Diagnostic Utility of PHOX2B in Primary and Treated
Neuroblastoma and in Neuroblastoma Metastatic
to the Bone Marrow
Jessica L. Hata, MD, MS; Hernan Correa, MD; Chandra Krishnan, MD; Adam J. Esbenshade, MD; Jennifer O. Black, MD;
Dai H. Chung, MD; Bret C. Mobley, MD, MS
 Context.—Neuroblastoma (NB) is the most common
extracranial tumor of childhood. Although most cases have
a distinctive histology, a subset of primitive cases require
immunohistochemical studies to distinguish them from
other small round blue cell tumors of childhood. Immu-
nohistochemistry is also used to detect small amounts of
tumor metastatic to the bone marrow and in posttreatment
samples with obscuring fibrosis, calcification, or inflam-
mation. The transcription factor PHOX2B is essential for
the differentiation and survival of sympathetic neurons and
chromaffin cells, and therefore is highly specific for the
peripheral autonomic nervous system.
Objective.—To determine the diagnostic utility of
PHOX2B immunohistochemistry as a marker of primary,
treated, and metastatic NB.
Design.—Neuroblastoma tissue microarrays were
stained with PHOX2B, CD57, and synaptophysin. Arrays
containing rhabdomyosarcoma, Ewing sarcoma, and
Wilms tumor were stained with PHOX2B, and negative
N euroblastoma (NB) is the most common extracranial
tumor of childhood.1 It has a diverse clinical behavior
ranging from metastatic disease with dismal patient
outcome to tumor maturation or spontaneous regression.1,2
Primary tumor morphology and age at diagnosis have an
impact on clinical outcome, with more differentiated tumors
and younger age at diagnosis considered favorable.2,3
Although most cases of NB show distinct and recognizable
histology, a subset of primitive neuroblastomas with small
round blue cell morphology require a panel of immunohis-
Accepted for publication May 19, 2014.
From the Departments of Pathology, Microbiology and Immunol-
ogy (Drs Hata, Correa, Black, and Mobley), Pediatrics (Dr
Esbenshade), and Pediatric Surgery (Dr Chung), Vanderbilt Univer-
sity Medical Center, Nashville, Tennessee; and the Department of
Pathology, Dell Children’s Medical Center, Austin, Texas (Dr
Krishnan).
The authors have no relevant financial interest in the products or
companies described in this article.
Presented in poster form at The Society for Pediatric Pathology fall
meeting; September 27, 2013; Salt Lake City, Utah.
Reprints: Bret Mobley, MD, Department of Pathology, Microbiol-
ogy and Immunology, Vanderbilt University Medical Center, 1161
21st Ave S, Medical Center North C-2318, Nashville, TN 37232 (e-
mail: bret.mobley@vanderbilt.edu).
Arch Pathol Lab Med—Vol 139, April 2015
bone marrow samples were stained with PHOX2B and
CD57.
Results.—PHOX2B and CD57 were similar to synapto-
physin in their ability to detect NB. PHOX2B and CD57
similarly showed robust staining in posttreatment NB and
NB metastatic to the bone marrow. In contrast to the
cytoplasmic staining pattern seen with synaptophysin and
CD57, clear and strong nuclear PHOX2B permitted
identification of individual tumor cells. PHOX2B staining
was absent in all cases of rhabdomyosarcoma, Ewing
sarcoma, and Wilms tumor, and in the negative bone
marrow.
Conclusions.—PHOX2B and CD57 are useful markers of
NB. PHOX2B is specific for NB in its differential diagnosis
with other small round cell tumors, and its nuclear staining
may be helpful for accurate bone marrow tumor quanti-
fication.
(Arch Pathol Lab Med. 2015;139:543–546; doi: 10.5858/
arpa.2014-0255-OA)
tochemical stains to permit distinction from other tumors of
childhood, such as rhabdomyosarcoma, Wilms tumor,
Ewing sarcoma, and lymphoma.
Neuroblastoma most commonly shows reactivity for some
or all of the following markers: synaptophysin, chromogra-
nin, neuron-specific endolase, CD56, and NB84.4,5 In
general, these markers are sensitive but not entirely specific,
and they can show background reactivity in nonneoplastic
cells, especially in the bone marrow (BM). New and specific
immunohistochemical markers will not only improve the
diagnostic process in cases of NB that lack characteristic
clinical findings (increased urinary catecholamines or meta-
iodobenzylguanidine uptake) and characteristic morphology
(neuronal differentiation and/or Schwannian stroma), but
they will also enhance our ability to detect small foci of BM
metastases and posttreatment residual disease.
