European Journal of Endocrinology (2007) 156 143153

ISSN 0804-4643

INVITED REVIEW

What are the markers of aggressiveness in prolactinomas?

Changes in cell biology, extracellular matrix components,
angiogenesis and genetics
Alper Gurlek1,2, Niki Karavitaki1, Olaf Ansorge2 and John A H Wass1
1
2

Department of Endocrinology, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, UK and
Department of Neuropathology, Radcliffe Infirmary, Oxford, UK

(Correspondence should be addressed to J A H Wass; Email: john.wass@noc.anglox.nhs.uk)

Abstract
Prolactinoma is the most common pituitary tumour in adults. Macroprolactinomas, particularly in
men, may occasionally exhibit a very aggressive clinical course as evidenced by progressive growth,
invasion through bone into the sphenoid sinus, cavernous sinus, suprasellar region or the
nasopharynx. Some may even progress to pituitary carcinoma with craniospinal or systemic
metastases. Aggressive tumours have low cure rates despite appropriate medical and surgical
treatment. The mechanisms underlying this aggressive biological behaviour have not yet been fully
clarified. Recent immunohistochemical, molecular and genetic studies have provided some insight in
this respect. Invasive prolactinomas may be associated with a high Ki-67/MIB-1 labelling index
indicating increased cell proliferation, although this is not a universal finding. The AA polymorphism
in the cyclin adenine (A)/guanine (G) gene is more frequently detected in invasive prolactinomas.
Increased expression of the polysialylated neural cell adhesion molecule (NCAM) and reduced
expression of the E-cadherin/catenin complex implies a contribution of altered cell-to-cell adhesion and
cellular migration. Extracellular matrix components (ECM), matrix metalloproteinases (MMPs) and
their inhibitors play important roles in the context of angiogenesis and invasion. The induction of
fibroblast growth factor and vascular endothelial growth factor via oestrogen-induced overexpression
of novel genes (PTTG, hst and Edpm5) enhance cell growth, proliferation and angiogenesis contributing
to invasiveness in prolactinomas. Although mutations in proto-oncogenes like Ras are uncommon,
loss of tumour suppressor genes at loci 11q13, 13q1214, 10q and 1p seem to be associated with
invasiveness. Of the described mechanisms, only reduced E-cadherin/catenin expression and
overexpression of hst gene seem to be relatively specific markers for prolactinoma invasiveness
compared with other pituitary adenomas. Further research is needed to clarify the molecular
mechanisms behind the aggressive course of some prolactinomas to predict those with a potentially
poor clinical outcome, and to devise treatments that will eventually enable the cure of these
challenging tumours.
European Journal of Endocrinology 156 143153

Introduction
Prolactinoma is the most common pituitary tumour in
adults accounting for 60% of all functioning pituitary
adenomas (1). Prolactinomas show various initial
presentations, radiological appearances and biological
behaviour. In general, prolactinomas in women of
childbearing age are indolent microadenomas
(!10 mm in diameter) (25). These patients come to
medical attention due to the effects of hyperprolactinaemia, for example, galactorrhea, oligomenorrhea,
loss of libido and sexual dysfunction (6). In contrast,
men and post-menopausal women frequently harbour a
macroprolactinoma (O10 mm in diameter) with or
without extrasellar extension (7, 8). Apart from signs of
hyperprolactinaemia, patients with macroadenomas
q 2007 Society of the European Journal of Endocrinology

with extrasellar extension generally seek medical

attention due to mass effect. These patients may present
with headache, visual disturbances, cranial nerve
palsies, hypopituitarism (9) or rarely spontaneous
cerebrospinal fluid leakage (10), epilepsy due to
temporal lobe compression and obstructive hydrocephalus (11).
The mechanisms of aggressive biological behaviour of
some prolactinomas, despite their mostly benign
histopathological features, have not yet been fully
defined. There is inconsistency in the definition of
aggressiveness in prolactinomas. From the clinical
point of view, the most important factors to take into
account are resistance to dopamine agonists and high
recurrence rates after surgery. From the radiological
point of view, the tumours are considered as aggressive
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A Gurlek and others

