Pathology of the Peripheral Neuroblastic Tumors - LabMedicine

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Received 3.31.06 | Revisions Received 5.4.06 | Accepted 5.9.06
Pathology of the Peripheral Neuroblastic Tumors
Hiroyuki Shimada, MD, PhD, 1 Atsuko Nakagawa, MD, PhD 2
( 1 Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles and University of Southern California Kech School
of Medicine, Los Angeles, CA, 2 Division of Pathology, Department of Clinical Laboratory Medicine, National Center for Child Health
and Development, Tokyo, Japan)
DOI: 10.1309/0506C1BM8GBVV224
Abstract
Peripheral neuroblastic tumors (pNTs;
neuroblastoma, ganglioneuroblastoma,
ganglioneuroma) in infants and children, once
called as “enigmatic,” are now considered as
Peripheral neuroblastic tumors (pNTs including neuroblastoma,
ganglioneuroblastoma, and ganglioneuroma) comprise
one of the most common groups of neoplastic disease in infants
and children. 1 They are derived from immature sympathetic
neuroblasts and diagnosed in the primary sites related to the embryonic
distribution of neural crest cells, such as adrenal medulla
and structures of the sympathetic nervous system in the thorax,
abdomen, and pelvic cavity. These tumors are well known to
show unique and often unpredictable clinical behaviors. Recent
advances in basic, translational, and clinical research lead us to
believe that the pNTs are heterogeneous and composed of biologically
different, favorable and unfavorable, tumors. 2,3 The
biologically favorable tumors have a potential of involution/
spontaneous regression or tumor maturation with or without
chemotherapy/irradiation therapy. In contrast, the biologically
unfavorable tumors progress aggressively and often bring a fatal
outcome to the patients despite intensive treatment. It is also
speculated that differences between the biologically favorable
and unfavorable tumors could be determined during the early
stage of the neoplastic process by molecular/genetic properties of
the individual cases. 2,3
Peripheral neuroblastic tumors are so unique that those
factors useful in the routine practice of surgical pathology, such
as positive surgical margins, tumor necrosis, vascular invasion,
and even hematogenous/lymphatic spread, are not necessarily
significant indicators of aggressive tumor progression and a
poor clinical outcome of the patients. Biologic properties are
often more important and critical than those conventional
prognostic factors for predicting clinical behavior of the tumor
in individual cases. The Children’s Oncology Group (COG)
Neuroblastoma Biology Study has been utilizing front-end
prognostic factors for patient stratification and protocol assignment
of this disease. Those prognostic factors include patient’s
age at diagnosis, clinical stage 4 (see Table 1), histopathology,
MYCN oncogene status, and DNA index. By combination of
these factors, 3 risk groups: low, intermediate, and high are
distinguished among the pNT cases (Table 2). 5
biologically heterogeneous. The unique
clinical/biological behaviors: such as,
involution, spontaneous regression, tumor
maturation, and aggressive progression, are
closely related to the molecular/genetic
properties of the tumors of individual cases. In
this article, we describe prognostic significance
and biological relevance of the International
Neuroblastoma Pathology Classification (INPC).
Since pNTs offer one of the best models for analysis/evaluation
of histologic features as phenotypical manifestation of
multiple factors including age and various molecular/genetic
properties of the tumors, the international committee of pathologists
established a histopathology classification [the International
Neuroblastoma Pathology Classification (INPC)] in
1999 6,7 and partly modified it in 2003. 8 In this article, we first
describe a definition of pNTs and then apply this histopathology
classification to 6 representative cases for illustrating prognostic
significance and biological relevance of the INPC system.
Discussion focuses on the close relationship between histologic
features and molecular/genomic alterations of (1) MYCN amplification,
an established marker indicating aggressive tumor
progression, 9,10 (2) DNA index separating near-triploid tumors
with a better prognosis from near-diploid tumors with a poor
prognosis, 11 and (3) trkA (a high-affinity nerve growth factor
receptor) expression reported to have anti-oncogenic effects 12,13
in pNTs.
Definition
The name peripheral neuroblastic tumors (pNTs) indicates
that the tumors in this group are composed of the neoplastic cells
with a neuronal phenotype of neuronal (neural crest) cellular origin.
Peripheral neuroblastic tumors should be clearly distinguished
from those having neuronal phenotype but of unknown (presumably
non-neuronal) cellular origin. For example, pPNET (peripheral
primitive neuroectodermal tumor), known to have a limited
neuronal differentiation, does not seem to have a proven neuronal
cellular origin. These 2 tumor entities, pNTs and pPNET, also
have distinct molecular properties: pNTs are often associated with
deletion of chromosome 1p and MYCN amplification, 2,3 whereas
pPNET has unique chromosomal translocation fusing EWS/ETS. 14
Furthermore, transfection of EWS/ETS fusion genes into NIH
3T3 fibroblasts is reported to induce epithelial and neuroectodermal
differentiation of pPNET-phenotype. 15,16
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Table 1_International Neuroblastoma Staging System (INSS)
Stage Definition
1 Localized tumor confined to the area of origin. Complete gross resection with or without microscopic residual disease; identifiable ipsilateral and
contralateral lymph node negative for tumor. Adherent lymph nodes in direct continuity with and removed with the tumor may be positive for the tumor.