The transcription factor PHOX2B is essential for the
differentiation and survival of sympathetic neurons and
chromaffin cells, and thus is highly specific for the
peripheral autonomic nervous system.6 It was first described
in an NB cell line.7 PHOX2B is mutated in congenital central
hypoventilation syndrome, a rare autosomal dominant
syndrome in which infants fail to breathe in response to
progressive hypercapnia and hypoxia, and die in their sleep.8
Congenital central hypoventilation syndrome is also asso-
PHOX2B in Neuroblastoma—Hata et al 543
ciated with tumors of neural crest origin, including NB, and
with Hirschsprung disease.
Bielle et al6 studied a variety of neoplasms and found
PHOX2B to be expressed specifically in tumors of auto-
nomic nervous system origin, including 6 of 6 undifferen-
tiated NBs. No other small round blue cell tumors showed
PHOX2B reactivity, highlighting the utility of this marker for
distinguishing undifferentiated NB from its mimics.
CD57 (Leu-7/HNK-1) is a carbohydrate epitope expressed
on adhesion molecules of migrating neural crest progenitor
cells,9,10 and NB has been shown to express this antigen by
flow cytometry.11 CD57 immunohistochemical studies have
demonstrated conflicting results, including restriction of
CD57 staining to ganglioneuromas (GNs)5 and expression
of this antigen by neuroblastomas, as well as neuroecto-
dermal tumors, Ewing sarcomas, and Wilms tumors.12 The
clinical diagnostic utility of CD57 has yet to be thoroughly
evaluated.
In this study we optimized commercially available
PHOX2B, CD57, and synaptophysin antibodies for immu-
noperoxidase staining of formalin-fixed, paraffin-embedded
material and used tissue microarrays (TMAs) to examine
their utility in the diagnosis of NB at various stages of
differentiation. We also investigated the ability of PHOX2B
and CD57 to identify NB metastatic to the BM and
posttreatment NB. PHOX2B expression was also examined
in other small round blue cell tumors of childhood.
MATERIALS AND METHODS
Paraffin-embedded TMAs, including 30 poorly differentiated
NBs, 18 differentiating NBs, 6 ganglioneuroblastomas, 28 GNs, and
34 posttreatment NBs, were constructed. Neuroblastoma cases
from 1992 to 2011 were obtained from the surgical pathology
database per institutional review board protocol. An additional
TMA was constructed from cases collected between 1995 and 2007;
these included 4 undifferentiated NBs, 15 rhabdomyosarcomas, 15
Wilms tumors, and 11 Ewing sarcomas. Original hematoxylin-
eosin stain slides and, when available, immunohistochemical
stains, were reviewed and the diagnosis was confirmed using
standard diagnostic criteria.13 A Beecher Instruments Manual
Tissue Arrayer (Sun Prairie, Wisconsin) was used to prepare tissue
cores from selected regions of archival tissue blocks. To create the
TMAs, three to four 1-mm cores were prepared for each tumor case
and re-embedded into a gridded paraffin block.
Paraffin-embedded blocks for 15 decalcified BM core biopsies
and particle preparations involved by metastatic NB were obtained
from the surgical pathology database spanning the years 1997 to
2012. The diagnosis was confirmed by review of original
hematoxylin-eosin slides and immunohistochemical stains, when
performed. Included were cases with large tumor volume and cases
with very small foci of tumor initially requiring immunohisto-
chemistry (IHC) for diagnosis. Nine negative BM samples obtained
for staging of non-NB sarcoma cases were used as negative
controls.
Immunohistochemical studies were performed using commer-
cially available antibodies and the automated Leica Bond Max
Immunohistochemistry stainer (Leica Microsystems Inc, Buffalo
Grove, Illinois). Primary antibodies directed against the following
proteins were used: PHOX2B (catalog No. sc-13224, Santa Cruz
Biotechnology Inc, Santa Cruz, California), synaptophysin (catalog
No. PA0299, clone 27G12, Leica Microsystems), and CD57 (catalog
No. RTU-NK1, clone NK-1, Leica Microsystems). Heat-induced
antigen retrieval was performed on the Bond Max using Leica’s
Epitope Retrieval solutions. Slides were then incubated with
antibodies (anti-PHOX2B at a 1:100 dilution; synaptophysin and
CD57 without further dilution). An anti-goat biotinylated second-
ary (Vector Laboratories Inc, Burlingame, California) at a 1:1000
dilution and the Bond Polymer Refine detection system (Leica)
544 Arch Pathol Lab Med—Vol 139, April 2015
were used for visualization of the PHOX2B antibody. Synaptophy-
sin and CD57 required only the Bond Polymer Refine detection
system for visualization. The TMAs and involved BM biopsies were
stained with PHOX2B, CD57, and synaptophysin. The negative
sarcoma margins were stained with PHOX2B and CD57.
