2022, Volume 38, Number 2, Page(s) 099-105
The Significance of Histopathologic Assessment in Bone Marrow Disease in Neuroblastoma
Sumeyye EKMEKCI1, Dilek INCE2, Nur OLGUN2, Erdener OZER3
1Department of Pathology, University of Health Sciences, Izmir Tepecik Research and Training Hospital, IZMIR, TURKEY
2Department of Clinical Oncology, Dokuz Eylul University, Institute of Oncology, IZMIR, TURKEY
3Department of Pathology, Dokuz Eylul University, School of Medicine, IZMIR, TURKEY
Keywords: Bone marrow biopsy, Neuroblastoma, Metastasis, Tumor differentiation, Tumor percentage
Neuroblastoma (NB) is the most common extracranial solid tumor in children and is responsible for 12% of cancer-related deaths.
The status of metastatic disease in the bone marrow (BM) is a predictor of poor outcome. The purpose of this study was to investigate the
predictive significance of histopathological examination of BM in NB.
Material and Method: The study included 61 cases with archival bone marrow biopsy tissues. The cases were evaluated regarding the percentage
of metastatic tissue and its differentiation. Primary tumor slides were also reviewed to perform the Shimada classification based on the
differentiation status and mitosis-karyorrhexis index. The patientsí age, gender, NMYC amplification, clinical risk group, and disease outcome
were also noted.
Results: Of the 61 cases, 17 had BM involvement. Of those, eight cases (47.1%) were refractory NB showing disease relapse. Based on BM
examination, five cases (29.4%) were categorized as complete response, seven (41.2%) as progressive disease, three (17.6%) as minimal disease,
and two (11.8%) as stable disease. The progressive disease category was significantly related with refractory disease and NMYC amplification
along with the high-risk category (p =0.002 and p= 0.003 respectively). Undifferentiated histology and presence of more than 20% of tumor tissue
in the BM biopsy at diagnosis were significantly associated with the progressive disease category (p=0.01 and p<0.001, respectively).
Conclusion: We conclude that evaluating the percentage of metastatic tumor tissue and tumor differentiation in BM biopsies is of clinical
importance in the management of neuroblastoma patients.
Neuroblastomas (NB) are the most common extracranial
solid tumors in children less than 15 years of age 1-9
They are responsible for approximately 15% of cancerrelated
deaths in this age group 2-10
. There are established
parameters in the risk stratification of NB patients such
as tumor differentiation and mitosis-karyorrhexis index
(MKI), NMYC amplification, the patientís age, and tumor
. According to the International Neuroblastoma
Risk Group (INRG) staging system, metastasis at the time
of diagnosis is considered a significant indicator of poor
. If the tumor has spread to distant sites such
as the lymph nodes, bone, liver, skin, bone marrow (BM),
or other organs, it meets the criteria for stage 4. BM is the
most common metastatic site in NB patients at the time of
diagnosis and one of the common localizations of disease
In the past few decades, a significant improvement has
been observed in the prognosis of certain well-defined NB
patient subsets, whereas only a modest improvement has
been reported in the prognosis of children of the highrisk
groups 15. The presence of tumor tissue in the BM
corresponds to not only advanced stage but also a high-risk
category. NB patients with bone marrow involvement carry
a high risk of refractory disease and are likely to show a poor
prognosis. Therefore BM aspiration and biopsy are quick,
easy and cost-effective methods and have become routine
standard procedures to evaluate the prognosis in newly
diagnosed patients and assess disease response without
having to wait for greater tumor burden to develop 1,10.
The information achieved by bilateral trephine BM biopsy
interpretation is also valuable for the clinical follow-up
strategy 1. We aimed in the present study to address the
clinical importance of BM involvement in the management
of NB patients, and also to test the significance of established
histopathological criteria in predicting the prognosis.