if they show invasion into adjacent structures such as

the cavernous sinus. The lack of any increase in tumour
size in the majority of patients with microadenoma is
compatible with the frequent incidental finding of these
tumours in unselected autopsy cases (12, 13). The risk
of progression from a microadenoma to a macroadenoma is estimated to be about 47% (1). This
observation suggests that aggressive macroadenoma
may represent a different and distinct entity (14, 15).
There are no clear-cut criteria to predict biological
behaviour in prolactinomas (16, 17). As mentioned
previously, some prolactinomas are able to invade the
surrounding structures and tissues. These are called
invasive prolactinomas. Presently, local invasion per se
is not considered a sign of malignancy. Only rare
pituitary tumours with distant metastases are termed
pituitary carcinomas, indicating truly malignant
behaviour. Their pattern of spread may be craniospinal,
systemic or both. Traditional histopathological features
of malignancy (nuclear polymorphism, high cellularity,
cytological atypia, the presence of necrosis and
nucleolar prominence) are not generally helpful to
discriminate the pituitary carcinomas from benign
adenomas (18). When mitotic figures are present,
they do provide some insight into the potentially
aggressive nature of the tumour, but their discriminating power is somewhat limited (17).
The World Health Organization (WHO) classification
of endocrine tumours has defined an atypical pituitary
adenoma as one having an elevated mitotic activity, a
Ki-67 labelling index O3%, and p53 (a nuclear
phosphoprotein whose immunohistochemical accumulation has served as an unfavourable prognostic factor
for a wide range of human neoplasms) immunoreactivity O3% (19, 20). These features are generally lacking
in non-invasive adenomas. Nearly, all pituitary carcinomas present the above-mentioned histological
features of atypical adenomas (21).
There is also a problem in assessing the invasiveness of
a particular tumour, since there are various methods
used to define it. For example, the spectrum of
invasiveness may encompass radiological invasion,
intraoperative observations by the surgeon or microscopical invasion. It has also been proposed that a
tumour, which extends into the adjacent structures
according to imaging, may not represent an invasive one
on microscopy (16). With the advancement of techniques in genetics, molecular biology and immunohistochemistry in the past two decades, more specific
markers have been identified which might more precisely
indicate an aggressive potential in these tumours.
In this review, after briefly reviewing the biochemical,
neuroradiological and clinical aspects of aggressiveness
(i.e. tumoural expansion and invasion through the
surrounding and even remote structures causing
difficulties in management and low cure rates), we will
address suggested markers which may help identify these
tumours in the context of cell proliferation, cell cycling,
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EUROPEAN JOURNAL OF ENDOCRINOLOGY (2007) 156

cell-to-cell adhesion, angiogenesis, alterations of extracellular matrix (ECM) components and growth factor
signalling. We will also summarise the genetic alterations leading to changes in proliferative capacity and
angiogenesis in prolactinomas. The contribution of some
of these markers to gender differences in prolactinoma
behaviour and the development of resistance to dopaminergic agonists will also be discussed. We should
acknowledge, however, that the studies that will be
reviewed in this paper are based on surgically removed
prolactinomas. As surgical therapy for these tumours is
performed in particular subsets of patients only, an
inevitable selection bias may be envisioned in all these
studies.

Biochemical and radiological assessment

of aggressive prolactinomas
Hormone-secreting pituitary adenomas are readily diagnosed by endocrine testing which can be routinely
performed. In giant prolactinomas, the diagnosis is
generally straightforward, since they present with
extreme elevation of prolactin (even O100 000 ng/ml).
Prolactinomas are unique in the sense that dopamine
agonists usually cause rapid tumour shrinkage with relief
in compressive symptoms (2224). So, it is important to
diagnose a prolactinoma patient before considering
surgery as a first step treatment modality. Clinicians
should also be aware of the high dose hook effect while
interpreting a low prolactin level in a patient with giant
invasive pituitary adenoma (25, 26).
Calle-Rodrigue et al. (27) have shown that preoperative serum prolactin concentrations were significantly
higher in invasive prolactinomas compared with the
non-invasive ones (median prolactin levels 705 vs
141 ng/ml respectively). When prolactin cut-off was
chosen as 3300 ng/ml, the specificity for predicting an
invasive prolactinoma was 91% (sensitivity was not
calculated). Similarly, Ma et al. (28) have reported that
prolactinomas invading the cavernous sinus had
significantly higher prolactin concentrations and
tumour sizes, and furthermore, that preoperative
prolactin levels were significantly associated with
invasiveness. No comparisons have been done in these
studies (27, 28), however, regarding plurihormonal
adenomas co-secreting prolactin versus pure prolactinsecreting adenomas in terms of aggressive behaviour as
evidenced by invasiveness. These data suggest that
prolactin levels correlate with tumour size and that
high preoperative prolactin levels indicate the presence
of an invasive prolactinoma.
Although magnetic resonance imaging (MRI) is the
best technique for assessing the extent of the tumour,
computerised tomography (CT) provides better information about interruption of the sellar floor and
intratumoural calcification. From the radiological point

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of view, aggressiveness in these tumours is probably best

reflected by cavernous sinus invasion.
Recently, Cottier et al. (29) defined MRI-based criteria
for the diagnosis of cavernous sinus invasion using the
surgical findings of 106 patients as standard-of-reference criteria for invasion. Invasion of the cavernous
sinus was certain (positive predictive value (PPV)
100%) if the percentage of the encasement of the
internal carotid artery (ICA) by tumour was 67% or
greater. Invasion was considered to be highly probable if
the carotid sulcus venous compartment was not
depicted (PPV 95%) or the line joining the lateral wall
of the intracavernous and supracavernous ICAs was
passed by the tumour (PPV 85%). If the percentage of
the encasement of the intracavernous ICA is lower than
25, or the line joining the medial wall of the
intracavernous and supracavernous ICAs was not
passed by the tumour, cavernous sinus invasion by the
tumour is excluded.

