Seminar
Neuroblastoma
Lancet 2007; 369: 2106–20
Children’s Hospital of
Philadelphia and University of
Pennsylvania School of
Medicine (J M Maris MD,
M D Hogarty MD) and the
Abramson Family Cancer
Research Institute (J M Maris),
Philadelphia, PA, USA;
University of Arizona Health
Sciences Center and Steele
Children’s Research Center,
Tucson, AZ, USA
(R Bagatell MD); and Comer
Children’s Hospital and
University of Chicago, Chicago,
IL, USA (Prof S L Cohn MD)
Correspondence to:
John M Maris, The Children’s
Hospital of Philadelphia,
Division of Oncology, ARC 902A,
Philadelphia, PA 19104-4318,
USA
maris@chop.edu
2106
John M Maris, Michael D Hogarty, Rochelle Bagatell, Susan L Cohn
The clinical hallmark of neuroblastoma is heterogeneity, with the likelihood of cure varying widely according to age at
diagnosis, extent of disease, and tumour biology. A subset of tumours will undergo spontaneous regression while
others show relentless progression. Around half of all cases are currently classified as high-risk for disease relapse,
with overall survival rates less than 40% despite intensive multimodal therapy. This Seminar focuses on recent
advances in our understanding of the biology of this complex paediatric solid tumour. We outline plans for the
development of a uniform International Neuroblastoma Risk Group (INRG) classification system, and summarise
strategies for risk-based therapies. We also update readers on new discoveries related to the underlying molecular
pathogenesis of this tumour, with special emphasis on advances that are translatable to the clinic. Finally, we discuss
new approaches to treatment, including recently discovered molecular targets that might provide more effective
treatment strategies with the potential for less toxicity.
Introduction Localised tumours
Neuroblastoma accounts for more than 7% of malignancies Around 40% of patients will present with localised
in patients younger than 15 years and around 15% of all disease that can range from an incidentally discovered
paediatric oncology deaths.1 It is the most common intra-adrenal mass discovered on prenatal ultra-
extracranial solid tumour in childhood and the most sonography to very large and locally invasive tumours
frequently diagnosed neoplasm during infancy.2 anywhere along the sympathetic chain. Primary thoracic
The disease is remarkable for its broad spectrum of tumours might be detected incidentally on chest
clinical behaviour. Although substantial improvement in radiographs, whereas cervical masses could be associated
outcome of certain well-defined subsets of patients has with Horner syndrome.9,10 Paraspinal tumours in the
been observed during the past few decades, the outcome thoracic, abdominal, and pelvic regions occur in
for children with a high-risk clinical phenotype has 5–15% of patients, and these can extend into the neural
improved only modestly, with long-term survival still less foramina causing symptoms related to compression of
than 40%.3,4 nerve roots and the spinal cord.11-13 In total, up to 5% of
In the 1980s, when technology that permitted detection newly diagnosed neuroblastoma patients will have
of tumour-derived catecholamines in spot urine samples neurological signs related to cord impingement
became widely available, screening programmes designed including motor weakness, pain, and sensory loss.11,12,14
to detect neuroblastoma in infants were developed. Studies These cases represent true oncological emergencies, but
in Japan have shown that neuroblastoma could be detected there remains some controversy about what type of
by screening urine at 6 months of age, and early results immediate intervention is best. Localised tumours are
suggested that preclinical detection led to improved generally chemotherapy responsive, and several
survival.5,6 However, two prospective population-based retrospective reports have documented equivalent
controlled trials in Germany and North America have neurological outcome for patients treated with chemo-
shown that screening does not reduce mortality.7,8 In both therapy or laminectomy.11–13,15 Since the long-term
of the studies, the incidence of neuroblastoma increased in orthopaedic consequences of laminectomy can be sub-
the screened population, and almost all tumours detected stantial,13,15 most investigators recommend emergent
by screening had favourable biological features. The cost of chemotherapy as the intervention of choice for
infant screening and the essentially unchanged mortality symptomatic paraspinal neuroblastoma.
rates argue against the public health usefulness of mass
screening of infants for neuroblastoma.
