2016, Volume 32, Number 2, Page(s) 099-104
The Relationship Between Apoptotic Activity and Prognostic Factors in Neuroblastomas
Sümeyye EKMEKCİ1, Nur OLGUN2, Erdener ÖZER1
1Department of Pathology, Dokuz Eylul University, School of Medicine, İZMİR, TURKEY
2Department of Pediatric Oncology, Dokuz Eylul University, School of Medicine, İZMİR, TURKEY
Keywords: Apoptosis, Bcl-2, Neuroblastoma, Prognosis
Prognostic parameters in determining risk groups for treatment in neuroblastoma are cellular differentiation, mitosis karyorrhexis
index (MKI), N-myc amplification and age. However, additional prognosticators are still needed because patients can show unpredictable
biological behavior. We aimed to study the prognostic significance of apoptotic activity in neuroblastomas.
Material and Method: Thirty-five primary neuroblastoma were evaluated. The data including stage, treatment protocol, metastatic disease,
survival, N-myc status, age and prognostic categorization were obtained from the clinical records. The differentiation and MKI were evaluated in
hematoxylin and eosin stained slides. Apoptotic activity was assessed by both bcl-2 immunohistochemical staining and the transferase-mediated
d-UTP-biotin nick end labeling (TUNEL) method. Data were correlated with established prognostic factors and clinical outcome.
Results: Twenty-five (71.4%) cases were located in the adrenal. Sixteen cases showed low and 19 high MKI. Thirty-three (%94) were
immunopositive for bcl-2. TUNEL staining was negative in 2 cases. Of the remaining positive 33 cases, 14 had an apoptotic index of ≤2%, 11
of 2-4% and 8 of ≥4%. Cases located in the adrenal showed higher scores of bcl-2 positivity than extra-adrenal tumors. There was no statistical
significance for both bcl-2 staining and apoptotic index in correlation with cellular differentiation, MKI, N-myc amplification, age and other
clinical parameters. Statistical significance was observed between bcl-2 scoring and tumor localization.
Conclusion: According to our results, apoptotic activity is unlikely to be a prognostic parameter in neuroblastomas. Some studies showing
significant correlations between clinical outcome and both bcl-2 immunoscoring and apoptotic index, as assessed by the TUNEL method,
differences in case numbers and methodology can explain these conflicting results. Larger series and different methodologies are needed to
evaluate the prognostic value of apoptotic activity in neuroblastomas.
Peripheral neuroblastic tumors including neuroblastomas
(NBs) are the most common extracranial solid tumors in
childhood and infancy that make up approximately 15%
of all tumors within the first four years of childhood1,2
The common localizations are adrenal medulla (40%) and
sympathetic ganglia of abdominal (25%), thoracic (15%),
cervical (5%) and pelvic (5%) sites3
NBs manifest themselves in a variety of clinical conditions
ranging from spontaneous regression and maturation to
aggressive progression1. The prognosis depends on the
patients age at the time of diagnosis, presence of metastasis,
histological subtypes, and genetic transformations in the
tumor tissue2. Prognostic histological parameters include
Schwannian stroma percentage, neuroblastic differentiation
and mitosis karyorrhexis index (MKI)1. The most
common genetic anomaly in NB is N-myc amplification
and deletion of the short arm of 1st chromosome4,5.
Although there are established prognostic parameters for
NBs, additional prognosticators are still needed because
patients can show unpredictable biological behavior.
