International Journal of Pediatric Otorhinolaryngology (2006) 70, 227234

www.elsevier.com/locate/ijporl

Proliferation, angiogenesis and hormonal markers

in juvenile nasopharyngeal angiofibroma
nerci a
Gu
leser Saylam a,*, O. Taskn Yu
cel a, Arzu Sungur b, Metin O
a
b

Department of Otorhinolaryngology, University of Hacettepe, Ankara, Turkey

Department of Pathology, University of Hacettepe, Ankara, Turkey

Received 12 April 2005; accepted 6 June 2005

KEYWORDS
Nasopharynx
angiofibroma;
Proliferation;
Angiogenesis;
Hormonal markers

Summary
Objectives: Juvenile nasopharyngeal angiofibroma (JNA) is a highly vascular and
locally invasive tumor that exclusively affects male adolescents. Sex hormones are
first discussed to clarify the etiology of JNA. Recently with the advances in the field of
cell biology angiogenetic markers, proliferation markers and growth factors are
investigated to identify the molecular basis of JNA as all neoplasm. In this study
we tried to evaluate the expression of proliferation, angiogenesis and hormonal
markers in JNA.
Methods: Immunohistochemical analysis were performed on paraffin-embedded 27
JNA samples which were obtained from the patients operated at University of
Hacettepe Department of Otorhinolaryngology, a tertiary care center. Estrogen
receptor (ER), progesterone receptor (PR), proliferating cell nuclear antigen (PCNA),
vascular endothelial growth factor (VEGF) and transforming growth factor b (TGF-b)
specific antibodies were used and evaluated by light microscopy
Results: Two of 27 cases were ER positive. Nine of 27 cases were positive for PR. All of
the cases were stained with PCNA. Twenty-four of 27 cases stained with VEGF. TGF-b
was positive in 14 of 27 cases. All recurrent cases were stained with PCNA and VEGF;
just three of them were stained with TGF-b.
Conclusions: Hormonal markers ER and PR did not seem to play a role in pathogenesis
of JNA. PCNA, VEGF and TGF-b may play a role in the pathogenesis of JNA by
promoting angiogenesis and proliferation, but this role did not seem to have a relation
with hormonal markers.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Presented at the RhinoIstanbul-XXIII. International symposium

on infection and allergy of the nose, 1823 June, 2004.
* Corresponding author. Tel.: +90 312 3441654/656;
fax: +90 312 2321604.
E-mail address: guleserkilic@yahoo.com (G. Saylam).

1. Introduction
Juvenile nasopharyngeal angiofibroma (JNA) is a
histologically benign, locally aggressive neoplasm
of the nasopharynx that exclusively affects male

0165-5876/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijporl.2005.06.007

228

G. Saylam et al.

adolescents, with an average age of onset being 14

years. It accounts for 0.5% of all head and neck
neoplasms with a high incidence of persistence
and recurrence. Evidence of intracranial spread
occurs in 1020% of cases [15].
Occasional reports of endocrine dependence,
malignant change, spontaneous regression and several case reports of females with disease have
stimulated increased interest in this unusual tumor
[610]. A review of the literature reveals controversy regarding almost every aspect of this tumor.
Many theories have been suggested to explain
pathogenesis of angiofibroma in the light of its
clinicopathological features. The sex selectivity
and some occasional association with sexual under
development suggested that steroid and sex hormones might be involved in the biology of the tumor.
Thus JNA has been considered as a tumor associated
with sex hormones, a number of recent studies have
dealt with hormonal receptors of JNA tissue. However, the findings are not in accordance with each
other and the concept of hormone receptors in JNA
tissue is still controversial [68,1113].
Despite being classified as benign, JNA may grow
considerably and cause significant structural and
functional damage. There are no clear explanations
onto why a histologically benign tumor should erode
bone and push into and through regional structures.
Current studies about stromal and vascular proliferation suggested that angiogenic growth factors
and cytokines might play a role in the pathogenesis
[1,3,14,15]. Schiff et al. [3] documented immunolocalization of basic fibroblast growth factor, a
strong proangiogenic cytokine, in vessel endothelium of JNA. This finding supported the role of the
growth factors in the etiology. Recently, Dillard
et al. [1] reported the expression of transforming
growth factor b (TGF-b) in JNA stroma and endothelial cells. Moreover, Brieger et al. [15] studied vascular endothelial growth factor (VEGF), the most
prominent proangiogenic growth factor in tumor

biology, and suggested the expression of VEGF by

stromal cells and association of VEGF with proliferation and increased vessel density. In their study they
have also suggested that a study that explores the
role of hormonal markers and growth factors with
angiogenic factors may help understanding of
pathogenesis of JNA.
In this sudy, we analyzed the role of and expression of hormonal markers with relation to proliferation, angiogenic and growth factors in JNA. Agents
for anti angiogenic activity are under investigation
and understanding of role of these markers could
provide a better way of treatment in these tumors.