Search strategy
Clinical presentation We identified reports using a Medline search through the
Neuroblastoma is a disease of the sympaticoadrenal lineage PubMed database (1986–2006) by combining the keyword
of the neural crest, and therefore tumours can develop neuroblastoma with tumorigenesis, angiogenesis, progression,
anywhere in the sympathetic nervous system. Most metastasis, pathology, pathobiology, pathophysiology,
primary tumours (65%) occur within the abdomen, with at molecular genetics, and genetics. We searched citation lists in
least half of these arising in the adrenal medulla. Other retrieved papers to identify additional references. Papers were
common sites of disease include the neck, chest, and selected on the basis of the best available evidence for each
pelvis.2 Presenting signs and symptoms are highly variable specific question discussed. To limit the number of references,
and dependent on site of primary tumour as well as the review articles, book chapters, or the latest publications in a
presence or absence of metastatic disease or paraneoplastic series of articles from the same laboratory were given
syndromes. Although there is extensive overlap, three preference. Only English language papers were included.
main clinical scenarios are commonly recognised.
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A
B cause bone pain, limping, or irritability. Additionally,
Figure 1: Neuroblastic tumours vary in degree of differentiation
(A) Schwann cells and ganglion cells (indicated by arrows) are prominent in
stroma-rich neuroblastoma. (B) Stroma-poor neuroblastoma consists of densely
packed small round blue cells with scant cytoplasm.
Two major paraneoplastic syndromes are seen much
more commonly in patients with localised tumours,
suggesting that the syndrome itself might modify the
malignant potential of the tumour. Secretion of
vasoactive intestinal peptide can result in intractable
watery diarrhoea and failure to thrive, which usually
resolves after tumour removal.16 Opsoclonus-myoclonus
syndrome, seen in 2–4% of patients with neuroblastoma,
consists of rapid eye movements, ataxia, and irregular
muscle movements.17 Most children with opsoclonus-
myoclonus syndrome have a favourable outcome with
respect to their malignancies.18,19 However, 70–80% of
these children will have long-term neurological
deficits.20–22 Some children have improvement of
symptoms after tumour removal or administration of
glucocorticoids, adrenocorticotropic hormone, intra-
venous immunoglobulin, or chemotherapy,21,23–26
although the best treatment strategy remains contro-
versial. The Children’s Oncology Group is doing a
prospective study to establish whether the addition of
intravenous gammaglobulin to chemotherapy and
corticosteroids will improve the neurological outcome
for patients with neuroblastoma and opsoclonus-
myoclonus syndrome.
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Seminar
Metastatic disease
About half of patients present with evidence of haema-
togeneous metastasis. Here we distinguish metastasis to
distant sites such as cortical bone, bone marrow, liver,
and non-contiguous lymph nodes from locoregional
spread to lymph nodes adjacent to the primary tumour.
Unlike children with localised disease, who often have a
normal performance status and few symptoms at
diagnosis, children with metastatic neuroblastoma often
have extensive tumour burdens and are typically quite ill
at presentation. This tumour has an unexplained
tendency to metastasise to the bony orbit, and thus
periorbital ecchymoses (raccoon eyes), proptosis, or
both, are classic signs of disseminated neuroblastoma.
Widespread bone and bone marrow disease can also
there can be bone marrow replacement and symptoms
of marrow failure.27 Patients have occasionally had
renin-mediated hypertension due to compromise of
renal vasculature,28 but symptoms of catecholamine
excess such as non-renin mediated hypertension and
flushing commonly seen in phaeochromocytoma are
rare. Dissemination in the central nervous system is not
often seen at diagnosis, but can occur with progression
or relapse.