Apoptosis plays a significant role in tumor formation
when it fails to regulate tissue homeostasis and causes an
imbalance between cell death and proliferation. Because
anticancer therapies aim to kill tumor cells through
induction of apoptosis, failure to induce apoptosis may
cause tumor resistance to cancer drugs administered in
Assessment of proteins playing a crucial role in molecular
events of apoptosis may enable finding additional
prognosticators in NBs. Bcl-2 is an oncoprotein with
antiapoptotic and proapoptotic functions and plays a
significant role in regulating apoptosis. Bcl-2 protein
inhibits apoptosis in two different ways: directly inhibiting
pro-caspases or inhibiting the degradation of apoptogenic
Several methods have been used to investigate apoptotic
activity. Of these methods, transferase-mediated d-UTPbiotin
nick end labeling (TUNEL) method enables
demonstrating DNA fragmentation in an in-situ
examination. The basic principle of this method is to insert
or add marked nucleotides to a single and/or doublestranded
DNA breakage7. In this study, we aimed to
evaluate the prognostic significance of apoptotic activity
in NBs by using the TUNEL method to assess apoptotic
activity and also the immunohistochemical method to
evaluate bcl-2 protein expression.
The present study comprised 35 NB patients
diagnosed and treated at the Dokuz Eylul University
Hospital. The clinical parameters such as age, gender,
tumor location, clinical tumor stage, presence of distant
metastasis, N-myc amplification status, treatment scheme,
prognosis and survey were determined from the patients
Histopathological examination: The archival hematoxylin
and eosin stained sections were reviewed by two pathologists
(SE and EO) and the differentiation and MKI of tumor cells
The tumors were subtyped into three groups according
to the degree of differentiation: undifferentiated, poorly
differentiated, or differentiating NB. If the tumor cells
did not show any cytological features indicative of
differentiation, such as vesicular appearance of nucleus,
development of nucleolus, and enlargement of eosinophilic
or amphophilic cytoplasm, a diagnosis of undifferentiated
NB was made, or if less than 5% of the tumor cells showed
features that were indicative of intermediate cells or
ganglion cells, a diagnosis of poorly differentiated NB was
made. If 5% or more tumor cells showed features indicative
of differentiation, a diagnosis of differentiating NB was
To evaluate MKI, 5000 tumor cells were counted in 10
or higher power fields depending on the cellularity. Sites
that included necrosis, hemorrhage, and autolysis were
excluded. The cases were categorized as low MKI if it was
less than 2%, moderate if 2-4% and high if more than 4%8.
Bcl-2 immunhistochemistry: The sections were cut from
archival tissue blocks and stained with Bcl-2 antibody
(Ventana, 1/50 dilution). The immunohistochemical
staining was carried out in Ventana BenchMark XT
IHC/ISH Staining Module using XT ultraView DAB v3 procedures. Lymph node tissue was used as a positive
control. Bcl-2 staining was classified as negative, when
positive cells were less than 5%. The positive cases were
considered as 1+ when the percentage of positive cells was
5-75% and 2+ when more than 75%9,10.
TUNEL staining: The sections were cut from archival tissue
blocks, stained using Millipore ApopTag® Plus Peroxidase
In Situ Apoptosis Detection Kit S7101 and underwent
the routine TUNEL protocol7. Lymph node tissue was
used as a positive control. The apoptotic index (AI) was
determined in hot spot areas by counting 1000 cells and
calculating the percentage of positive tumor cells. The cases
were categorized as low AI if less than 2%, moderate if 2-4%
and high if more than 4%.
Statistical Analysis: Interobserver variability between two
researchers was assessed by using Kappa test. The statistical
relation for both Bcl-2 immunoscores and TUNEL AI with
morphologic, genetic and clinical parameters was evaluated
by performing chi-square test and Mann Whitney U test. A
p-value of < 0.05 was considered statistically significant.
Overall 35 cases of NB were enrolled into the study. The
ages at the time of diagnosis ranged from 1 to 26 years
with a median age of 4.1 years. Nine (25.7%) patients were
younger than 18 months, 19 (54.3%) between 18 months
and 5 years old and 6 (17.1%) older than 5 years old with
one case of unknown age (2.9%). Nineteen (54.3%) patients
were female and 16 (45.7%) male. Twenty-five (71.4%)
cases were located in the adrenal, and 10 (28.6%) in the
extra-adrenal organs (Table I
Four (11.4%) cases were stage I, two (5.7%) stage II, seven
(20%) stage III, 15 (42.9%) stage IV and one (2.9%) stage
IVS. Fourteen (40%) patients received radiotherapy,
whereas 26 (74.3%) cases had chemotherapy. All the
patients who received radiotherapy also had chemotherapy.