The study initially included 86 NB cases diagnosed and
treated at our institution. Of those, 25 cases were excluded
because archival bilateral BM trephine biopsy materials were
unavailable. Histological slides of the remaining 61 patients
were reviewed and tumor involvement at diagnosis and
during follow-ups was confirmed in 17 cases. These cases
were categorized according to the percentage of metastatic
tumor by two expert pathologists (EO, SE), as proposed in
the literature 1,16
. Additional sections of hematoxylin
and eosin (H&E)-stained and immunostained slides with
at least three antibodies (synaptophysin, chromogranin and
PGP 9.5) were used to determine the amount of tumor 1,17,18
. The surface area involved by the neuroblastic tumor
was given as a percentage in the BM tissue. Based on the
percentage of tumor tissue in BM biopsy at diagnosis and
repeated biopsies during follow-ups, the disease status of
the cases was categorized according to the criteria in Table
. The differentiation status of the tumor in BM at diagnosis
was grouped as well-differentiated, poorly-differentiated
and undifferentiated, along with the percentage of the
tumor tissue as <5%, 5-20% and >20% (Figure 1A-D
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|Table I: The categorization of response status based on bone marrow involvement.
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|Figure 1: Neuroblastoma metastasis in bone marrow biopsy (involvement ratio: 15%). H&E, A x40, B x100, C x400, D: synaptophysin
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|Figure 2: Neuroblastoma infiltration in bone marrow biopsy (involvement ratio: 70%). H & E; A x40, B x100, C x400, D: synaptophysin
Archival histological slides of the primary tumor tissues
of 17 patients were also reviewed by two pathologists
(EO, SE) to assess the Shimada category based on tumor
differentiation and MKI. Demographic data such as
patientsí age and gender, clinical data including tumor
NMYC amplification status, stage, patient risk group and
refractory status were obtained from the patient records.
Clinical and demographic characteristics of the patients
were defined by using descriptive statistics. The statistical
relationship of both BM metastatic tumor percentage
category and differentiation with clinical and demographic
characteristics was evaluated with the chi-square test. A probability level of 0.05 or less was chosen to represent
statistical significance. All p-values were two-sided and
denoted by p. Fisherís exact test was used to calculate
p values, as the cell frequencies were too small for the
standard chi-squared test to be accurate.
Approval for the study was granted by the Local Medical
Ethics Committee (approval No. 2021/5-2). All study
procedures were performed according to the Declaration
of Helsinki principles.
A total of 86 neuroblastoma cases were examined for the
study, and 61 cases had BM data. Of the 61 cases, 17 had
BM involvement. Eleven of these cases were boys, and six
were girls. Nine patients were older than 5 years old, one
younger than 18 months, and seven between 18 months
and 5 years old. Sixteen (54.1%) cases were stage IV and one
case (5.9%) was stage IVS. Eleven patients (64.7%) were in
the high-risk category and six (35.3%) in the intermediate
risk group. MYCN amplification was detected in 11 (64.7%)
cases. The median follow-up was 28 months. Refractory
disease was observed in eight (47.1%) of the cases.
Histologically, four tumors were undifferentiated NBs,
and 13 were poorly differentiated. Four cases had low, one
case had moderate MKI and 12 cases had high MKI. All
tumors had unfavorable Shimada histology. Of the tumors
with BM involvement, five (29.4%) were undifferentiated
NBs and 12 (73.6%) were poorly differentiated. There
were eight cases (47.1%) with BM involvement less than
5%, three (17.6%) with 5-20%, and six (35.3%) with more
than 20%. Based on BM involvement criteria, seven cases
(41.2%) were progressive disease, five (29.4%) complete
response, three (17.6%) minimal disease, and two (11.8%)
Table II demonstrates the statistical relationship between
BM disease status and the prognostic parameters. There
was no statistically significant relationship between disease
status and gender, age, tumor differentiation and MKI.
A statistically significant relationship was found between
progressive disease and BM involvement percentage more
than 20% (p<0.001), lack of differentiation of the tumor
(p=0.001), relapse (p=0.002), high-risk category (p=0.003),
and N-MYC amplification (p=0.003). Progressive disease
and NMYC amplification were significantly related with
refractory disease (p=0.002 and p=0.003 respectively).
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|Table II: The relation between bone marrow status and clinicopathological characteristics of the study group.