4S disease
D’Angio and colleagues29 first described the striking clinical
phenotype of stage 4S (S=special) disease that occurs in
about 5% of cases. These infants have small localised
primary tumours with metastases in liver, skin, or bone
marrow that almost always spontaneously regress.
However, infants younger than 2 months frequently show
extensive and rapidly progressive intrahepatic expansion of
neuroblastoma that can result in respiratory compromise.
Diagnosis
The diagnosis of neuroblastoma is based on the presence
of characteristic histolopathological features of tumour
tissue or the presence of tumour cells in a bone marrow
aspirate or biopsy, accompanied by raised concentrations
of urinary catecholamines (figure 1). High-risk patients
often have raised concentrations of serum lactate dehydro-
genase, ferritin, or chromagranin, but these are relatively
non-specific for the population as a whole and do not seem
to be independently prognostic of outcome in light of
modern biological co-variates. Tumour-specific genetic
markers and histopathological assessment are crucial
determinants of treatment planning, especially for children
younger than 18 months. Tumour biopsies sufficient to
provide enough materials for molecular genetic analyses
are therefore highly encouraged at the time of diagnosis.
Clinical assessment of disease
Computed tomography (CT) is the preferred method for
assessment of tumours in the abdomen, pelvis, or
mediastinum. Magnetic resonance imaging is better for
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paraspinal lesions, and is essential when assessing

intra-foraminal extension with the potential for cord
compression. ⁹⁹mTc-diphosphonate scintigraphy (bone
scan) has been traditionally used to survey for occult bony
metastases. Enhanced sensitivity and specificity for
detecting bone metastases as well as occult soft tissue
disease is provided by metaiodobenzylguanidine (MIBG)
scintigraphy. Because MIBG is selectively concentrated in
more than 90% of neuroblastomas, MIBG scintigraphy is
a highly specific method for assessment of the primary
tumour and metastatic disease (figure 2).30,31 ¹²³I is the
isotope of choice because of its enhanced image resolution
compared with the ¹³¹I isotope previously used. Because a
subset of highly undifferentiated neuroblastomas might
not concentrate MIBG, Tc-diphosphonate bone scan is
only recommended when MIBG is negative or unavailable.
MIBG is recommended for re-assessment both during
and after therapy in high-risk patients with MIBG-avid
disease at diagnosis.32 The sensitivity and specificity of
positron emission tomography with fluorine-18 fluoro-
deoxyglucose or other novel positron emitters for
detection and follow-up of metastatic disease is currently
being assessed and compared to MIBG scintigraphy.33,34
Bone marrow disease should be assessed via bilateral
bone marrow aspirates and biopsies, and standard
histological analyses should be done. There is a large body
of published work regarding the use of immunocyto-
chemical and PCR-based technologies to detect neuro-
blastoma cells and neuroblastoma-specific transcripts such
as tyrosine hydroxylase, GD2 synthase, and PgP9.5 in
marrow or blood samples at diagnosis and after treatment
to assess minimal residual disease.35–39 Although these
techniques can increase the sensitivity for detecting
neuroblastoma by one or two orders of magnitude, whether
this level of sensitivity provides prognostic information
about the likelihood of disease relapse is not yet clear.

Risk stratification
Risk assessment depends on several clinical and
biological features. Because approaches to risk
stratification have varied greatly throughout the world,
efforts have been made in the past 2 years to develop a
consensus approach that will permit comparison of
outcomes for patients with neuroblastoma treated in
centres around the world. In an effort to develop an
International Neuroblastoma Risk Group (INRG)
classification system, a working group, representing the
major paediatric cooperative groups around the world, Figure 2: Metaiodobenzylguanidine (MIBG) avid neuroblastoma
met in 2005 to review data obtained for 11 054 patients Increased uptake of radio-labelled tracer can be detected in multiple sites of
treated in Europe, Japan, USA, Canada, and Australia disease, including bone and soft tissue. Figure provided by K Matthay, University
between 1974 and 2002. Consensus was reached to of California, San Francisco.