Resistance to dopaminergic agonists: not

a sine qua non of aggressiveness
There is no consistency in the literature about what
constitutes resistance to dopamine agonists. Failure to
normalise prolactin levels independently of the baseline
value, or failure to reduce prolactin levels below 50% of
baseline have been considered as resistance. Similar
arguments are valid for tumour size reduction as well.
There may be discordance in the response of a given
patient with respect to prolactin reduction and tumour
size reduction (for review, see 30). While dopamine
agonists or surgery cure most non-invasive prolactinomas, invasive ones may not be cured by surgery alone
requiring additional treatments like radiotherapy (31).
Furthermore, they may have a propensity to be resistant
to dopamine agonists. However, dopaminergic agonists
should still be considered as the first-line treatment in
most patients with giant prolactinomas. Wu et al. (26)
have recently demonstrated that bromocriptine treatment of invasive giant prolactinomas after a mean
follow-up of 37 months results in improvement of
clinical symptoms in almost all patients, as well as
tumour volume reduction by a mean of 93.3%. Failure
to normalise prolactin levels is found in about 25% of
patients treated with bromocriptine and in 1015% of
those treated with pergolide or cabergoline in prolactinomas regardless of size or invasiveness (30). Resistance
of the tumour to shrinkage with bromocriptine is seen
in about 50% of the tumours, while this figure is about
1015% with cabergoline and pergolide (30). Another
important drug, which has been used in bromocriptineresistant tumours and in patients with intolerance to
bromocriptine, is the non-ergot dopamine agonist
quinagolide. It is very difficult to verify the incidence
of primary quinagolide resistance in prolactinomas. In a
recent crossover longitudinal study, Di Sarno et al. (32)

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145

have shown that quinagolide treatment normalised

prolactin levels in all microprolactinomas, and 87.5%
of macroprolactinomas. These figures were almost the
same for treatment with cabergoline (32).
Drug resistance is more common in macroadenomas
compared with microadenomas. For example, in a
retrospective analysis, a group of patients who were
initially being treated with bromocriptine was switched
to cabergoline (33). Bromocriptine resistance in terms of
prolactin normalisation was more frequent in macroprolactinomas (54%) than microprolactinomas (43%).
Resistance to cabergoline was 18 and 10% in macroand microadenomas respectively. The underlying
mechanism leading to the resistance in most tumours
has been attributed to decreased cellular D2 receptors
with lower affinities to their ligands (34).
Macroprolactinomas in men tend to be more
frequently resistant to bromocriptine therapy than
those in women (8). Delgrange et al. (8) have shown
significant differences in frequencies of bromocriptine
resistance between males (30%) and females (5%). The
correlation of drug resistance with other markers of
aggressiveness, in particular the ones related with
proliferative capacity, will be discussed in the following
part to provide an explanation to this gender difference
in biological behaviour of these tumours.

Specific markers implicated in

aggressiveness in prolactinomas
Implications of proliferative capacity,
cell cycling and cell-to-cell adhesion in
prolactinoma behaviour
It is well known that tumour expansion and invasion
primarily relate to several mechanisms, including
increases in cell proliferation, changes in cell cycling
and cell-to-cell adhesion. Several markers have been
investigated in prolactinomas in an attempt to relate
these mechanisms with aggressive tumour biology.
Some of the studies that may be relevant are given in
Table 1, and are further discussed in the following
paragraphs taking into account the inconsistent and
controversial issues in the literature.
Markers of proliferation Nuclear proteins, such as Ki67 and proliferating cell nuclear antigen (PCNA) are
often used to assess proliferative activity of tumour cells
(16). Ki-67 is recognised by the MIB-1 monoclonal
antibody and it is expressed throughout the cell cycle in
G1, S, G2 and M phases, but not the quiescent G0 phase
(28, 35). The percentage of cells immunostained for this
protein (Ki-67/MIB-1 labelling index) represents a
reliable marker of proliferative activity (36). PCNA is a
protein that accumulates in the nucleus during the cell
cycle. It has a higher specificity for the S phase than
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Table 1 Detected markers of aggressiveness related to cell proliferation, cell cycling and cell-to-cell adhesion.
Category

Marker

Status of expression and clinical relevance

Ref.