We could not reach the data regarding whether five of the
patients had received chemotherapy or not.
The median survival time of the cases was 30 months. Six
cases (17.1%) died of disease and 29 cases (82.8%) were
alive whilst 27 of them had no recurrence and 2 of the alive
patients (5.7%) were being followed up by different centers.
Table II demonstrates the histopathological findings. The
Kappa statistics revealed a substantial agreement (Kappa
value = 75%) between two investigators (EÖ and SE).
The final scores in the disagreement cases were decided
after two observers reached a consensus after reviewing
the cases together. The histological examination revealed that 12 (34.3%) cases were undifferentiated, 13 (37.1%)
poorly differentiated and 10 (28.6%) differentiated. Sixteen
(45.7%) cases had low MKI and the remaining 19 (54.3%)
cases high MKI. A genetic analysis was conducted in only
22 patients. Of those, six (17.1%) patients had positive
N-myc amplification and 16 (45.7%) were negative (Table
The bcl-2 score was negative in two (5.7%) patients, 1(+)
in six (17.1%) and 2(+) in 27 (77.1%) (Figure 1, 2). AI was
low in 14 (40%) patients, moderate in 11 (31.4%) and high
in 8 (22.8%) (Figure 3, 4). TUNEL staining, which was used
to measure AI, was negative in two cases (5.7%) (Table II).
There was no statistically significant difference for
bcl-2 staining and TUNEL AI in correlation with age,
differentiation, MKI, N-myc amplification, stage, treatment
protocol and prognosis. A statistically significant difference was observed between bcl-2 scoring and tumor localization
(p=0.04). NB cases located in the adrenal showed higher
scores of bcl-2 positivity compared to extra-adrenal tumors
(88% versus 50%). AI assessed by TUNEL was found to be
significantly lower in girls (p=0.01). Low AI was found in
17 (89.5%) female cases in contrast to 8 (50%) males (Table
Click Here to Zoom
|Table III: Apoptotic index and bcl-2 immunohistochemical
scores for statistically significant parameters
Neuroblastomas are clinically evident and biologically
heterogeneous tumors. While advanced stage NBs are
likely to metastasize, stage IVS tumors are regressed. In
addition some early stage tumors may have a bad prognosis4
. When localized cases (stage I-III) undergo surgical resection after adjuvant chemotherapy, the 10-year survival
rate may increase to 80%. On the other hand, the 20-year
survival rate is not more than 20% in metastasized cases
(stage IV) even after aggressive chemotherapy. Established
histological parameters and biochemical test results cannot
explain these biological divergences and the worse prognosis
is often explained with molecular test results11
N-myc amplification is characterized by a worse prognosis,
NBs with negative N-myc amplification may have a better
. Therefore different parameters are
still needed to explain the different biological behaviors
and define the risk groups for treatment protocols.
Apoptosis is a one of the key mechanisms for tumor
development in NBs as in many other tumors. This fact has
been already evaluated in several studies in the literature.
There are several factors that activate and inhibit apoptosis.
The Bcl-2 family is a protooncogene situated outside the
mitochondrial membrane and endoplasmic reticulum.
Members of the Bcl-2 family serve to sustain cell life and
apoptosis of cells. Expression of bcl-2 normally exists in
many lymphoid tissues and epithelium cells as well as in
the majority of malignancies6,9,13.
