Olfactory neuroblastoma (ONB) is a rare neoplasm of the sinonasal area with neuroendocrine differentiation. ISL-1, TTF-1 and PAX5 are transcription factors that are frequently upregulated in tumors showing neuroendocrine differentiation. The aim of our study was to evaluate these markers in a group of ONBs. We included 11 ONBs from 4 large university hospitals. Immunohistochemical expression of TTF-1, PAX5 and ISL-1 was evaluated. TTF-1, ISL-1 and PAX5 were expressed in 3/11 cases (27.27%, h-score: 3-45), 7/11 cases (63.64%, h-score: 23-200), and in 3/11 cases (27.77%, h-score 3-85), respectively. The patient with the strongest PAX5 reactivity exhibited an aggressive clinical course with rapid dissemination to the spine and death shortly after the diagnosis. No significant correlation in the expression of PAX5 and TTF-1 (ρ = 0.43; p = 0.18) was observed. ISL-1 is widely expressed in tumors with neuroendocrine differentiation and therefore of limited value in their differential diagnosis. TTF-1 positivity does not exclude the diagnosis of primary ONB, although usually only a small percentage of cells are positive. PAX5 expression is infrequent (27.27%) in ONB; however, if present it can be associated with a very aggressive clinical course

Adam Gorczyński

Jarosław Dzierżanowski

Joanna Karczewska

Joanna Reszeć

Johannes Haybaeck

Okoń

Piotr Czapiewski

Wojciech Biernat

Wojciech Skrzypczak

CiteSeerX

130
Original paper
expressiOn pattern Of isl-1, ttf-1 and pax5  
in OlfactOry neurOblastOma
Piotr CzaPiewski1, adam GorCzyński1, Johannes haybaeCk2, krzysztof okoń3,  
Joanna reszeć4, woJCieCh skrzyPCzak5, Jarosław dzierżanowski6, miChał kunC7,  
Joanna karCzewska1, woJCieCh biernat1
1Department of Pathomorphology, Medical University of Gdansk, Gdansk, Poland
2Department of Neuropathology, Institute of Pathology, Medical University of Graz, Graz, Austria
3Department of Pathomorphology, Jagiellonian University Collegium Medicum, Krakow, Poland
4Department of Medical Pathomorphology, Medical University of Bialystok, Bialystok, Poland
5Department of Otolaryngology, Medical University of Gdansk, Gdansk, Poland
6Department of Neurosurgery, Medical University of Gdansk, Gdansk, Poland
7Faculty of Medicine, Medical University of Gdansk, Gdansk, Poland
Olfactory neuroblastoma (ONB) is a rare neoplasm of the sinonasal area with neuro- 
endocrine differentiation. ISL-1, TTF-1 and PAX5 are transcription factors that are 
frequently upregulated in tumors showing neuroendocrine differentiation. The aim 
of our study was to evaluate these markers in a group of ONBs. 
We included 11 ONBs from 4 large university hospitals. Immunohistochemical 
expression of TTF-1, PAX5 and ISL-1 was evaluated. 
TTF-1, ISL-1 and PAX5 were expressed in 3/11 cases (27.27%, h-score: 3-45), 
7/11 cases (63.64%, h-score: 23-200), and in 3/11 cases (27.77%, h-score 3-85), 
respectively. The patient with the strongest PAX5 reactivity exhibited an aggres-
sive clinical course with rapid dissemination to the spine and death shortly after 
the diagnosis. 
No significant correlation in the expression of PAX5 and TTF-1 (ρ = 0.43;  
p = 0.18) was observed.
ISL-1 is widely expressed in tumors with neuroendocrine differentiation and there-
fore of limited value in their differential diagnosis. TTF-1 positivity does not ex-
clude the diagnosis of primary ONB, although usually only a small percentage of 
cells are positive. 
PAX5 expression is infrequent (27.27%) in ONB; however, if present it can be 
associated with a very aggressive clinical course. 
Key words: olfactory neuroblastoma, esthesioneuroblastoma, ISL-1, TTF-1, 
PAX5.
doi: 10.5114/PJP.2016.61448 Pol J Pathol 2016; 67 (2): 130-135
Introduction
Olfactory neuroblastoma (ONB), also known as 
esthesioneuroblastoma, was originally described in 
1924 by the French physicians Berger and Luca in 
1924. With its frequency of 0.4/million/year [1], it 
accounts for 3% of all sinonasal malignancies [2].
ONB is believed to be derived from olfactory ep-
ithelium, but its pathogenesis is largely unknown. 
Data on viral pathogenesis in some cases of animal 
ONB were not confirmed in humans [3]. ONB is 
frequently hormonally active and can thus result in 