In the evaluation of NBs, the International Neuroblastoma
Staging System (INSS) defines these tumors by resectability
and tumor location in relation to the presence or absence of
regional or distant metastatic spread 17,19,20
. According to this definition, the stage of the disease, the age of the
patient and the tumor biological factors form the basis
of the therapeutic risk classification 19
. When the case
distribution in the risk classification of NB is taken account,
about half of the cases have a high-risk phenotype and a
low survival. Five-year survival is 30-40% in the high-risk
group, whereas NBs in the low or intermediate risk group
have excellent survival 21
Almost half of NBs have metastases at the time of
diagnosis 3,17. Although the most common localization
of metastases is the BM, metastases can be observed in
the skeleton, lymph nodes, liver, and intracranial and
orbital regions 3,4,6,7,17,21,22. Despite aggressive
multimodality therapy, the long-term survival rate at
diagnosis of metastatic disease for patients older than 18
months is still less than 30% 15.
BM is the major site of metastasis in advanced
neuroblastoma, and detection of even minimal residual
neuroblastoma cells in this region is associated with a poor
prognosis 9,17. However, the potential for metastatic
spread may not be equal among all patients with NB
19. Although it is a well-known tumor with its different
behaviors and the connections between these behaviors and molecular patterns, the metastatic propagation
mechanisms of NBs are not fully understood. Studies have
shown that some tumors have a higher risk of metastatic
spread than others as a result of searching the factors
associated with metastatic spread and BM infiltration 19.
Since the determination of BM metastasis is a key point in
evaluating the course of NB, core biopsies are frequently taken as part of routine bone marrow sampling in pediatric
patients in many institutions. An advantage of this sampling
is that it allows the use of immunohistochemistry on these
samples for detection of tumor cells 17. Previous studies
have shown that immunohistochemical analysis is superior
to routine H&E assessment 17 and it is recommended to
use at least three immunohistochemical markers such as
synaptophysin, chromogranin, PGP 9.5 and cyclin-D1 to
demonstrate tumor tissue 1,13,17. Moreover, neuroblastic
differentiation and the percentage of tumor tissue should
be stated in the pathology report 1.
Amplification of the MYCN gene is observed in
approximately 20-35% of all NBs and is an important
molecular marker for identifying high-risk patients
3,4,14. The combination of MYCN overexpression and
caspase-8 deletion significantly increases BM metastasis 3,4. However, it has been reported that BM metastasis is
associated with poor prognosis in patients older than 1 year,
regardless of MYCN amplification 10,13. In addition,
BM status beyond other known predictive factors such as
MYCN copy number was shown to provide independent
prognostic information for patients over the age of 1 and
with stage 4 NBs 18. In a study evaluating the MYCN
status by using FISH and amplification, BM involvement
was reported in 6% of the cases. In this study, all cases were
high-risk and stage 4, and the criterion for positive BM
involvement was the presence of neuroblastoma in at least
20% of the biopsy 14.
In the present study, the majority of the cases were
in the high-risk group older than 18 months. MYCN
amplification was observed in 70% of the cases with BM
metastasis. In a study by Jo et al. 8, similar results were reported. Kuroda et al. 13 investigated the presence of
circulating tumor cells (CTC) in the peripheral blood and/
or BM micrometastases during treatment in advanced stage
NB cases receiving chemotherapy. MYCN amplification
was detected in 12 of these cases. However, neither CTC
nor persistent BM micrometastases were associated with
MYCN amplification. In our study, a statistically significant
relationship between progressive disease based on BM
involvement and NMYC amplification was found.