consider age (dichotomised around 18 months), stage

(assessed before treatment), and MYCN status in the
risk-group schema. The specific criteria that will be
included in the final INRG classification system will be
identified once the results of continuing statistical
analyses are completed.
Clinical variables
Various imaged-based and surgery-based systems were
used for assigning disease stage before the 1990s. In an
effort to facilitate comparison of results obtained
throughout the world, the International Neuroblastoma

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 Seminar

Staging System (INSS) was developed (panel), and it

remains in use in numerous European and North Panel: INSS staging system
American countries.40 However, because surgical 1 Localised tumour with complete gross excision, with or
approaches differ from one institution to another, INSS without microscopic residual disease; representative
stage for patients with locoregional disease can also vary ipsilateral lymph nodes negative for tumour
substantially. To define extent of disease at diagnosis in microscopically (nodes attached to and removed with the
a uniform manner, a pre-surgical INRG staging system primary tumour could be positive)
has been proposed. This system is based on an approach 2A Localised tumour with incomplete gross excision;
developed by the European International Society of representative ipsilateral non-adherent lymph nodes
Paediatric Oncology Neuroblastoma Group.41 Studies by negative for tumour microscopically
this cooperative group have shown that the radiological 2B Localised tumour with or without complete gross
characteristics of the primary tumour are useful in excision, with ipsilateral non-adherent lymph nodes
predicting successful and safe surgery. In the proposed positive for tumour. Enlarged contralateral lymph nodes
INRG staging system, extent of disease will be defined should be negative microscopically
by imaging studies and bone marrow morphology. 3 Unresectable unilateral tumour infiltrating across the
Radiological features will be used to distinguish loco- midline, with or without regional lymph node
regional tumours that do not involve local structures involvement; or localised unilateral tumour with
(INRG stage L1) from locally invasive tumours (INRG contralateral regional lymph node involvement; or
stage L2). Stages M and MS are proposed to categorise midline tumour with bilateral extension by infiltration
tumours that are widely metastatic or have an INSS 4S (unresectable) or by lymph node involvement
pattern of disease, respectively. 4 Any primary tumour with dissemination to distant lymph
Traditionally, age has been analysed as a binary nodes, bone, bone marrow, liver, skin, or other organs
variable, with 365 days used as the age cutpoint in most (except as defined by stage 4S)
risk stratification matrices. However, analysis of data 4S Localised primary tumour in infants younger than
for 3666 patients with neuroblastoma suggests that 1 year (as defined for stage 1, 2A, or 2B), with
this cutpoint might be too low, and that an age cutpoint dissemination limited to skin, liver, or bone marrow
around 15–18 months of age might be preferable.42 (<10% malignant cells)
Additional support for a modified age cutoff is provided
by two studies that showed that intensively-treated
children aged 12–18 months with INSS stage 4 tumours
without MYCN amplification had more favourable Age MYCN Ploidy Histology Other Risk group
outcomes than had previously been reported.43,44 The 1 Low
risk stratification scheme used by the Children’s 2A/2B Not amplified >50% resection Low
Oncology Group has recently been revised in light of Not amplified <50% resection Intermediate
these findings, as shown in the table. Upcoming Not amplified Biopsy only Intermediate
clinical trials will test the safety of therapy reduction in Amplified High
children between 12 months and 18 months of age with 3 < 547 days Not amplified Intermediate
INSS stage 3 or 4 disease and favourable biological ≥ 547 days Not amplified Favourable Intermediate
characteristics. Amplified High
≥ 547 days Not amplified Unfavourable High
Tumour histology 4 <365 days Amplified High
In 1984, Shimada and colleagues45 devised a classification <365 days Not amplified Intermediate
schema that relates the histopathological features of the 365–547 days Amplified High
tumour to clinical behaviour. Tumours are classified as 365–547 days DI=1 High
favourable or unfavourable depending on the degree of 365–547 days Unfavourable High
neuroblast differentiation, Schwannian stroma content, 365–547 days Not amplified DI>1 Favourable Intermediate
mitosis-karyorrhexis index, and age at diagnosis. In ≥547 days High
subsequent studies, tumours have been classified using 4S <365 days Not amplified DI>1 Favourable Asymptomatic Low
the International Neuroblastoma Pathology Classification <365 days Not amplified DI=1 Intermediate
System (INPC), a modification of the Shimada system.46
<365 days Missing Missing Missing Intermediate
The use of age in the Shimada and INPC systems has
<365 days Not amplified Symptomatic Intermediate
confounded efforts to establish the independent
<365 days Not amplified Unfavourable Intermediate
prognostic value of tumour histology in multivariable
<365 days Amplified High
analyses. In the INRG classification schema, the
histopathological features differentiation and mitosis- Data do not change risk grouping.