paraneoplastic syndromes [4].
131
ISL-1, TTF-1 and PaX5 In oLFacTory neurobLaSToma
ONB presents with neuroendocrine differentia-
tion, and primary as well as metastatic tumors can 
mimic other tumor entities with this differentiation 
pattern. Therefore, a continuous search for new 
markers that broaden differentiation with other 
neuroendocrine tumors is ongoing. Recent evidence 
shows that ISL-1, TTF-1 and PAX5 occur in a large 
percentage of neuroendocrine tumors [5, 6, 7, 8, 9]. 
However, the frequency of their expression and their 
expression pattern have not been studied in ONB.
No data are available about the functional relation-
ship at the molecular level of TTF-1, PAX5 and ISL-1 
in the literature. Also no relationship in their immu-
nohistochemical expression has been studied despite 
their frequent presence in neuroendocrine tumors. 
The aim of our study was to evaluate immuno- 
histochemical expression of TTF-1, ISL-1 and PAX5 
in a cohort of ONB collected from 4 large university 
hospitals in order to show their potential diagnostic 
usefulness. Additionally we assessed the potential 
reciprocal relationship between expression of these 
proteins in order to give some suggestions about the 
potential correlation in their biological activity and 
neoplastic transformation.
Material and methods
Patients
Databases of the departments and institutes of pa-
thology in 4 large university hospitals were searched 
for the diagnosis of ONB or esthesioneuroblastoma. 
The retrieved cases were sent to the central laborato-
ry (Medical University of Gdansk) for reevaluation. 
It was performed according to the criteria defined by 
the WHO classification of head and neck tumors. 
Immunohistochemistry
Expression of PAX5, TTF-1 and ISL-1 was evalu-
ated by immunohistochemistry. For TTF-1 and PAX5 
ready-to-use antibodies from Dako (Denmark) were 
used and the staining was performed using an auto-
mated tissue stainer (Dako, Denmark). The analy-
sis of ISL-1 (1 H9, Abcam, 1 : 1500) was performed 
manually. 
Intensity of immunohistochemical reaction was eval-
uated using the h-score system. In h-score, the intensi-
ty of reaction and the percentage of positive cells were 
multiplied and summed up (1× n%+2×n%+3× 
n% = y), i.e., the score ranged from 0 to 300. 
Each case was evaluated for each protein by two experi-
enced pathologists (P.C. and A.G.).
Statistics
Spearman’s rank correlation test was performed 
using Statistica 12 in order to evaluate the correlation 
in expression of ISL-1, PAX5 and TTF-1.
Results
Patients
The study group included 11 cases of ONB: 
4 males and 7 females, aged 20-86 (mean: 52.3). 
Basic clinicopathological characteristics of the pa-
tients are given in Table I. 
Expression of TTF-1
TTF-1 was expressed in 3 cases (27.27%). Expres-
sion was nuclear and occurred in 1-27% of cells. In all 
cases there were at least single cells with strong immu-
noreactivity; the h-score of positive cases were in the 
range 3-45. Representative staining is shown in Fig. 1.
Expression of ISL-1
ISL-1 was expressed in 7/11 cases (63.63%). Ex-
pression was nuclear and it was observed in 15-90% 
of cells. The h-score of positive cases ranged between 
23 and 200. Representative staining is shown in Fig. 2.
Expression of PAX5
PAX5 was expressed in 3/11 (27.27%) cases. One 
of them showed only single weakly positive cells 
(h-score: 3). The other tumors showed higher expres-
sion of PAX5 (16% and 35% of positive cells, h-score: 
24 and 85, respectively). The patient with the highest 
level of PAX5 expression developed lymph node me-
tastases at the primary diagnosis, and these recurred 
shortly afterwards with dissemination to the spine. She 
died 3 months after the initial diagnosis. PAX5 immu-
nohistochemical staining is shown in Fig. 3.
Correlations in the expression of the 
markers
We did not observe statistically significant correla-
tion between the expression of investigated proteins. 
However, taking into account the low number of cas-
es it can be stated that there is a trend toward a mod-
erate correlation between expression of TTF-1 and 
Table I. Basic clinicopathological data of ONB cohort
sex Male 4 (36%)
Female 7 (64%)
age (years)