A grossly resected midline tumor without ipsilateral (with: stage 2A) or contralateral (with: stage 2B) lymph node involvement is considered stage 1
2A Unilateral with incomplete gross resection; identifiable ipsilateral and contralateral lymph node negative for tumor
2B Unilateral with complete or incomplete gross resection; with ipsilateral lymph node positive for tumor; identifiable contralateral lymph node negative for tumor
3 Tumor infiltrating across midline with or without regional lymph node involvement; or unilateral tumor with contralateral lymph node involvement or midline
tumor with bilateral lymph node involvement
4 Dissemination of tumor to distant lymph nodes, bone marrow, liver, or other organs except as defined as stage 4S
4S Localized primary tumor as defined for stage 1 or 2 with dissemination limited to liver, skin, and bone marrow (

Review
of neuroblastic differentiation and MKI to the nodular (neuroblastomatous)
component.
Case Presentation
Case 1: A 4-month-old female with a clinical stage 4S disease
of the adrenal primary and liver involvement.
Microscopic sections from the adrenal tumor and liver
biopsy sample showed the same appearance of neuroblastoma
(Schwannian stroma-poor), poorly differentiated subtype with a
low mitosis-karyorrhexis index (MKI) (Image 1A). Because of
the age of the patient at diagnosis, the case was classified into a
favorable histology group (Table 2). The tumor sample had nonamplified
MYCN and high trkAI/II (high-affinity nerve growth
factor receptor, a receptor tyrosine kinase that binds nerve
growth factor) expression, and the DNA index, as determined
by flow cytometry, was 1.25. According to the COG risk-grouping
scheme, the patient was placed in the low-risk protocol
(Table 1). The tumor regressed spontaneously, and she is alive
and well 4 years after diagnosis.
Comment: This was an example of spontaneous regression
of a widely spread disease. Favorable histology, non-amplified
MYCN, and a hyperdiploid DNA pattern all supported that the
tumor of this case was biologically favorable. A high level of
trkAI/II expression also suggested a potential of either maturation
or apoptosis (see description in Discussion) of the neuroblastic
cells. 12,13 Stage 4S (S=special) is defined as a disease
condition in patients

of neuroblastic cells expressing lower level of trkAI/II, and do
not seem to have a potential of age-appropriate maturation.
Case 5: A 5-year and 2-month-old male with a clinical
stage 1 tumor located in the pelvis.
Microscopic sections from the completely resected tumor
showed the appearance of ganglioneuroblastoma, intermixed
A B
C D
E F
Review
(Schwannian stroma-rich) (Image 1E). The case was classified
into a favorable histology group. The tumor sample had nonamplified
MYCN and a diploid pattern of DNA content. Level
of trkAI/II expression was not tested. He was a low-risk patient
(Table 2), and is now alive and well 10 years after the surgery
without chemotherapy/irradiation therapy.
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Review
Comment: More than 50% of tumor tissue in this case was
composed of Schwannian stroma. Microscopic nests of neuroblasts,
differentiating neuroblasts, and maturing ganglion cells in a
background of neuropil were clearly recognizable, but well
blended in the surrounding Schwannian stromal tissue. This is a
transitional form to ganglioneuroma, [ie, fully mature form of
pNTS where the tumor tissue is predominantly composed of
Schwannian stroma (Schwannian stroma-dominant) with individually
scattered ganglion cells]. Both ganglioneuroblastoma,
intermixed and ganglioneuroma, are commonly diagnosed in
older children and young adults, and always associated with an
excellent prognosis of the patients. They do not have MYCN
amplification, and flow cytometry test on those tumors usually
detect a diploid pattern of Schwannian stromal cells rather than
a hyperdiploid (near-triploid) pattern of biologically favorable
neuroblastic/ganglion cells.
Case 6: A 1-year and 11-month-old female with a clinical
stage 2a disease of the mediastinal primary.
The tumor was composed of 2 different components: one
component was solid and tan-yellow, and the other was hemorrhagic
and fragile. The former component showed an appearance
of ganglioneuroblastoma, intermixed (Image 1F, left side).
The latter was neuroblastoma, poorly differentiated subtype with
a low MKI (Image 1F, right side). This tumor was placed into a
category of ganglioneuroblastoma, nodular (composite,
Schwannian stroma-rich/stroma-dominant, and stroma-poor).
Because of the histologic features of the neuroblastoma component
and the age of the patient at diagnosis, the case was classified
into an unfavorable subset (unfavorable histology) of
ganglioneuroblastoma, nodular. 8 The tumor sample had nonamplified
MYCN and the DNA index of 1. Level of trkAI/II
expression was not tested. This is a low-risk case according to the
COG scheme. However, she died of progressive disease with
bone metastasis 2 years after the diagnosis.