Many investigators have so far studied the Bcl-2 family
and the significance of bcl-2 expression on prognosticators
of NBs such as histological differentiation, stage, N-myc
amplification, prognosis, and treatment. Reed et al.14 suggested that increased expression of Bcl-2 in NBs
prevented apoptosis and prolonged cellular life cycle as
well as being able to induce cellular death. In addition,
high levels of bcl-2 expression may result in poor response
to treatment interacting with many chemotherapeutics
with cellular death mechanisms. Castle et al. suggested a strong correlation between high bcl-2 expression and both
poor differentiation and advanced stage15. In a study by
Hoehner et al., there was a similar correlation between poorly
differentiated NB cells and cells with higher immunoscores
for bcl-2 immunohistochemistry16. Similarly Mejia et al.
reported that increased Bcl-2 expression was significantly
correlated with advanced tumor stage9.
In our study, 33 of the 35 cases were bcl-2 positive. We
found that NBs located in the adrenal had significantly
higher immunoscoring for bcl-2 positivity than extraadrenal
NBs. There are no data in the literature showing a
prognostic difference between adrenal NBs and others with
extra-adrenal locations to the best of our knowledge and the
correlation between the primary tumor site and biological
behavior still remains unexplained. Furthermore, it is
interesting to note that cervical NBs are rarely correlated
with distant metastasis. In contrast, if the primary site of the
tumor is abdomen, 90% of the patients become metastatic17.
In contrast to findings in the relevant literature13-16,18,
our study did not reveal any statistically significant relation
between bcl-2 expression and established prognostic
factors in NBs such as cellular differentiation, tumor stage
and N-myc amplification. This contrast may be explained
by the different numbers of patients enrolled in the studies
and different methodologies used for investigating bcl-2
expression. In addition, cumulative data obtained from
studies investigating different proteins and genes involved
in the regulation of apoptosis are needed. For example
Adida et al. carried out a study to investigate expression
of survivin, an antiapoptotic gene, and found a correlation
between its expression and progression of the disease19.
Besides bcl-2 immunohistochemistry, we also assessed
AI with the TUNEL method in our study and TUNEL
staining was positive in most cases (94.3%). AI was low
in 40% of the patients, moderate in 31.4% and high in
22.8%. We did not find any statistically significant relation
between AI and established prognostic factors and other
parameters. However, AI was significantly lower in the
tumors of female patients. Because of the limited number
of cases enrolled in the study, further studies using survival
and multivariate analysis are needed to investigate the
prognostic significance of apoptotic activity in NB.
In a study by Diensthuber et al., the percentage of TUNEL
positivity in NB was similar. Similar to our study, they did
not suggest a correlation between AI and prognosis, age,
and gender20. The increased sensitivity of the TUNEL
method in comparison with other apoptosis detection methods results from its ability to demonstrate apoptotic
cells in micro-sizes that histochemical methods or
sophisticated DNA methods commonly fail to demonstrate.
Mejia et al. analyzed apoptotic activity and chromosomal
breakages in NBs using in situ methods9. They conducted
their study with 111 cases. The mean age of the patients
was 2.1 years and the majority of the patients were male.
In contrast, the number of patients in our study was 35 and
the mean age 4.1 years. Most of our patients were female.
These characteristic differences between the two studies
may explain the divergent results.
In summary, based on our results we conclude that
apoptotic activity assessed by TUNEL staining and bcl-
2 immunohistochemistry is unlikely to be a prognostic
parameter in NBs. However further studies with larger
series and different methodologies are needed to evaluate
the prognostic significance of apoptotic activity in NBs and
establish it as an evidence-based prognosticator.
1) Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B,
Stram DO, Gerbing RB, Lukens JN, Matthay KK, Castleberry RP.
The International Neuroblastoma Pathology Classification (the
Shimada system). Cancer. 1999;86:364-72.
2) Schroeder H, Wacher J, Larsson H, Rosthoej S, Rechnitzer C,
Petresen BL, Carlsen NL. Unchanged incidence and increased
survival in children with neuroblastoma in Denmark 1981-2000:
A population-based study. Br J Cancer. 2009;100:853-7.
3) Joshi VV. Peripheral neuroblastic tumors: Pathologic
classification based on recommendations of international
neuroblastoma pathology committee (Modification of shimada
classification). Pediatr Dev Pathol. 2000;3:184-99.