karyorrhexis index will be separated from age for risk
Table: Proposed Children’s Oncology Group risk stratification schema, by stage
stratification.

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 Seminar
Figure 3: MYCN amplification demonstrated by fluorescence in situ hybridisation (FISH)
The presence of multiple copies of MYCN can be readily detected in tumour cells using a labelled MYCN probe.
Figure provided by L Moreau and A Tom Look, Dana Farber Cancer Institute.
Biological variables
The genetic aberration most consistently associated
with poor outcome in neuroblastoma is genomic
amplification of MYCN.47–49 MYCN amplification occurs
in roughly 20% of primary tumours and is strongly
correlated with advanced stage disease and treatment
failure (figure 3).50,51 Its association with poor outcome
in patients with otherwise favourable disease patterns
such as localised tumours or INSS stage 4S disease
underscores its biological importance.52–54
Deletions of the short arm of chromosome 1 (1p) can be
identified in 25–35% of neuroblastomas. These deletions
correlate not only with MYCN amplification, but also with
advanced disease stage.55–59 However, the gene or genes
within chromosome 1p involved in the pathogenesis of
neuroblastoma have not been identified despite intensive
investigation. Whether the loss of heterozygosity due to
deletion of alleles from 1p is an independent indicator of
prognosis remains controversial. However, evidence
suggests that allelic loss at 1p36 predicts an increased risk
of relapse in patients with localised tumours.56,60–62
Upcoming clinical trials by the Children’s Oncology Group
and trials in Germany will stratify treatment according to
1p allelic status.
Allelic loss of 11q is present in 35–45% of primary
tumours.63–65 Notably, this genomic aberration is rarely
seen in tumours with MYCN amplification, yet remains
highly associated with other high-risk features. In a study
of almost 1000 patients registered with Children’s
Oncology Group studies, unbalanced deletion of 11q
(11q loss with either retention or gain of 11p material) was
independently prognostic for outcome in a multivariate
2110
analysis.66 In the next intermediate-risk trial by the
Children’s Oncology Group, treatment duration will be
established by tumour 1p and 11q allelic status.
A gain of 1–3 additional 17q copies, often through
unbalanced translocation with chromosome 1 or 11, can
also correlate with a more aggressive phenotype.67 The 17q
breakpoints vary, but gain of a region from 17q22-qter
suggests that a dosage effect of one or more genes provides
a selective advantage.68 Candidate genes include BIRC5
(survivin), NME1, and PPM1D, which are overexpressed
in this subset of tumours.69–71 The prognostic significance
of 17q gain relative to other genetic and biological markers
will be studied in large prospective clinical trials.
DNA index is also a prognostic marker for patients
younger than 2 years who have disseminated disease.72–74
The DNA content of neuroblastomas fall into two broad
categories: near-diploid or hyperdiploid (often near triploid).