In our study, BM involvement over 20% and
undifferentiated histology were related to refractory
disease. In a study by Russell et al. 19, low-stage NB was
unlikely to have metastatic disease in the BM, and therefore
they recommended further investigation of the genetic
factors such as MYCN amplification and chromosome
1p deletion in order to better predict which tumors are
at risk of metastatic spread. In contrast, the presence of
permanent BM micrometastasis after chemotherapy was
reported as a predictor of poor prognosis 13. In addition
MYCN amplification should be evaluated both in the
primary tumor and BM metastases although the tumor may
show genetic heterogeneity. It should be kept in mind that
genetic differences that can be found in the primary tumor,
and the metastatic tumor may also affect the treatment
In the study by Tian et al. 10, the presence of BM
metastasis was seen in 75% of the cases at the time of
diagnosis and 45% of the cases after treatment. Five-year
event-free survival was found to be statistically different
in patients with residual metastatic disease compared to
patients without 15. In our study, a statistically significant
relationship was found between the presence of more than
20% of tumor tissue in the BM biopsy at diagnosis and
progressive disease. In conclusion, BM metastasis status at
diagnosis and during follow-up appears to be a significant
prognostic factor apart along with poor prognostic
factors. The crucial cut-off point for BM involvement is
20% and a value over this ratio is significantly related to
refractory disease. Our study demonstrates the prognostic
and predictive significance of determining the tumor
percentage and differentiation of NBs in the BM biopsies at
diagnosis and during follow-up.
CONFLICT of INTEREST
The authors have no conflicts of interest to declare.
The authors have declared that they did not receive any
financial support for this study.
Concept: SE, EO, Design: SE, EO, Data collection or
processing: SE, DI, NO, EO, Analysis or Interpretation: SE,
DI, NO, EO, Literature search: SE, EO, Writing: SE, EO,
Approval: SE, DI, NO, EO.
1) Burchill SA, Beiske K, Shimada H, Ambros PF, Seeger R, Tytgat
GA, Brock PR, Haber M, Park JR, Berthold F. Recommendations
for the standardization of bone marrow disease assessment
and reporting in children with neuroblastoma on behalf of the
International Neuroblastoma Response Criteria Bone Marrow
Working Group. Cancer. 2017;123:1095-105.
2) Park JR, Bagatell R, Cohn SL, Pearson AD, Villablanca JG,
Berthold F, Burchill S, Boubaker A, McHugh K, Nuchtern JG,
London WB, Seibel NL, Lindwasser OW, Maris JM, Brock P,
Schleiermacher G, Ladenstein R, Matthay KK, Valteau-Couanet
D. Revisions to the international neuroblastoma response
criteria: A consensus statement from the national cancer institute
clinical trials planning meeting. J Clin Oncol. 2017;35:2580-7.
3) Morandi F, Corrias MV, Pistoia V. Evaluation of bone marrow
as a metastatic site of human neuroblastoma. Ann N Y Acad Sci.
4) Teitz T, Inoue M, Valentine MB, Zhu K, Rehg JE, Zhao W,
Finkelstein D, Wang YD, Johnson MD, Calabrese C, Rubinstein
M, Hakem R, Weiss WA, Lahti JM. Th-MYCN mice with
caspase-8 deficiency develop advanced neuroblastoma with bone
marrow metastasis. Cancer Res. 2013;73:4086-97.
5) Ekmekci S, Olgun N, ÷zer E. The relationship between apoptotic
activity and prognostic factors in neuroblastomas. Turk Patoloji
6) Osman J, Galli S, Hanafy M, Tang X, Ahmed A. Identification
of novel biomarkers in neuroblastoma associated with the risk
for bone marrow metastasis: A pilot study. Clin Transl Oncol.
7) Yoshino K, Tanabe M, Ohnuma N, Takahashi H. Histopathologic
analysis of bone marrow and bone metastasis in murine
neuroblastoma. Clin Exp Metastasis. 1996;14:459-65.
8) Jo JH, Ahn SD, Koh M, Kim JH, Lee SW, Song SY, Yoon SM, Kim
YS, Kim SS, Park JH, Jung J, Choi EK. Patterns of recurrence after
radiation therapy for high-risk neuroblastoma. Radiat Oncol J.
9) Ulrich H, Ratajczak MZ, Schneider G, Adinolfi E, Orioli
E, Ferrazoli EG, Glaser T, CorrÍa-Velloso J, Martins PCM,
Coutinho F, Santos APJ, Pillat MM, Sack U, Lameu C. Kinin and
purine signaling contributes to neuroblastoma metastasis. Front
10) Tian X, Cao Y, Wang J, Yan J, Tian Y, Li Z, Wang H, Duan X, Jin
Y, Zhao Q. A single center clinical analysis of children with highrisk
neuroblastoma. Oncotarget. 2017;8:30357-68.