4) Hiyama E, Hiyama K, Ohtsu K, Yamaoka H, Ichikawa T, Shay
JW, Yokoyama T. Telomerase activity in neuroblastoma: Is
it a prognostic indicator of clinical behaviour?. Eur J Cancer.
5) Schwab M, Westermann F, Hero B, Berthold F. Neuroblastoma:
Biology and molecular and chromosomal pathology. Lancet
6) Tsujimoto Y. Role of Bcl-2 family proteins in apoptosis:
Apoptosomes or mitochondria? Genes Cells. 1998;3:697-707.
7) Kressel M, Groscurth P. Distinction of apoptotic and necrotic cell
death by in situ labelling of fragmented DNA. Cell Tissue Res.
8) Shimada H, Umehara S, Monobe Y, Hachitanda Y, Nakagawa
A, Goto S, Gerbing RB, Stram DO, Lukens JN, Matthay KK.
International neuroblastoma pathology classification for
prognostic evaluation of patients with peripheral neuroblastic
tumors: A report from the Childrens Cancer Group. Cancer.
9) Mejia C, Navarro S, Llombart-Bosch A. Apoptosis in peripheral
neuroblastic tumors. Immunohistochemical expression of bcl-2
and p53 is related to DNA fragmentation. Histol Histopathol.
10) Ikeda H, Hirato J, Akami M, Matsuyama S, Suzuki N, Takahashi
A, Kuroiwa M. Bcl-2 oncoprotein expression and apoptosis in
neuroblastoma. J Pediatr Surg. 1995;30:805-8.
11) Poremba C, Willenbring H, Hero B, Christiansen H, Schäfer KL,
Brinkschmidt C, Jürgens H, Böcker W, Dockhorn-Dworniczak
B. Telomerase activity distinguishes between neuroblastomas
with good and poor prognosis. Ann Oncol. 1999;10:715-21.
12) Hiyama E, Hiyama K, Yokoyama T, Ishii T. Immunohistochemical
analysis of N-myc protein expression in neuroblastoma:
Correlation with prognosis of patients. J Pediatr Surg.
13) Negrini M, Silini E, Kozak C, Tsujimoto Y, Croce CM. Molecular
analysis of mbcl-2: structure and expression of the murine gene
homologous to the human gene involved in follicular lymphoma.
14) Reed JC, Meister L, Tanaka S, Cuddy M, Yum S, Geyer C,
Pleasure D. Differential expression of bcl2 protooncogene in
neuroblastoma and other human tumor cell lines of neural
origin. Cancer Res. 1991;51:6529-38.
15) Castle VP, Heidelberger KP, Bromberg J, Ou X, Dole M, Nuñez
G. Expression of the apoptosis-suppressing protein bcl-2, in
neuroblastoma is associated with unfavorable histology and
N-myc amplification. Am J Pathol. 1993;143:1543-50.
16) Hoehner JC, Hedborg F, Wiklund HJ, Olsen L, Påhlman S.
Cellular death in neuroblastoma: In situ correlation of apoptosis
and bcl-2 expression. Int J Cancer. 1995;62:19-24.
17) Cheung NKV, Cohn SL. Neuroblastoma. 1st ed. Berlin,
Heidelberg, NewYork:Springer; 2005
18) del Carmen Mejía M, Navarro S, Pellín A, Ruíz A, Castel V,
Llombart-Bosch A. Study of proliferation and apoptosis in
neuroblastoma. Their relation with other prognostic factors.
Arch Med Res. 2002;33:466-72.
19) Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC.
Anti-apoptosis gene, survivin, and prognosis of neuroblastoma.
20) Diensthuber M1, Potinius M, Rodt T, Stan AC, Welkoborsky HJ,
Samii M, Schreyögg J, Lenarz T, Stöver T. Expression of bcl-2 is
associated with microvessel density in olfactory neuroblastoma. J