Genetic models of neuroblastoma suggest that less
aggressive tumours have a fundamental defect in mitosis
associated with whole chromosome gains and losses, which
could explain why near-triploidy seems to be favourable.
Conversely, more malignant neuroblastomas have a
fundamental defect in genomic stability, resulting in
chromosomal rearrangements, unbalanced translocations,
and maintenance of a near-diploid DNA content.74
Principles of initial therapy
The treatment methods used in the management of
neuroblastoma include surgery, chemotherapy, radio-
therapy, and biotherapy, as well as observation alone in
carefully selected circumstances. Most paediatric oncology
clinical trials groups stratify patients into risk groups at
diagnosis based on many of the risk factors reviewed here.
The risk stratification system of the Children’s Oncology
Group incorporates patient age at diagnosis and INSS
stage, as well as tumour histopathology, DNA index, and
MYCN gene status to assign patients to one of three
risk-groups (low-risk, intermediate-risk, or high-risk) and
to stratify treatment intensity accordingly. As shown in
figure 4, risk stratification schemes used in the past 15 years
have successfully identified groups of patients with
markedly different outcomes. A major goal of continuing
clinical trials, in addition to improving overall survival, is
to prospectively validate existing risk stratification schemas
and to integrate newly discovered prognostic markers into
future algorithms in an effort to further refine the
risk-group classification system.
A comprehensive summary of current therapies across
neuroblastoma risk groups is beyond the scope of this
Seminar. However, a simplified approach requires two
important issues to be addressed. First, whether residual
tumour remains after surgical resection must be
established. If so, integration of biological and clinical data
is crucial to facilitate predictions about the behaviour of
any residual tumour. This distinction is of importance
because in many instances, biological parameters seem to
be more important than traditional clinical features as
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predictors of outcome. Neuroblastoma has a remarkable
propensity to regress or differentiate. Therefore, there are
many circumstances in which the presence of macroscopic
residual tumour, even at metastatic sites, is not an
indication for adjuvant treatment. Conversely, some
localised tumours can be near-totally resected, yet the
presence of unfavourable biological features supports the
need for intensive adjuvant therapy.
Locoregional tumours
Most localised neuroblastomas have favourable biological
features and most are successfully treated with surgery
alone.53,75–79 Studies suggest that a subset of localised
tumours will spontaneously regress, and these patients
can be safely observed without any treatment.80–83 Local
recurrences can typically be managed surgically. Metastatic
recurrences are rare and often treated successfully with
chemotherapy. The treatment of patients with localised
tumours with unfavourable biological features, especially
MYCN amplification, remains controversial. Although
such children have substantially worse outcome than
patients with localised disease that lacks MYCN
amplification, a subset could achieve long-term remission
after surgery alone.52,53,79 These rare cases require continued
prospective assessment to clarify optimum management.
The management of more invasive locoregional tumours
(INSS stage 3) also remains controversial.84 Traditionally,
patients with favourable biology INSS stage 3 tumours
have received moderately intensive chemotherapy with the
goal of facilitating subsequent surgical resection. In view of
the favourable outcome for these children, most
investigators avoid the use of radical surgery or radiotherapy.
In fact, there is a gradual movement toward the reduction
of adjuvant therapy in these cases despite the presence of
gross residual tumour,85 although these approaches need
prospective validation. By contrast, for invasive locoregional
tumours with unfavourable biological features, intensive
multimodality therapy is often required to achieve cure.