Comment: This was an example of composite tumor having
2 different clones. Based on our experience, prognosis of the
patients with the composite tumor like this one is determined by
the behavior of neuroblastomatous component. The same criteria
of age-appropriate evaluation of histologic features as used in
Table 3_International Neuroblastoma Pathology Classification
the FH and UH distinction for neuroblastoma tumors are applied
to the neuroblastomatous component to distinguish favorable
and unfavorable subset in this tumor category. 8
Discussion
The International Neuroblastoma Pathology Classification
(INCP) significantly distinguishes 2 prognostic groups (Table
3), (ie, favorable histology group and unfavorable histology
group). 6-8 As for the tumors in the categories of neuroblastoma
(Schwannian stroma-poor) and ganglioneuroblastoma, nodular
(composite, Schwannian stroma-dominant/stroma-rich and
stroma-poor), the INPC employs an age-linked evaluation system
on histologic features (grade of neuroblastic differentiation
and MKI) for determining 2 prognostic groups. Tumors in the
categories of ganglioneuroblastoma, intermixed, and ganglioneuroma
are always classified into the favorable histology group.
As illustrated above Cases 1, 2, 5 were the examples of the
favorable histology group and all the patients are alive and well.
In contrast, Cases 3, 4, 6 were classified into an unfavorable histology
group, and the patients died ofthe disease.
The following is a summary of clinically as well as biologically
important relationships between histologic features and
molecular/genomic alterations in pNTs.
(1) It is well known that patients with amplified MYCN
tumor have a very poor clinical outcome. 9,10 The MYCN amplification
followed by excessive MYCN protein expression with subsequent
myc-max heterodimer formation in the neuroblastoma
cells24 is considered to be a powerful driving force for preventing
cellular differentiation and increasing mitotic and karyorrhectic
activities. Accordingly, MYCN amplified tumor typically shows
an appearance of neuroblastoma (Schwannian stroma-poor) of
either undifferentiated or poorly differentiated subtype with a high
MKI. 21,22
Favorable histology tumors are almost always MYCN nonamplified.
Among the UH tumors, about 40% are MYCN amplified
with an extremely poor clinical outcome of the patients,
and 60% are MYCN non-amplified also with a poor prognosis
of the patients.
Category Favorable Histology Unfavorable Histology
Neuroblastoma Grade of Differentiation MKI Grade of Differentiation MKI
(Schwannian stroma-poor)
Age

(2) Patients with tumors having near-diploid DNA contents
have a significantly worse prognosis than those having neartriploid
DNA contents. 11 The near-diploid tumors tend to have
structural aberrations, such as MYCN amplification and 1p
deletion, more frequently than the near-triploid tumors. It is
noted that the neuroblastic cells in the favorable histology group
are usually near-triploid, and Schwannian stromal cells are always
diploid. It is also noted that favorable histology tumors can
promote Schwannian stromal development, and come up to the
advanced maturational stages of ganglioneuroblastoma, intermixed
(Schwannian stroma-rich), and ganglioneuroma
(Schwannian stroma-dominant). Because of the difference of
ploidy patterns, we assume that Schwannian stromal cells
(diploid) and neuroblastic cells (near-triploid) do not have a
common cellular origin, and the stromal cells are recruited by
the neuroblastic tumor cells. 18,19
(3) TrkA (high-affinity nerve growth factor receptor),
mapped to chromosome 1q21-q22 and organized into 17 exons,
has 2 splice variants, trkAI (lacking mini-exon 9 in the extracellular
domain) and trkAII. It is reported that patients with tumors
expressing higher levels of trkAI/II have a significantly
better prognosis than those expressing lower levels of trkAI/II. 13
It is also reported that NGF(Nerve Growth Factor)/trkA(I/II)/
Ras/MAPK signaling pathway is anti-oncogenic causing cellular
differentiation, growth arrest, apoptosis, and anti-angiogenesis. 12
However, our recent analysis shows that the levels of trkA expression
did not add any additional prognostic information significantly
to the risk model currently used by the COG
Neuroblastoma Biology Study that is created by the combination
of clinical stage, histopathology, and MYCN status. 25 It is
also noted that neuroblastic cells having non-amplified MYCN
and high levels of trkAI/II expression have a potential of agedependent
differentiation after a certain in vivo latent period
within a framework of the INPC system. 25
Recently, Tacconelli and colleagues reported another splice variant,
trkAIII, and its oncogenic/tumor-promoting effect in
pNTs. 26,27 This variant does not have exons 6, 7, and 9 in the extracellular
domain, is NGF-unresponsive, and seems to antagonize
NGF/trkA(I/II) signaling. Further investigation should be required
especially for clinical significance of trkAIII expression in pNTs.
Summary
Histopathology classification is a very useful tool for distinguishing
the pNT patients who require aggressive treatment protocols
from those who need no or reduced treatment modalities
after biopsy/surgery. Pediatric tumors, especially pNTs, provide
us with an excellent opportunity to investigate a biologically significant
relationship between the genetic/molecular alterations
and their clinical behaviors. This is one of the most exciting
fields where we pathologists can play a significant role of bridging
between the laboratory bench and clinical bedside. We can
recognize unique morphologic features as the manifestation of
various genetic/molecular changes, known or still unknown,
and also analyze the prognostic significance of those morphologic
features for the sake of the patients. LM
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