2. Material and method

The medical charts of 27 patients with histologically
proven nasopharyngeal angiofibroma seen at the
Department of Otorhinolaryngology, Hacettepe University Hospital, a tertiary referral center, between
1983 and 2002 were retrospectively reviewed.
Patients ages, tumor stages, incidence of recurrence and follow up were recorded from charts.
All patients were males; their ages ranged from
10 to 34 years (mean 17.8). The patients were
followed up for an average of 42 months. The
patients were classified according to Radkowski
staging system [2].

2.1. Immunohistochemical study

Immunohistochemical study was performed on 4 mm
formalin-fixed, paraffin-embedded tissue sections
using the avidin-biotin-peroxidase complex (ABC)
method using monoclonal primary antibodies
against proliferating cell nuclear antigen (PCNA,
NeoMarkers), VEGF (NeoMarkers), TGF-b (NeoMarkers), estrogen receptor (ER, NeoMarkers), progesterone receptor (PR, NeoMarkers). Section of 4 mm
thickness were cut from the paraffin blocks and than

Table 1 Stage recurrence and incidence of stained cases with estrogen receptor (ER), progesterone receptor (PR),
proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), transforming growth factor b
(TGF-b)
Stage

Recurrence

Incidence of positivity
ER(+)

IA
IIA
IIB
IIC
IIIA
IIIB
Total

1
1
2
1
5

6
1
5
5
7
3
27

1/1
1/5

PR(+)

PCNA(+)

VEGF(+)

TGF-B(+)

1/6

6/6
1/1
5/5
5/5
7/7
3/3
27

4/6
1/1
4/5
5/5
7/7
3/3
24

3/6

1/5
3/5
2/7
2/3
9

3/5
3/5
3/7
2/3
14

Proliferation, angiogenesis and hormonal markers in juvenile nasopharyngeal angiofibroma

Fig. 1

229

Estrogen immunostain in juvenil nasopharingeal angiofibroma (immunohistochemistry  460).

they were deparaffinized and rehydrated through a

graded series of alcohol. Sections were pretreated
for antigen retrieval by autoclaving or proteinase K
treatment. The sections were incubated 1 h at room
temperature with primary antibodies. Detection
was done using a streptavidin-biotin-peroxidase
kit (NeoMarkers) and then staining with freshly
prepared diaminobenzidine solution and counter-

Fig. 2

staining with hematoxylin were done. Immunoreactivity for PCNA, ER, PR in the sections was evaluated
by counting the number of positively stained nuclei
in at least 1000 cells and was scored according to the
proportion of negative cells observed: (+++) 50
100%, (++) 2550%, (+) less than 25% and ( ) absent
for PCNA and (+++) 50100%, (++) 1050%, (+) less
than 10% and ( ) absent for ER and PR. Immunor-

Progesterone staining is illustrated (immunohistochemistry  460).

230

G. Saylam et al.

eactivity for VEGF and TGF-b in the sections staining

intensities were scored from 0 to 3 (+). In addition
Hematoxylin-eosin-stained slides from each case
were assessed for cellularity.

3. Results
Classification of tumors was illustrated in Table 1.

Table 2 Incidence of positive stained cases with

estrogen receptor (ER), progesterone receptor (PR),
proliferating cell nuclear antigen (PCNA)
Staining pattern

(+)
(++)
(+++)
Total

Incidence of positive stained

cases
ER

PR

PCNA

1/27
1/27
2/27

6/27
3/27

9/27

1/27
3/27
23/27
27/27

3.1. Recurrence
Three of 27 patients had a recurrent tumor following
primary surgical resection previously in another
hospital. Five cases recurred following surgery in
our institute. One of these five cases was the patient
that was previously operated in another hospital.
The other two patients referred as recurrent angiofibroma were operated and no recurrence was
observed in these during 12 months period of follow
up. Thus in our institute a total of five cases were
evaluated as recurrent tumor and consequently
recurrence rate was 18.5%.

3.2. Immunohistochemical analysis

We studied two hormonal markers, estrogen receptor (ER) and progesterone receptor (PR). Two of 27
cases were ER positive. These positive cases were
stained (++) and showed 12%, 30% cell staining,
respectively (Fig. 1). For PR staining nine of 27 cases

Fig. 3

were positive (Fig. 2). Six of them demonstrated

lower than 10% of cell staining. 1050% of cell
staining was observed in the remaining three cases
(Table 2).
All of the cases stained with PCNA both for the
stromal and endothelial cells (Fig. 3). According to
cell staining 3.7% (n: 1) of cases were stained lower
than 25%, 11.1% (n: 3) of cases were stained 2550%
and 85.2% (n: 23) of all cases were stained more than
50% (21 of PCNA stained cases showed 70100% of
cell staining.) (Table 2).
Twenty-four of all cases (88.9%) stained with VEGF
(Fig. 4). Cases were determined according to intensity. 1+ staining was seen in 11 cases (40.7%), 7
(25.9%), 6 (22.2%) cases were stained in 2+ and 3+
intensity, respectively (Table 3). We observed diffuse
staining in 45.8% (n: 11) cases, remaining 13 (54.2%)
cases were showed focal staining.
Fourteen (40.7%) cases were stained with TGF-b
(Fig. 5). Eight of stained cases showed 2+ intensity

Nuclear staining with proliferating cell nuclear antigen (immunohistochemistry  230).