Metastatic tumours
Almost half of all patients presenting with neuroblastoma
have disease dissemination at diagnosis. Most patients with
stage 4S disease (infants with hyperdiploid, favourable
histology, single-copy MYCN tumours) fall into the low-risk
category with an overall survival probability of 85% to 92%.86
However, patients diagnosed in the first 2 months of life
seem particularly vulnerable to respiratory compromise
secondary to rapidly progressive hepatomegaly.54,86,87
Additionally, a subset of stage 4S patients have unfavourable
biological features such as MYCN amplification and often
have rapid tumour progression or eventual disease relapse,
similar to classic stage 4 disease.54,86,88 These differences
emphasise the importance of biological assessment of
tumour tissue in patients with stage 4S disease.86
Treatment of older children with widely disseminated
neuroblastoma (stage 4) remains one of the greatest
challenges for paediatric oncologists. In the past two
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Seminar
100
Low
80 Intermediate
Event-free survival rate (%)
60
40
20 High
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Years since enrolment
Figure 4: Survival of patients with neuroblastoma based on risk group
Patients treated between 1986 and 2001 in Children’s Cancer Group, Pediatric Oncology Group, and Children’s
Oncology Group studies were classified as low-risk, intermediate-risk, and high-risk at diagnosis based on clinical
and biological features. Kaplan-Meier survival analysis shows marked differences in event-free survival for these
groups of patients. Data courtesy of W London, Children’s Oncology Group statistical office.
decades, attempts have been made to improve outcomes in
high-risk patients by delivering intensive induction therapy.
Commonly used agents include cisplatin, etoposide,
doxorubicin, cyclophosphamide, and vincristine.89 The
combination of topotecan and cyclophosphamide has been
used in the relapse setting for more than a decade, and a
recent pilot study has shown the feasibility of integrating
this combination into an aggressive induction regimen.90
The Children’s Oncology Group is planning a Phase III
study with a topotecan-containing induction. During
induction therapy, stem cells are harvested in preparation
for the consolidation phase of therapy.
The goal of consolidation is to eliminate any remaining
tumour, usually with myeloablative cytotoxic agents and
stem cell rescue. Delayed surgical resection of the primary
tumour and external beam radiotherapy to the primary
and major metastatic sites are provided. The idea of
eliminating resistant tumour clones with supralethal
chemotherapy has been studied in neuroblastoma since
the early 1980s, and the results of a randomised phase III
cooperative study by the Children’s Cancer Group4 has
shown improved event-free survival with autologous
transplantation. An intention-to-treat analysis of a
randomised clinical trial of high-risk neuroblastoma
patients by the German Society of Paediatric Oncology and
Hematology also showed an improvement in 3-year
event-free survival with myeloablative therapy and
autologous stem-cell rescue compared with maintenance
chemotherapy.91 Similar to the Children’s Cancer Group
study, however, overall survival rates were not statistically
different. Prolongation of the disease free interval is
clinically important, but clearly much remains to be done
to improve overall survival for high-risk patients.
Since relapse is a frequent occurrence after autologous
transplantation, biological therapy to treat persistent
minimal residual disease has been added to current
treatment regimens. Several novel agents specifically
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targeted to the unique biology of neuroblastoma might be
effective at eliminating minimal residual disease. The
retinoids are a class of compounds known to induce
terminal differentiation of neuroblastoma cells in vitro.92
The use of 13-cis-retinoic acid in the post-transplant setting
was tested in a randomised phase III trial done by the
Children’s Cancer Group. The cohort of patients assigned
to receive post-transplant therapy with 13-cis-retinoic acid
had an improved event-free survival and toxicity was
acceptable.4 Thus, retinoid-based biotherapy in the
post-transplant setting is now widely used. Other retinoids
such as 9-cis retinoic acid,93 all trans-retinoic acid,94 or
fenretinide,95 might also have activity for minimal residual
disease in high-risk neuroblastoma patients and warrant
further study.
Assessment of response to treatment
Establishing overall response requires the assessment of
both primary and metastatic sites using the methods
discussed in this Seminar. On completion of treatment,
urinary catecholamines and imaging studies can be used to
monitor for tumour recurrence. In the relapse setting,
assessment of response to additional therapy can be
particularly challenging, especially when disease involving
cortical bone or bone marrow is detectable but difficult to
quantify. Scoring systems for quantifying MIBG positive
disease have been developed to allow assessment of
therapeutic response in patients with bony metastases.96–98
Additionally, several neuroblastoma-specific phase II
studies have been designed with strata for patients with
disease measurable by CT or MRI and for patients with
disease only involving bone marrow, cortical bone, or both.