Proliferation

Ki-67/MIB-1 LI

Cell cycling

PCNA
Cyclin D1

(8)
(28)
(21)
(36)
(37)
(41)

Cell-to-cell adhesion

Cyclin E
PNCAM

Associated with resistance to DA

No association with tumour size
Increases may indicate malignant progression
If O3%, highly specific for invasiveness
Increased in parallel with tumour recurrence
Increased in aggressive pituitary tumours as a whole,
no increase in macroadenomas
Increased in macroprolactinomas
Highest in invasive rat prolactinomas and metastatic lesions
No correlation with invasiveness in human prolactinomas
Decreased in invasive prolactinomas

E-cadherin/catenin/p120 complex

(42)
(5356)
(57)
(63)

LI, labelling index; DA, dopamine agonist; PCNA, proliferating cell nuclear antigen; PNCAM, polysialylated neural cell adhesion molecule.

Ki-67, and labels only those cells that passed the

important G1/S boundary (37). A comparison of Ki-67
and PCNA proliferation in pituitary tumours has shown
that PCNA overestimates the cell proliferation in these
tumours when tumours with a labelling index !0.1%
were considered to be negative for proliferation (38).
Concerning prolactinomas, DelGrange et al. (8) found no
significant difference in PCNA index between invasive
and non-invasive prolactinomas. Similar to the PCNA
index, numerous studies have been performed on the Ki67/MIB-1 labelling index in pituitary adenomas with
inconsistent results in terms of correlation with clinical
presentation. With respect to non-functioning adenomas, no significant relationship has been found between
the clinical course and the labelling index (39). The
predictive ability of the Ki-67/MIB-1 labelling index for
aggressive behaviour is greater in functioning pituitary
adenomas, including prolactinomas. In a series reported
by Thapar et al. (36), hormone-secreting adenomas had
a significantly higher mean labelling index (3.25%) than
non-functioning adenomas (2.06%). The highest labelling indices were in prolactinomas. A cut-off of 3% of
labelling as an indicator of invasiveness had a specificity
of 97.3% and a sensitivity of 73% (36).
Two studies have shown higher levels of the labelling
indices in males with macroprolactinomas when
compared with similar tumours in female patients (8,
28). Regarding the association of Ki-67/MIB-1 labelling
index with tumour behaviour, these studies (8, 28) did
not find consistent differences between invasive and
non-invasive macroprolactinomas except in a small
group of males (nZ4) with dopamine agonist-resistant
invasive prolactinomas. The mean labelling index was
4.7% compared with !1% in all tumours that are
responsive (8). PCNA labelling yielded similar results
(8). On the other hand, one recent study by Ma et al.
(28), conducted in 123 Japanese prolactinoma patients,
found no association between Ki-67 labelling index and
clinical parameters. In this study, half of the cases were
pure prolactin-producing tumours while the other half
was plurihormonal prolactinomas. No significant
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association could be detected between proliferative

capacities and tumour size. The impact of patient age
and gender in both plurihormonal tumours and pure
prolactinomas was not assessed (28). Recently,
Scheithauer et al. (21) found no statistically significant
difference between invasive and non-invasive adenomas
(including prolactinomas) with respect to Ki-67 and
PCNA labelling indices. The level of p53 expression was
also similar between the groups. Ki-67 labelling and
p53 were not statistically significant in determining
adenomas prone to recur. It should be emphasised that
the difference between the Ki-67 results of Scheithauer
et al. (21) and Thapar et al. (36) may relate to different
techniques used for the interpretation of immunohistochemical stainings (manual cell counting versus digital
image analysis respectively). The clinical value of the
recently introduced atypical adenoma category in
predicting recurrence or malignant progression, therefore, remains to be determined.
Proteins implicated in cell cycling Cyclins and cyclindependent kinases (CDK) are essential for cell cycle
regulation in eukaryotes. Active cyclinCDK complexes
drive cells through cell cycle phases by phosphorylating
the protein substrates that are essential to achieve
transition to the next phase. The levels of cyclins are
regulated at the level of transcription and also by targeted
degradation via the ubiquitin pathway (40). Jordan et al.
(41) have found that non-functioning adenomas and
functioning invasive tumours, including prolactinomas
significantly express cyclin D1 to a greater extent than
non-invasive tumours and the normal pituitary gland.
Pituitary carcinomas did not appear to be different from
the benign tumours in terms of cyclin expression (41).
We have previously observed that it was only cyclin E
expression that was higher in macroprolactinomas than
microprolactinomas (42). Since cyclin E is expressed
maximally at G1/S transition of the cell cycle and thought
to be important in the initiation of DNA replication, it
may have an implication in growth and expansion of
prolactinomas (43). This warrants further analysis in