Proliferation, angiogenesis and hormonal markers in juvenile nasopharyngeal angiofibroma

Fig. 4

Positive staining with vascular endothelial growth factor (immunohistochemistry  230).

staining; five of them stained with 1+ intensity and

just one case was stained with 3+ intensity (Table 3).
We also evaluated the H-E stained slides of all
specimens for cellularity. 70.4% (n: 19) cases were
determined as highly cellular, and cellularity was
low in 29.6% (n: 8) cases.
All recurrent cases were stained with PCNA and
VEGF; just three of them were stained with TGF-b.
Three of the recurrent cases were stained with PR
and none of the cases was stained with ER.

4. Discussion
JNA is still a particularly interesting tumor because
of its association with significant morbidity and
occasional mortality. Pathophysiology of this tumor
still needs clarification and recent studies have
showed that especially angiogenic factors could
have role in this issue.
Table 3 Staining patern with vascular endothelial
growth factor (VEGF), transforming growth factor b
(TGF-b)
Staining pattern

1(+)
2(+)
3(+)
Total

231

Incidence of positive
stained cases
VEGF

TGF-b

11/27
7/27
6/27
24/27

5/27
8/27
1/27
14/27

Angiogenesis is an essential process required for

growth and metastasis in solid tumors. Angiogenesis
is regulated by angiogenic factors such as VEGF
located on vascular endothelial cells, which are a
key regulator of angiogenesis. The authors suggest a
role for VEGF in tumor lymph angiogenesis and
vascular angiogenesis, promoting tumor growth
and propagation of cancer cells [16,17]. In a recent
study they showed the effect of VEGF on proliferation and suggested the promotion of vascularisation
by VEGF [15]. High ratio of VEGF stained cases
(88.9%) in our study can be considered that angiofibroma is a vascular and proliferative tumor. Brieger
et al. [15] also observed this finding in their study
and they have seen high vessel densities associated
with stromal VEGF and Ki67 expression. They
remarked that the participation of hormonal receptors and angiogenic growth factors has been suggested in the growth of JNA. However the results of
antiandrogen therapy and of immunohistochemical
analyses of androgen receptors remain conflicting.
They have concluded that more studies for investigation of impact and action of sex hormones with
relation to angiogenic factors were needed.
It was suggested by some authors that the occurrence of these rare tumors almost exclusively in
adolescent males supports the hypothesis that an
alteration of the pituitary androgenestrogen axis
contributes to the pathogenesis of JNA [18]. Exhaustive studies of the pituitarygonadal axis in these
patients, however, have failed to identify any endocrinologic abnormality [2]. Reports on hormonal

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G. Saylam et al.

Fig. 5

Transforming growth factor-b positivity is illustrated (immunohistochemistry  230).

receptors of angiofibroma are still controversial.

Some reports suggested that the fibroblasts of
JNA tissue possess estradiol receptors and they
considered that JNA is associated with estradiol
[6]. Brentani et al. [7] examined estrogen, progesterone, androgen and glucocorticoid receptors in
cytosol and reported that progesterone receptor
was positive in 58% with clearly predominating
25% for estrogen and androgen receptors. Kumagami
[6] studied testesteron and estradiol in five cases
and reported that estradiol was positive in all cases
beside testosterone negativity in all cases. The sex
selectivity and some occasional association with
sexual underdevelopment, as well as variable
responses to estrogens, suggested that estrogens
might be involved in the biology of these benign

tumors. On the other hand, Farag et al. [8] studied

dehydrotestesterone, testosterone and estradiol17B levels in the serum and reported that serum
levels were normal but dehidrotestesterone receptor showed higher specific affinity. Antonelli et al.
[19] reported that estrogen and progesterone
receptors were negative but dehydrotestesterone
was highly positive in cytosol. ER, PR and androgen
were studied immunohistochemically by Hwang
et al. [20] and they reported 75% positive staining
with androgen and just 8.3% staining with PR. They
did not observed ER staining in any patients. Therefore androgen dependency was suggested by these
and the other researchers who confirmed the androgen receptor positivity [8,1113,19,20] and treatment with estrogen, the physiological antagonist of

Table 4 Summary of reported studies [estrogen receptor (ER), progesterone receptor (PR), proliferating cell nuclear
antigen (PCNA), vascular endothelial growth factor (VEGF), transforming growth factor b(TGF-b)]
Incidence of positive stained cases
Jhons et al. [11]
Lee et al. [12]
Kumagami [6]
Hwang et al. [20]
Gatalica [26]
Dillard et al. [1]
Liang et al. [13]
Brieger et al. [15]
a

ER

6
8
5
24
8
19
25
10

0
0
5
0
0

Only three cases were studied.