PHOX2B staining for cellular samples was scored on a 4-point
scale as follows: 0, no staining of any cells within the tumor; 1þ,
weak, focal nuclear staining (,5% of cells) or very faint staining
diffusely; 2þ, strong nuclear staining in 5% to 50% of cells; and 3þ,
strong nuclear staining in more than 50% of cells. For diagnostic
purposes, scores 2þ and 3þ were considered ‘‘positive,’’ whereas
scores 0 and 1þ were considered ‘‘negative.’’ For GN samples or
posttreatment samples with a low relative volume of neuronal cells,
a 2-point scale was used as follows: 0, no staining of any cells
within the tumor; and 1þ, any nuclear staining. Samples composed
entirely of Schwannian stroma were excluded.
Synaptophysin and CD57 staining were scored on a 4-point scale
as follows: 0, no labeling of any cells within the tumor; 1þ, weak,
focal cytoplasmic labeling of cells (,5%) or granular cytoplasmic
blush in many cells; 2þ, strong cytoplasmic staining in 5% to 50%
of cells; and 3þ, strong cytoplasmic staining in more than 50% of
cells. For diagnostic purposes, scores 2þ and 3þ were considered
‘‘positive,’’ whereas scores 0 and 1þ were considered ‘‘negative.’’
RESULTS
PHOX2B showed strong staining across all stages of NB
differentiation and in lesions metastatic to the BM, with
essentially the same expression pattern as CD57 and
synaptophysin (Figure 1). PHOX2B was positive in neuro-
blasts and ganglion cells and showed reactivity in 75% of
undifferentiated NBs, 100% of poorly differentiated NBs,
and 96% of differentiating NB/ganglioneuroblastomas. This
marker provided a crisp nuclear signal that allowed
confident identification of tumor cells. No PHOX2B
reactivity was identified in negative BM margins or in cases
of rhabdomyosarcoma, Ewing sarcoma, or Wilms tumor
(Table).
Ganglioneuromas, although usually diagnosed without
the aid of IHC, were also tested for PHOX2B expression.
Ganglioneuromas showed reactivity, in agreement with a
previous report,6 but it was slightly decreased in compar-
ison with other stages of NB differentiation, with only 74%
of tumors labeling with PHOX2B. Low numbers of
ganglion cells per GN core, variable PHOX2B nuclear
reactivity, or loss of transcription factor expression in fully
mature cells may have been responsible for this reduced
staining rate.
Synaptophysin showed granular, cytoplasmic staining in
all tumors, and CD57 showed strong cytoplasmic staining in
100% of poorly differentiated NBs, 96% of differentiating
NBs/ganglioneuroblastomas, and 93% of GNs (Table).
PHOX2B, similar to synaptophysin and CD57, showed
robust staining in decalcified BM biopsies. PHOX2B stained
88% of involved BMs, compared with 100% staining by
synaptophysin and CD57. In contrast to the cytoplasmic
staining pattern seen with synaptophysin and CD57,
PHOX2B staining provided a discrete nuclear signal that
permitted better discrimination of tumor cells from back-
ground hematopoietic precursors and stromal cells (Figure
2). In BM samples, PHOX2B was more specific for NB than
CD57 was; although strong CD57 cytoplasmic staining was
seen in 22% (2 of 9) of negative BM margins, no PHOX2B
staining was seen in negative control tissue. CD57 also
showed strong membranous staining in scattered small
cells, likely natural killer cytotoxic T lymphocytes.
PHOX2B in Neuroblastoma—Hata et al

Figure 1. A case of poorly differentiated neuroblastoma stained with
(A) synaptophysin, (B) PHOX2B, and (C) CD57 (original magnification
3600).
In posttreatment NB samples, synaptophysin, CD57, and
PHOX2B performed similarly. Synaptophysin reactivity was
seen in 100% of cases, CD57 staining was seen in 91% of
cases, and posttreatment neuroblasts and ganglion cells
showed PHOX2B reactivity in 94% of cases. The staining
intensity was as robust for these markers as it was in
untreated cases.