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larger prolactinoma groups categorised according to their

invasiveness or recurrence rate.
The cyclin D1 gene contains a frequent A/G
polymorphism within the splice donor region of exon
4/intron 4 (44). The cyclin D1 genotype is associated
with clinical outcome, particularly the survival after
diagnosis, in a significant number of neoplasms (44,
45). In a cohort of 294 (20% were prolactinomas)
pituitary adenomas, Simpson et al. (46) examined the
distribution of cyclin D1 A/G polymorphisms across the
Hardy grade of the tumours. Grade 3 and 4 tumours
were considered as invasive. When the data were
analysed separately in the prolactinoma subgroup, the
authors observed a progressive increase in AA genotype
from grade 1 (11%) through grade 4 (66%), while the
GG genotype progressively decreased from grade 1
(43%) through grade 4 (0%) (46). Two patients out of
eleven in the invasive group showed recurrence after
the operation, while none of the grade 1 patients
recurred. Therefore, AA genotype in cyclin D1 gene
may be a marker for invasiveness of prolactinomas, and
may also contribute to tumour recurrence. Overexpression of high-mobility group A non-histone chromosomal proteins (HMGA) play a significant role in the
determination of chromatin structure. These proteins
have been found overexpressed in several cancer types
(47). It has previously been demonstrated that transgenic mice overexpressing HMGA2 develop growth
hormone (GH) and prolactin-secreting tumours (48).
With respect to human prolactinomas, aggressive
tumours with unresponsiveness to dopamine agonists
are associated with higher expression level of this
protein (49).
Role of adhesion molecules The neural cell adhesion
molecules (NCAMs), member of the immunoglobulin
superfamily, contribute to cell-to-cell or cell substrate
adhesion through homophilic and heterophilic
mechanisms (50, 51). They can be post-translationally
modified by polysialylation giving rise to PNCAMs that
are implicated in cell growth and migration (52).
PNCAM expression has been noted in aggressive
tumours, such as small-cell lung carcinomas, and also
some neuroendocrine tumours (5355). The expression
of PNCAM has been investigated both in animal models
and in human pituitary adenomas. Daniel et al. (56)
have investigated the PNCAM expression in four
lineages of rat pituitary transplantable tumours
(SMtTW) primarily secreting prolactin. They observed
the highest PNCAM expression in the lineage SMtTW4
that was characterised by invasion and metastasis. The
expression level was correlated with growth rate in
different tumour lineages. Embarking from this observation, Trouillas et al. (57) have recently investigated
the expression of PNCAMs in a variety of human
pituitary tumours in relation to extrasellar invasion

Markers of aggressive prolactinomas

147

using immunohistochemistry, western blot analyses

and ELISA. They have observed a positive correlation
between tumour invasiveness and PNCAM expression,
particularly in the GH-secreting adenomas. This was
not the case in the prolactinoma subgroup (nZ14),
although some of them expressed PNCAM (57). The
discrepancy between animal and human prolactinomas
may be related to differences in tumour invasion
mechanisms between species, and the small number of
prolactinomas studied in humans.
Tumour cell invasion is a multistep process that
requires complex changes in adhesive interactions
among cells. The detachment of tumour cells from the
primary lesion is thought to be an important initiative
step leading to invasion. E-cadherin is an important
molecule implicated in tumoural invasion processes
(58). The functions of E-cadherin are mediated through
its cytoplasmic linkage to the actin cytoskeleton via
certain cytoplasmic plaque proteins known as the
catenins (a, b and g) (59, 60). P120, by binding to
E-cadherin as a regulatory factor, enables it to function
to promote intercellular adhesion (61). It is believed
that the cadherin/catenin/p120 complex acts as an
invasion suppressor system. There are several observations in various cancers indicating its abnormal or
reduced expression (62). Qian et al. (63) have shown
that expression of E-cadherin and b-catenin seems to be
significantly lower in invasive prolactinomas compared
with non-invasive ones, and the complex is significantly
less expressed in macroadenomas than microadenomas.
The tumours derived from men showed a significantly
lower level of expression than tumours from women.
The decrease in cadherin/catenin expression predicted
increased MIB-1 labelling indicative of high proliferative
activity. The results of this study indicate that low
cadherin/catenin expression may be a useful marker in
predicting tumour aggressiveness in prolactinomas
(63). In contrast, Kawamoto et al. (64) have found no
significant difference in E-cadherin expression between
invasive and non-invasive, non-functioning and
GH-secreting pituitary adenomas. It seems therefore
that loss of E-cadherin expression may be a marker
of invasiveness for prolactinomas but not GH-producing or
non-functioning adenomas. The molecular mechanisms
responsible for loss of cadherin/catenin expression in
invasive prolactinomas are not presently known.