PR

Androgens

3a
0
18
8 (weak)

2
0

VEGF

TGF-b

19
0

0
8

PCNA

Proliferation, angiogenesis and hormonal markers in juvenile nasopharyngeal angiofibroma

androgens, is likely to reduce the vascularity and

arrest the size of these tumors [8]. In our study only
7.4% (n: 2) cases were ER positive and progesterone
receptor positivity was detected in 33.3% of our
cases similar to the studies in literature.
The studies that try to clarify the role of angiogenesis, proliferation and growth factors in pathogenesis of JNA are scarce and a list of these studies
can be seen in Table 4.
The massive amount of extracellular matrix that
is present in JNA is another hallmark of the disease.
Because of the contents, extracellular matrix is
influenced by estrogen and growth factors. JNA
may be the first example of a growth factor
mediated disease that is due to the failure of the
extracellular matrix to regulate its activity. Therefore any therapeutic approach that enhances the
quality of the endogenous matrix, like estrogen,
may have profound effects on cell growth and differentiation and hence, progression of the tumor
[3].
TGF-b helps to regulate the cell cycle and induces
angiogenesis. TGF-b positivity in all angiofibroma
cases was identified therefore authors concluded
that TGF-b may play a significant role in the pathogenesis of JNA. They also suggested that blocking
the activity of TGF-b may be used to arrest the
growth of extensive tumors to permit safer extirpation of the more extensive tumors [1]. In another
study, it is considered that TGF-b may play a role in
the development of the fibrous component of this
tumor, and also insulin like growth factor II were
suggested as a potential growth regulator of JNA
[14]. In our study, we found the TGF-b positivity rate
as 51.8% and also 78.7% of positive cases were
stained moderately. This result revealed that TGFb may have a regulatory effect on JNA but there
must be supplementary factors that influenced the
tumor growth. So TGF-b has an indirect role in
pathogenesis of JNA.
In our study the other result that attracted attention was the three cases, which were not stained
with VEGF, were not stained with TGB-b also. Consequently we considered that evaluating these two
growth factors together might provide more useful
data.
PCNA is a marker that indicates proliferation of
tumors and may be a useful indication to the clinician to predict tumor behavior and prognosis. It can
possibly be used for risk assessment and surveillance
of local recurrence [21]. This proliferation marker
was studied in almost all head and neck tumors.
These results suggest that PCNA expression can be
used in decision making for treatment and assessment of prognosis in laryngeal carcinomas, nonsmall cell lung cancer, parotid tumors, papillary

233

thyroid cancer [22]. Also authors mentioned that

PCNA is valuable in predicting those people with a
higher potential for metastasis and recurrence after
surgery; thus, the evaluation is helpful in planning
reasonable adjuvant therapy, even in the earlier
stages [23]. In this study PCNA positivity in all cases
show that angiofibroma is a proliferative tumor with
high tendency to recurrence. However as there was
not a difference between recurrent cases and primary cases this finding was not of much help in
prognosis.
According to these findings it is suggested that
inhibitors of angiogenesis could become effective
therapeutic options for JNA.
Recurrence rate for JNA remains around 20% but
recurrence rates such as 30%, 36% and 46% were also
observed [2,4,24,25]. We observed recurrence in
18.5% of our cases. Recurrent cases were classified
in different stages. All recurrent cases were stained
with PCNA and VEGF, staining of these cases with
PCNA was strongly positive. Only three of recurrent
five cases were stained with TGF-b.

5. Conclusion
Pathogenesis and clinical behavior of JNA, which is
an uncommon tumor, is still controversial. In our
study PCNA and VEGF expressed in almost in every
tumor and TGF-b in most of them, indicating the
nature of this tumor with an aggressive and angiogenic character. These findings indicated a role for
PCNA, VEGF and TGF-b in the pathogenesis of JNA
but these findings could not help us in differentiating between recurrent and non-recurrent tumors
and aggressivity of tumors. Estrogen and Progesterone receptor expression was low to indicate a relation to angiogenesis and etiology of this disease.
Drugs that have inhibitory action on angiogenesis
and proliferation will be introduced in the future
could be useful in the treatment of JNA, but further
studies were needed to clarify their role in pathogenesis of JNA.

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