11) Cohn SL, Pearson AD, London WB, Monclair T, Ambros PF,
Brodeur GM, Faldum A, Hero B, Iehara T, Machin D, Mosseri
V, Simon T, Garaventa A, Castel V, Matthay KK; INRG Task
Force. The International Neuroblastoma Risk Group (INRG)
classification system: An INRG Task Force report. J Clin Oncol.
12) Popov A, Druy A, Shorikov E, Verzhbitskaya T, Solodovnikov
A, Saveliev L, Tytgat GAM, Tsaur G, Fechina L. Prognostic value
of initial bone marrow disease detection by multiparameter flow
cytometry in children with neuroblastoma. J Cancer Res Clin
13) Kuroda T, Morikawa N, Matsuoka K, Fujino A, Honna T,
Nakagawa A, Kumagai M, Masaki H, Saeki M. Prognostic
significance of circulating tumor cells and bone marrow
micrometastasis in advanced neuroblastoma. J Pediatr Surg.
14) Yue ZX, Huang C, Gao C, Xing TY, Liu SG, Li XJ, Zhao Q, Wang
XS, Zhao W, Jin M, Ma XL. MYCN amplification predicts poor
prognosis based on interphase fluorescence in situ hybridization
analysis of bone marrow cells in bone marrow metastases of
neuroblastoma. Cancer Cell Int. 2017;17:43.
15) Cai JY, Pan C, Tang YJ, Chen J, Ye QD, Zhou M, Xue H,
Tang JY. Minimal residual disease is a prognostic marker for
neuroblastoma with bone marrow infiltration. Am J Clin Oncol.
16) Monclair T, Brodeur GM, Ambros PF, Brisse HJ, Cecchetto G,
Holmes K, Kaneko M, London WB, Matthay KK, Nuchtern JG,
von Schweinitz D, Simon T, Cohn SL, Pearson AD; INRG Task
Force. The International Neuroblastoma Risk Group (INRG)
staging system: An INRG Task Force report. J Clin Oncol.
17) Parsons LN, Gheorghe G, Yan K, Simpson P, Jarzembowski
JA. Improving Detection of Metastatic Neuroblastoma in Bone
Marrow Core Biopsies: A Proposed Immunohistochemical
Approach. Pediatr Dev Pathol. 2016;19:230-6.
18) Seeger RC, Reynolds CP, Gallego R, Stram DO, Gerbing RB,
Matthay KK. Quantitative tumor cell content of bone marrow
and blood as a predictor of outcome in stage IV neuroblastoma:
A Childrenís Cancer Group Study. J Clin Oncol. 2000;18:4067-76.
19) Russell HV, Golding LA, Suell MN, Nuchtern JG, Strother DR.
The role of bone marrow evaluation in the staging of patients
with otherwise localized, low-risk neuroblastoma. Pediatr Blood
20) Wang Z, Sun H, Li K, Yao W, Dong K, Ma Y, Zheng S. Prognostic
factor analysis of stage 4S neuroblastoma in infant patients: A
single center study. J Pediatr Surg. 2019;54:2585-8.
21) Morgenstern DA, London WB, Stephens D, Volchenboum SL,
Simon T, Nakagawara A, Shimada H, Schleiermacher G, Matthay
KK, Cohn SL, Pearson AD, Irwin MS. Prognostic significance of
pattern and burden of metastatic disease in patients with stage 4
neuroblastoma: A study from the International Neuroblastoma
Risk Group database. Eur J Cancer. 2016;65:1-10.
22) Manenq C, Lesesve JF, Dreumont N, Massin F, Salignac S, Mansuy
L, Chastagner P, Latger-Cannard V, Brosťus J. Combined use of
multiparametric flow cytometry and cytomorphology to enhance
detection of neuroblastoma metastatic cells in bone marrow. Int J
Lab Hematol. 2020;42:52-60.
23) McCarthy LC, Chastain K, Flatt TG, Taboada E, Garola R, Herriges
J, Cooley LD, Ahmed AA. Neuroblastoma in Adolescents and
Children Older than 10 Years: Unusual Clinicopathologic and
Biologic Features. J Pediatr Hematol Oncol. 2019;41:586-95.