Role of extracellular matrix components

(ECM) and degradation of ECM
Components of the extracellular matrix are implicated in
pituitary tumourigenesis (65). Laminin, for example,
inhibits the production of prolactin and cell proliferation.
Differential expression of laminin has been observed in
D2 receptor knockout mice that spontaneously develop
aggressive prolactinomas (66). From the initial formation of hyperplasia, a dramatic reduction occurs in
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laminin expression with the advancement of tumour

progression. The expression of laminin is confined to
basal membranes surrounding blood vessels despite the
fact that it is abundantly expressed in the normal
adenohypophysis parenchyma (66). The significance of
laminin remains to be investigated in human prolactinoma behaviour.
The ECM can be degraded and reorganised by specific
enzymes from the family of matrix metalloproteinases
(MMPs). The activities of various MMPs are balanced by
tissue inhibitors of metalloproteinases (TIMPs) (67).
MMP-2 and MMP-9 are both type IV collagenases that
are able to promote tumour invasion by breaking down
the basement membrane (68). The secretion of MMP is
also an important early step contributing to endothelial
cell migration through degraded ECM enabling angiogenesis to occur (69). Serine proteases and their
modulators (plasminogen activators and inhibitors) are
also involved in ECM remodelling by initiating a cascade
of events, involving MMP-9 proteolytic activity (70).
In the context of the biological behaviour of pituitary
adenomas, studies investigating the contribution of
MMP-9 have yielded conflicting results. Our group has
previously demonstrated that MMP-9 expression by
macrophages in invasive macroprolactinomas was
significantly higher than in non-invasive macroprolactinomas, while expression levels of MMP-9 in pituitary
carcinomas was similar to that of invasive adenomas
(71). We have also found a positive correlation between
MMP-9 expression and angiogenesis. However, more
recently, Knappe et al. (70) found contradictory results
with respect to MMP-9 expression and invasiveness in
pituitary adenomas. Contrary to our observations, they
could not demonstrate a relationship between aggressive behaviour and MMP-9 expression (70). However,
they identified that TIMP-2 was overexpressed in noninvasive as compared with invasive adenomas. These
observations suggest that inhibitors of MMPs may play
an important role in the suppression of aggressive
behaviour in pituitary tumours. The discrepancies in
MMP-9 data among the cited studies above may have
resulted from different techniques used, diversity of
tumour subtypes, selected criteria for aggressiveness,
and different number of cases studied.
Local growth factor expression In the pituitary,
various growth factors are expressed and released into
the extracellular matrix (65). Fibroblast growth factors
(FGF) and vascular endothelial growth factor (VEGF) are
implicated in angiogenesis. Another local factor that
may be operative in aggressive prolactinoma behaviour
is epidermal growth factor (EGF). Anterior pituitary cells
normally express EGF, and it has binding sites mainly
confined to lactotroph and somatotroph cells (72, 73). In
pituitary tumours, EGF-binding sites are present in
76.5% of prolactinomas and 62.5% of gonadotrophinomas (74). EGF binding is more frequent in the invasive
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EUROPEAN JOURNAL OF ENDOCRINOLOGY (2007) 156

than non-invasive pituitary adenoma group which

includes prolactinomas (exact number not specified),
especially in adenomas invading the cavernous sinus. In
addition, adenomas including prolactinomas with
supra/extrasellar extension show higher EGF-binding
sites compared with those without (74). It seems that
EGF is a key factor contributing to pituitary tumour
aggressiveness possibly by its ability to modulate cell
proliferation. However, since the correlation of EGFbinding sites with prolactinoma invasiveness has not
been specifically addressed in this study (74), more data
in prolactinomas are clearly needed to state that EGF
receptor expression levels reflect the invasiveness in this
particular type of pituitary adenomas.

Role of tumour angiogenesis

Angiogenesis is the process of new vessel development
from existing ones and this has been shown to be
involved in tumour growth and spreading. In many
cancers, increased vascularisation of the tumour has
been correlated with poor prognosis and metastatic
spread (75).
Investigations related to angiogenesis in normal
pituitary tissues and pituitary adenomas have opened
new avenues to understand the biology of aggressiveness. Contrary to expectations, normal pituitary gland is
more vascular than tumours as exemplified by low
microvessel counts in immunohistochemistry for CD31
and ulex europaeus agglutinin I (UEAI) (76, for review,
see 77). It seems possible that pituitary tumours may
induce the development of new blood vessels directly
from the systemic circulation (77). Although pituitary
tumours are less vascular than normal pituitary tissue,
there is a distinction between specific tumour types in
respect of vascular density according to tumour size. We
have demonstrated that micro- and macroadenomas
that secrete GH or ACTH have comparable vascular
densities, whereas macroprolactinomas are significantly more vascular than microprolactinomas (76).
The relationship of angiogenesis with pituitary tumour
behaviour has also been studied by our group (78). We
have noticed that preoperative serum hormone levels
were directly proportional to microvessel density in
prolactinomas, but not in GH-secreting tumours. A
significantly increased microvessel density was observed
in invasive prolactinomas compared with non-invasive
ones (78). These data support the view that microvessel
density as assessed by CD31 and UEAI staining is a
marker of aggressive behaviour in prolactinomas
affecting the clinical outcome (78). In another study,
we have identified a positive relationship between antiapoptotic protein Bcl-2 expression and microvessel
density in a group of pituitary adenomas, including
prolactinomas (79). Thus, prevention of programmed
cell death and switch to an angiogenic phenotype seem
to be associated with tumour progression and invasion.