20-86
Average 52.3
Median 50.5
grade I 2 (18%)
II 5 (45%)
III 3 (27%)
IV 1 (9%)
132
Piotr CzaPiewski, adam GorCzyński, Johannes haybaeCk et al.
PAX5. The Spearman rank correlation test resulted 
in the following values for the investigated proteins:
• ISL-1 and TTF-1 (ρ = 0.0029; p = 0.99),
• ISL-1 and PAX5 (ρ = 0.0828; p = 0.81),
• TTF-1 and PAX5 (ρ = 0.43; p = 0.18).
Discussion
PAX5 is a member of the paired box (PAX) family 
of genes that are proteins involved in the regulation 
of development and differentiation of multiple tis-
sue types [10]. PAX5 is a transcription factor that 
controls B-cell development and is present on B-cells 
from early to late stages of B-cell differentiation [11]. 
Initially it was believed to be a specific marker of 
B-cell neoplasms, but it has also been identified in 
many other types of tumors with neuronal and neu-
roendocrine differentiation [12, 13, 14].
PAX5 is activated in the mesencephalon during 
murine embryogenesis [10], and knock-out of this 
gene results in malformations of the midbrain [15]. 
The role of PAX5 in the development and function of 
the olfactory epithelium is not known.
We showed for the first time that PAX5 is ex-
pressed in a subpopulation of ONB (3/11; 27.27%). 
In one of them only 3% of cells showed a weak reac-
tion, which from a diagnostic point of view is mean-
ingless. Two other cases revealed stronger immuno-
expression in 16 and 35% of cancer cells, respectively. 
This observation adds ONB to the large group of 
neuroendocrine tumors that may show PAX5 reac-
tivity. 
The patient with the highest PAX5 expression 
exhibited a very aggressive clinical course of disease: 
lymph node metastases at the diagnosis, rapid pro-
gression with spinal involvement and death 3 months 
after the onset of therapy. Unfortunately, the clini-
cal course of the remaining two PAX5-positive cases 
could not be retrieved from the clinical files. There-
fore, it is not possible to evaluate the role of PAX5 
expression regarding the clinical course and outcome. 
Its evaluation in an independent and larger cohort of 
PAX5-positive ONBs may answer this question. 
Fig. 1. Nests of olfactory neuroblastoma (A, HE, magnification 40×) with small, vesicular nuclei (B, HE, magnification 
200×) showing moderate to strong immunohistochemical reaction to TTF-1 (C, magnification 100×; D, magnification 
200×)
A
C
B
D
133
ISL-1, TTF-1 and PaX5 In oLFacTory neurobLaSToma
TTF-1 is a marker of pulmonary and thyroid or-
igin of tumors [16]. However, up to 40% of high-
grade neuroendocrine tumors of extrapulmonary ori-
gin show expression of this transcription factor [8, 9]. 
In the human fetal brain, TTF-1 expression has been 
reported in the infundibular anlage and ependymal 
and subependymal cells of the third ventricle [17]. 
Its role in the development and function of the olfac-
tory epithelium remains unknown. 
In the primary tumors, 3/11 cases were positive, 
although in one case only single cells were strongly 
positive (h-score = 3), while the other two cases were 
positive in 20-27% of cells. 
Some authors believe that TTF-1 expression ex-
cludes the diagnosis of ONB. However, we think 
that TTF-1 expression is just a marker of neuroen-
docrine differentiation, as it may be present in a sub-
stantial group of neuroendocrine tumors at various 
locations. Therefore, it does not exclude per se diagno-
sis of ONB. On the other hand, in none of our cases 
was strong, diffuse expression observed, so the differ-
ential diagnosis with the most common neuroendo-
crine malignancy, small cell carcinoma of the lung, 
which is usually strongly TTF-1 positive, should not 
be problematic in this setting. 
ISL-1 was originally described by Karlsson et al. 
as a protein that binds to an insulin gene enhancer 
and regulates its expression [18]. Its crucial role 
in the development and maturation of the endo-
crine cells in the pancreas was confirmed [19]. 
Initially expression of ISL-1 was believed to be re-
stricted to neuroendocrine tumors of pancreatic origin. 
However, recent data show that it can be expressed in 
the majority of neuroendocrine tumors of extrapancre-
atic origin, including Merkel cell carcinomas, pulmo-
