausal ovary as an androgen­

producing gland; hypothesis on the etiology of endometrial cancer. Maturitas 2002; 43 : 77-85.

Jongen VHWM, Hollema H, Santema JG, van der Zee AGJ, Heineman MJ. Ovarian stromal hyperplasia and ovarian vein steroid levels in relation to endometrioidendometrial cancer.

Br J Obstet Gynaecol 2003; 1 10: 690-5.

Jongen VHWM, Thijssen JHH, Hollema H, Donker GH, Santema JG, van der Zee AGJ, Heineman MJ. Is aromatase P450 involved in pathogenesis of endometrioid endometrial cancer?

Int l Gynecol Cancer 2005; 15 : 529-36.

Jongen VHWM, Hollema H, van der Zee AG, Heineman MJ. Aromatase in the context of breast and endometrial cancer. A review. Minerva Endocrinol 2006; 3 1 : 4 7-60.

Jongen VHWM, van RoosmalenJ. Een kraamvrouw met opisthotonus en strekkrampen. Ned Tijdschr voor Geneeskunde2007; 15 1(16):9 14.

185

Jongen VHWM, Briet JM, de Jong RA, ten Hoor KA, Boezen M, van der Zee AGJ, Nijman H, Hollema H. Expression of estrogen and progesterone receptors alpha and beta in a large cohort of patients with endometrioid endometrial cancer. Submitted.

Jongen VHWM, Briet JM, Joppe E, de Jong RA, ten Hoor KA, Boezen M, Evans DB, Hollema H, van der Zee AGJ, Nijman HW. Aromatase, COX-2, HER-2/neu and P53 as prognostic factors in endometrioid endometrial cancer. Submitted.

Abstracts

Deutsche Gesellschaft fur Gynekologie und Geburtshilfe/ Tropengynekologie; 3e Workshop Heidelberg Geburtshilfe unter einfachen Bedingungen, dee 1996: 3 .Rundbrief 13. 12. 1996:47. "Threatening threads of cervical cerclage: is a cervical cerclage appropriate in rural Africa?" Oral presentation.

22nd Annual Meeting te Amsterdam van de International Urogynecological Association, aug

1997. Int Urogynecol J 1997;8( 1): S3. "Comparison of the modified Pereyra procedure and Burch urethropexy". Oral presentation.

GynaecongresGroningen, mei 2003. NTOG 2003 ; 1 16:27 1-3 . "Postmenopauzaleovariumfunctieen de pathogenese van endometriumcarcinoom".

Oral presentation. Winnaaragio-prijsNVOG.

15 th International meeting van de European Society of Gynaecological Oncology (ESGO) te

Berlijn, oktober 20007. Abstract nr. 1804. "Estrogen and progesterone receptors alpha and beta are strong prognostic factors in endometrioid endometrial cancer". Oral presentation.

15 th International meeting van de European Society of Gynaecological Oncology (ESGO) te Berlijn, oktober 2007. Abstract nr. 1809. "The role ofaromatase, COX-2, HER-2/neu and P53

as prognostic factors in endometrioid endometrial canrer". Poster presentation.

186

Curriculum vitae

Vincent Jongen werd op 23 mei 1963 geboren als zoon van Maria Bender en Ir.

Hubertus Jongen. Na het behalen van het diploma Gymnasium beta aan het

Hertog Jan College te Valkenswaard en het vervullen van de militaire

dienstplicht bij het Garderegiment Jagers werd in 1982 gestart met de studie

Geneeskunde te Utrecht. In 1989 werd het artsexamen gehaald. In het kader van

de opleiding tot tropenarts werkte hij in het Ziekenhuis Overvecht te Utrecht en

het Ikazia Ziekenhuis te Rotterdam als arts-assistent chirurgie, inteme

geneeskunde/ cardiologie, gynaecologie/verloskunde en kindergeneeskunde. Van

1992 tot 1996 was hij als tropenarts via Memisa Medicus Mundi uitgezonden

naar Ndala Hospital, Tanzania. Na terugkeer in Nederland werkte hij 2 jaar in

het Medisch Centrum Leeuwarden als agnio gynaecologie, totdat aldaar in april

1998 met de opleiding tot gynaecoloog gestart kon warden (opleider Dr. W.A.

Brouwer). De opleiding gynaecologie werd daama voortgezet in het

Academisch Ziekenhuis Groningen (opleider Prof. Dr. J.G. Aalders) en de Isala

Klinieken te Zwolle (opleider Dr. J. van Eijck). Vanafjuli 2003 is hij werkzaam

binnen de maatschap gynaecologie van het Diaconessenhuis te Meppel (in

associatie met mw. A.B. Koster, G.J. Pricker en Dr. G.M. Vermeulen).

Hij is getrouwd met Dr. Anke van Vuuren. Samen kregen zij vijf kinderen,

namelijk Marije (1991), Karlijn (1993), Renske (t l997), Hielke (1998) en

Jolien (2000). In zijn vrije tijd houdt hij zich graag bezig met de paarden op de

boerderij.

187