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Genetic alterations leading to aggressiveness

A considerable number of genetic alterations have been
implicated in the pathogenesis of prolactinomas. Those
that may be associated with aggressive behaviour, are
summarised in Table 2, and discussed below.
The Ras proto-oncogene Pituitary tumours arise from
either gain of function mutations, or the overexpression
of proto-oncogenes that function in proliferative
pathways (80). The Ras gene codes for a GTP-binding
protein that is important in cellular proliferation and
differentiation and involved particularly in early stages
of tumourigenesis (81). A case has been reported with a
point mutation (Gly12Val) in the Ras gene presenting
with recurrent invasive prolactinoma with dopamine
agonist resistance (82). Despite the fact that Ras
mutations have been implicated in various human
malignancies, they are uncommon in pituitary adenomas (83) and probably not relevant as genetic markers
of tumour progression or malignancy.
Common and pituitary-specific tumour suppressor
genes The role of tumour suppressor genes in the
pathogenesis of pituitary tumours in animal models is
well known. In rodent models, knockout of the
retinoblastoma gene (RB) or p27Kip1 gene (a gene
involved in cell cycle arrest at G1 stage) has been
identified as causing pituitary adenomas (84, 85). In
humans, loss of heterozygosity (LOH) on chromosome
13q, the location of the RB gene, has been identified in a
small number of pituitary carcinomas (86). Bates et al.
(87) assessed the impact of allelic deletion of various
genes by LOH studies. LOH was found at loci 11q13 (the
site for the tumour suppressor gene of multiple
endocrine neoplasia type 1, MEN-1), 13q12-14, 10q
and 1p. About half of the invasive tumours, which
included prolactinomas, had at least one allelic deletion
at these sites. Of the six tumours with only one deletion,
five involved the 11q13 locus suggesting that this is an
early event taking place in transition from a noninvasive state into an invasive one (87). Although the

149

loss of alleles on chromosome 11q13 has been identified

in about 1020% of both sporadic GH-secreting
adenomas as well as prolactinomas (88, 89), subsequent studies have failed to reproduce similar results
in terms of mutations in the MEN-1 gene (90, 91). In a
recent study, MEN-1-associated prolactinomas have
been found to be more aggressive compared with the
sporadic ones (92), although other studies have
previously failed to show significant differences in this
regard (93, 94). Since mutations and deletions thus far
identified account for only a small percentage of
pituitary tumours, including prolactinomas, it is
presently difficult to infer that there is a generalised
genetic mechanism for aggressive tumour behaviour.
Genes related to growth factor expression, angiogenesis and tumour progression The relevance of the
locus 11q13 with respect to tumourigenesis and tumour
progression in the pituitary gland may also be related to
the hst gene that was initially identified as a transforming gene in solid malignant tumours (95). DNA derived
from human prolactinomas express transforming
activity in heterologous cells and had sequences in
close resemblance with those of hst gene (96). Overexpression of hst gene leads to increased production of
FGF-4 (97). Shimon et al. (97) demonstrated the
function of the hst gene in rat lactotroph tumour
formation and prolactin secretion. They were able to
show that lactotrophs in 5 of 14 prolactinomas stained
strongly with anti-FGF-4 monoclonal antibodies.
Immunoreactive hst product in adenoma cells was
observed in three of five invasive prolactinomas, whereas
only two of nine non-invasive ones were positive.
Interestingly, most of the non-functioning adenomas
and other types of functioning adenomas did not present
any staining for hst gene product. Furthermore,
immunostaining for proliferative marker Ki-67 showed
a greater proliferative status in hst-positive adenomas
compared with those that are immunonegative for hst
(97). These findings imply a role of hst gene, and its
product FGF-4, in cellular proliferation, growth and
aggressive behaviour in prolactinomas.

Table 2 Detected genetic alterations in invasive prolactinomas.