nary small cell neuroendocrine carcinoma, medullary 
thyroid carcinomas, paragangliomas/pheochromocy-
tomas, and adrenal neuroblastomas [6].
Recently we have described the expression of ISL-1 
in 44% (11/25) of medulloblastomas (Czapiewski, 
Pathology Research and Practice, in press). In the 
developing brain ISL-1 promotes striatonigral and 
represses striatopallidal genetic programs [20]. The 
role of ISL-1 in the development and function of ol-
factory epithelium is not known. 
In a report regarding ISL-1 expression in a small 
group of 4 cases of ONB, only 1 case was positive [6]. 
In our cohort of ONB patients expression of ISL-1 
was present in the majority of cases (63.63%). All of 
the positive cases were at least moderately immuno-
Fig. 2. Nests of monomorphic neoplastic cells (A, HE, magnification 100×; B, HE, magnification 200×) with moderate 
to strong expression of ISL-1 (C, magnification 100×; D, magnification 200×)
A
C
B
D
134
Piotr CzaPiewski, adam GorCzyński, Johannes haybaeCk et al.
reactive, and in 3 cases immunoexpression was very 
strong (h-score 170-200). 
These results increase the group of tumors with 
neuroendocrine differentiation and at the same time 
make this marker even less specific for the diagnosis 
of pancreatic neuroendocrine tumors than previous-
ly thought. Positive reaction with this antibody does 
not rule out and does not confirm the diagnosis of 
ONB. This can be of diagnostic value in metastases 
of neuroendocrine neoplasm to the sinonasal area. We 
do not know the expression of this marker in ONB 
metastases, but probably it may also be misleading 
under these clinical circumstances. 
 To sum up, ISL-1 can show a positive reaction in 
ONB and should be taken into consideration in the 
differential diagnosis of neuroendocrine tumors of the 
sinonasal area. 
We did not observe a statistically significant cor-
relation in expression of PAX5, ISL-1 and TTF-1 in 
our cohort of ONB. However, we detected a trend 
toward a moderate correlation between expression of 
PAX5 and TTF-1. The lack of significant correlation 
may be due small number of cases. It cannot be ex-
cluded that no relationship exists. On the other hand, 
this may be a real connection which could point to 
a common role of TTF-1 and PAX5 in biological ac-
tivity and neoplastic transformation of these two pro-
teins. However, no data confirming this dependency 
in neuroendocrine or any other tumor exist. This as-
pect requires further studies on larger ONB cohorts 
and in other neuroendocrine tumors.
Conclusions
Our results shed light on the diagnostic role of 
ISL-1, PAX5 and TTF-1 in ONB. Frequent ISL-1 
positivity in ONB increases the spectrum of ISL-1 
positive tumors with neuronal and neuroendocrine 
differentiation and proves that ISL-1 should not be 
considered as a marker of pancreatic origin in pan-
creatic tumors.
TTF-1 positivity does not exclude the diagnosis of 
ONB. PAX5 expression is infrequent in ONB, but 
strong reactivity can be associated with high grade 
tumors and very aggressive clinical course with rapid 
skeletal dissemination. 
PAX5 and TTF-1 show a trend toward a correla-
tion in expression which might be secondary to a yet 
Fig. 3. Cohesive nests of olfactory neuroblastoma cells (A, HE, magnification 100×; B, HE, magnification 200×) with 
strong expression of PAX5 (C, magnification 100×; D, magnification 200×)
A
C
B
D
135
ISL-1, TTF-1 and PaX5 In oLFacTory neurobLaSToma
undescribed molecular cooperation during the pro-
cess of neoplastic transformation of cells.
The authors declare no conflict of interest.
This study was supported by the ST-95 grant from the 
Medical University of Gdansk. 
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Address for correspondence 
Piotr Czapiewski
Department of Pathomorphology 
Medical University of Gdańsk 
Dębinki 7  
80-952 Gdansk, Poland
tel. +48 58 349 27 40 
e-mail: czapiewskipiotr@gumed.edu.pl


Original paper expressiOn pattern Of isl-1, ttf-1 and pax5 in OlfactOry neurOblastOma

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Original paper expressiOn pattern Of isl-1, ttf-1 and pax5 in OlfactOry neurOblastOma

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