Arch Pathol Lab Med—Vol 139, April 2015
Expression of PHOX2B, CD57, and Synaptophysin in
Neuroblastoma (NB) and PHOX2B in Selected Small
Round Blue Cell Tumors of Childhooda
PHOX2B CD57 Synaptophysin
Undifferentiated NB 3/4 — —
Poorly differentiated NB 30/30 30/30 30/30
Differentiating NB and 23/24 23/24 24/24
ganglioneuroblastoma
Ganglioneuroma 20/27 25/27 27/27
Posttreatment NB 31/33 31/34 34/34
Bone marrow involved 14/16 17/17 17/17
by NB
Negative bone marrow 0/9 2/9 —
Rhabdomyosarcoma 0/15 — —
Ewing sarcoma 0/11 — —
Wilms tumor 0/15 — —
a
— indicates stain was not performed.
COMMENT
The PHOX2B gene encodes a paired homeodomain
transcription factor with expression limited to the auto-
nomic nervous system.14 PHOX2B is essential for the
differentiation and survival of neurons, is present during
growth and development, and persists into adulthood.
Neuroblasts are derived from sympathoadrenal lineage
neural crest cells and therefore require and constitutively
express PHOX2B. PHOX2B IHC, as a marker of neural crest
derivation, has been shown to be sensitive and specific for
undifferentiated NB in the differential diagnosis with other
small round blue cell tumors of childhood.6 In this study we
confirm PHOX2B to be a sensitive marker of NB in all stages
of differentiation. We find the neural crest progenitor cell
surface protein CD57 to be a sensitive marker of NB as well.
A striking finding in this study is the specificity of
PHOX2B for NB in the small round blue cell tumor
differential of childhood. Cases of Wilms tumor, rhabdo-
myosarcoma, and Ewing sarcoma were uniformly and
unequivocally negative for this marker. Considered together
with the pediatric tumor panel performed by Bielle et al,6 the
accumulating evidence shows that this marker is specific for
autonomic nervous system tumors and will be clinically
valuable in this differential diagnosis.
The identification of NB metastatic to the BM is critical for
accurate staging and disease surveillance. Using IHC for
quantification of NB in the BM at diagnosis and during
induction chemotherapy provides prognostic information
that can identify patients with very high-risk disease who
may then be considered for experimental therapy.15
Evaluating BM biopsies for metastatic NB with synaptophy-
sin IHC can be difficult because of nonspecific background
staining and heterogeneous expression within tumor cells.
Synaptophysin may occasionally stain subsets of erythroid
precursors in a granular cytoplasmic and nuclear fashion
mimicking clusters of tumor,16 and can also show reactivity
within plasma cells, osteoclasts, and osteoblasts. Nuclear
PHOX2B staining permits discrimination of tumor cells from
BM hematopoietic precursors or fibroblasts. While synapto-
physin staining may lead to tumor quantity overestimation,
the strong nuclear signal provided by PHOX2B may be
better suited for quantifying the degree of marrow
involvement by NB. In BMs with very small amounts of
tumor, however, stains including synaptophysin and CD57
PHOX2B in Neuroblastoma—Hata et al 545

Figure 2. While synaptophysin staining may in some cases overstate
the degree of marrow involvement by tumor (A), PHOX2B provides a
discrete nuclear signal, permitting discrimination of tumor cells from
background hematopoietic and stromal cells (B) (original magnification
3400).
may highlight cytoplasm/neuropil when no nuclei are
available to be labeled by PHOX2B.
Dense fibrosis, prominent inflammatory infiltrates, and/or
diffuse calcification may be found in posttreatment NB
samples, and can make identification of residual tumor
difficult. Crush artifact can render groups of inflammatory
cells indistinguishable from tumor by hematoxylin-eosin
alone. We find that CD57 and PHOX2B label neuroblasts
and ganglion cells in posttreatment samples, and may
therefore be helpful in confirming the presence of post-
treatment residual disease.
Synaptophysin can show focal staining in alveolar
rhabdomyosarcoma17 and a small proportion of cases of
NB are negative for this marker.5 Evaluating PHOX2B
expression in synaptophysin-negative tumors would be of
interest; however, the cohort of NBs studied here were all
strongly synaptophysin positive. PHOX2B can detect
undifferentiated NB that lacks classic immunohistochemical
markers, such as synaptophysin, chromogranin, and tyro-
sine hydroxylase.6
546 Arch Pathol Lab Med—Vol 139, April 2015
SUMMARY
Neuroblastoma is a common tumor of childhood for
which multiple immunohistochemical stains can be em-
ployed to aid in diagnosis. PHOX2B is sensitive and specific
for NB and shows diagnostic utility for detecting this disease
at all stages of differentiation. CD57 shows a similar staining
profile to PHOX2B and synaptophysin. The crisp nuclear
staining provided by PHOX2B allows confident discrimina-
tion of tumor cells from nonspecific background staining.