Affected gene
(locus)

Detected alteration

Biological effect

Ref.

Retinoblastoma
(RB) (11q13)
hst (11q13)
PTTG (5q33)

Deletion (LOH)

Transition to an invasive state

(87)

Oestrogen-induced overexpression
Oestrogen-induced overexpression

(97)
(101)

Edpm5

Variability between rat strains

Induction of FGF-4, [ growth, [ proliferation, [ angiogenesis

[ bFGF expression, [ VEGF expression, tumour progression,
[ angiogenesis
Change in angiogenic switch, alterations in tumour
angiogenesis upon oestrogen stimulation

(106)

LOH, loss of heterozygosity; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PTTG, pituitary tumour-transforming gene.

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Oestrogen promotes tumourigenesis in the pituitary

gland via induction of a novel pituitary tumourtransforming gene (PTTG) that is located on chromosome 5q33 (98). PTTG has been shown to be
tumourigenic in vivo, by regulating basic fibroblast
growth factor (bFGF) secretion and inhibiting chromatid separation (99, 100). bFGF modulates angiogenesis,
tumour formation and progression in several tissues,
including the pituitary (101). Oestrogen induces overexpression of rat PTTG leading to increased bFGF and
VEGF production (VEGF) in the pituitary gland (101).
Moreover, bFGF itself has an ability to modulate VEGF
expression (101). This finding implies that oestrogen is
important in pituitary adenoma formation and progression via a paracrine mechanism involving angiogenesis. In accordance with these effects of oestrogen,
lactotroph tumours tend to enlarge considerably during
pregnancy (102). As noted earlier, prolactinomas in
men tend to exhibit a more aggressive course. This
seems to be counterintuitative concerning the oestrogenPTTG pathway, since men have lower oestrogen
levels than women. However, this discrepancy may be
explained by the observation that although men have
lower oestrogen levels, their tumours express more
oestrogen receptors than those in women (103). It
should be noted, however, that PTTG overexpression is
not specific for prolactinomas, and seen in all classes of
hormone-secreting pituitary adenomas (104, 105).
Recently, a novel gene locus named Edpm5 has been
shown to prevent the angiogenic switch in prolactinomas after chronic oestrogen treatment in rats (106).
The contribution of Edpm5 gene to angiogenesis and
aggressive behaviour in human prolactinomas,
however, is yet to be defined.
Genes implicated in resistance to dopamine agonists It is well known that dopaminergic receptor-2
(D2R) gene-deficient mice develop lactotroph cell
hyperplasia and tumours capable of massive invasion
(107). No mutation in the D2R gene has been reported
in 79 invasive and dopamine agonist-resistant prolactinomas (108). However, the impact of dopamine
receptor gene polymorphisms on prolactinoma
behaviour warrants further investigations.

Summary and conclusions

Aggressive prolactinomas represent a group presenting
with invasion into surrounding and remote structures
and causing challenges in treatment. Invasive tumours
are usually associated with lower cure and higher
recurrence rates as compared with non-invasive ones.
Prolactin levels at diagnosis are higher in these tumours
in correlation with their sizes, and nave or acquired
resistance to dopamine agonists are more frequently
encountered. Invasive prolactinomas may be associated
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EUROPEAN JOURNAL OF ENDOCRINOLOGY (2007) 156

with increased Ki-67/MIB-1 labelling indices indicating

increased cell proliferation, although this is not a
universal finding. Increased expression of polysialylated
neural cell adhesion molecule and reduced expression of
E-cadherin/catenin complex may imply a contribution
of changes in cell-to-cell adhesion and cellular
migration. The reduced E-cadherin/catenin expression
seems to be relatively specific for invasive prolactinomas, but not other functioning and non-functioning
pituitary tumours. ECM and MMPs with their inhibitors
play important roles in the context of angiogenesis, a
key switch factor leading to invasion. The induction of
local FGF and VEGF activity via the oestrogen-induced
overexpression of novel genes (PTTG, hst and Edpm5)
enhance prolactin cell growth, proliferation and
tumoural angiogenesis in prolactinomas. hst gene
overexpression is relatively specific for invasive prolactinomas. Loss of tumour suppressor genes at various loci,
including 11q13 has been found in invasive pituitary
adenomas, including prolactinomas. Since only a few
specific markers have been identified thus far, more
investigations into the mechanisms of aggressive
behaviour in prolactinomas are needed to eventually
predict cases with poor clinical outcome. New treatment
modalities specifically targeting these mechanisms may
promise a cure for these challenging tumours.

Acknowledgements
Current institutional address of Dr Alper Gurlek is:
Hacettepe University School of Medicine, Department of
Internal Medicine, Division of Endocrinology, Ankara,
Turkey.

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Received 29 September 2006

Accepted 20 November 2006

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