This is especially helpful in posttreatment samples, where
treatment effect may make detection of residual tumor
difficult by hematoxylin-eosin alone, and in the small foci of
tumor metastatic to the BM. PHOX2B, in combination with
a cytoplasmic stain such as synaptophysin, can help detect
NB and will likely help accurately quantify the tumor burden
in primary, metastatic, and posttreatment lesions.
We wish to thank Melissa Downing, Anthony Frazier, and the
Vanderbilt University Medical Center Translational Pathology
Shared Resource for outstanding technical expertise in preparing
the neuroblastoma TMAs and in immunohistochemical staining.
This work was supported in part by NIH K12 grant CA 09062513.
References
1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;
369(9597):2106–2120.
2. Goodman M, Gurney JG, Smith MA, et al. Sympathetic nervous system. In:
Ries LAG, Smith MA, Gurney JG, et al, eds. Cancer Incidence and Survival
Among Children and Adolescents: United States SEER Program 1975–1995. 99th
ed. Bethesda, MD: National Cancer Institute; 1999. http://seer.cancer.gov/
publications/childhood/sympathetic.pdf. Accessed June 29, 2013.
3. Shimada H, Ambros IM, Dehner LP, et al. The International Neuroblastoma
Pathology Classification (the Shimada system). Cancer. 1999;86(2):364–372.
4. Miettinen M, Chatten J, Paetau A, Stevenson A. Monoclonal antibody
NB84 in the differential diagnosis of neuroblastoma and other small round cell
tumors. Am J Surg Pathol. 1998;22(3):327–332.
5. Wirnsberger GH, Becker H, Ziervogel K, Hofler H. Diagnostic immuno-
histochemistry of neuroblastic tumors. Am J Surg Pathol. 1992;16(1):49–57.
6. Bielle F, Freneaux P, Jeanne-Pasquier, C et al. PHOX2B Immunolabeling: a
novel tool for the diagnosis of undifferentiated neuroblastomas among childhood
small round blue cell tumors. Am J Surg Pathol. 2012;36(8):1141–1149.
7. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. Expression and
interactions of the two closely related homeobox genes Phox2a and Phox2b
during neurogenesis. Development. 1997;124(20):4065–4075.
8. Marion TL, Bradshaw WT. Congenital central hypoventilation syndrome
and the PHOX2B gene mutation. Neonatal Netw. 2011;30(6):397–401.
9. Bronner-Fraser M. Analysis of the early stages of trunk neural crest
migration in avian embryos using monoclonal antibody HNK-1. Dev Biol. 1986;
115(1):44–55.
10. Bronner-Fraser M. Perturbation of cranial neural crest migration by the
HNK-1 antibody. Dev Biol. 1987;123(2):321–331.
11. Schlitter AM, Dorneburg C, Barth TFE, et al. CD57 high neuroblastoma
cells have aggressive attributes ex situ and an undifferentiated phenotype in
patients. PLoS ONE. 2012;7(8):e42025.
12. Michels S, Swanson MS, Robb JA, Wick MR. Leu-7 expression in small cell
neoplasms: an immunohistochemical study with ultrastructural correlations.
Cancer. 1987;60(12):2958–2964.
13. Qualman SJ, Bowen J, Fitzgibbons PL, Cohn SL, Shimada H. Protocol for
the examination of specimens from patients with neuroblastoma and related
neuroblastoma tumors. Arch Pathol Lab Med. 2005;129(7):874–883.
14. Brunet JF, Pattyn A. PHOX2 genes—from patterning to connectivity. Curr
Opin Genet Dev. 2002;12(4):435–440.
15. Seeger RC, Reynolds P, 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 Children’s Cancer Group Study. J Clin
Oncol. 2000;18(24):4067–4076.
16. Krishnan C, Twist CJ, Fu T, Arber DA. Detection of isolated tumor cells in
neuroblastoma by immunohistochemical analysis in bone marrow biopsy
specimens: improved detection with use of beta-catenin. Am J Clin Pathol.
2009;131(1):49–57.
17. Miettinen M, Rapola J. Immunohistochemical spectrum of rhabdomyosar-
coma and rhabdomyosarcoma-like tumors. Am J Surg Pathol. 1989;13(2):120–
132.
PHOX2B in Neuroblastoma—Hata et al