Molecular pathogenesis
Neuroblastoma detection in early life or in utero suggests
that early disruption of normal developmental processes
plays a part in tumour initiation. Insight into the molecular
regulation of noradrenergic development has shed light on
many facets of neuroblast biology and might lead to the
identification of novel targets. Similarly, an emerging
appreciation for the role of cancer stem cells in propagating
malignant disease could prove especially helpful in
understanding and treating embryonal tumours such as
neuroblastoma.
Neuroblastoma predisposition
A family history of neuroblastoma is identified in 1–2% of
cases.99–101 These rare pedigrees support autosomal
dominant inheritance with incomplete penetrance, with
patients often diagnosed at an earlier age or with multifocal
primary tumours consistent with Knudson’s two-mutation
hypothesis.102–104 Remarkable disease heterogeneity exists
within pedigrees considering affected individuals share
the same predisposing genetic lesion,99 suggesting that the
pattern of acquired secondary genetic alterations ultimately
defines the tumour phenotype.74 Traditional genetic
analyses have identified the short arm of chromosome 16
2112
(16p12-13) as a likely predisposition locus, though no causal
gene has been identified.105 Neuroblastoma has also been
seen in several patients with constitutional chromosomal
rearrangements, including deletions overlapping putative
tumour suppressor loci at chromosome bands 1p36 and
11q14-23.106–108 Thus, neuroblastoma predisposition is
genetically heterogeneous, and initiation of tumouri-
genesis could need multiple alterations.
Comorbidities of the autonomic nervous system occur
occasionally in neuroblastoma patients, suggesting
common genetic causes. Neuroblastoma can be seen with
Hirschsprung disease, congenital central hypoventilation
syndrome, phaeochromocytoma, and neurofibromatosis
type 1.109–114 Therefore, genes causal in these disorders have
been studied for a role in tumourigenesis. Recently, loss of
function mutations in PHOX2B, a neurogenesis regulator
that is mutated in congenital central hypoventilation
syndrome,115,116 have been detected in neuroblastoma,116–118
implicating this pathway in tumour initiation in at least a
subset of cases. Taken together, the multiplicity of potential
initiating events suggests that neuroblastoma is a complex
genetic disease in which interaction of effects from multiple
genetic alterations might be needed for tumourigenesis.
Co-opting neurotrophin pathways
The neurotrophin receptors (NTRK1, NTRK2, and NTRK3
encoding TrkA, TrkB, and TrkC) and their ligands (NGF,
BDNF, and neurotrophin-3, respectively) are important
regulators of survival, growth, and differentiation of neural
cells.119 Their temporospatial expression patterns affect
sympathetic development, and their co-opted signalling
correlates with neuroblastoma phenotype (figure 5). High
TrkA expression is seen in favourable neuroblastomas with
good outcome.120,121 Explanted neuroblastoma cells with
high TrkA expression differentiate when exposed to NGF
or undergo apoptosis in the absence of NGF.121 Thus,
NGF/TrkA signalling could provoke differentiation or
regression in favourable neuroblastomas depending on the
particular microenvironment. More recently, a neuro-
developmentally regulated oncogenic splice variant of TrkA
(TrkAIII) has been identified that antagonises the
anti-oncogenic NGF/TrkA signalling and promotes neuro-
blastoma tumour growth.122 TrkB is commonly expressed in
biologically unfavourable neuroblastomas. Although a
truncated isoform lacking the catalytic tyrosine kinase
domain could be expressed in favourable tumours, full-
length TrkB is expressed along with its ligand, BDNF,
predominantly in tumours with MYCN amplification.123
Co-expression might comprise an autocrine or paracrine
survival pathway that additionally promotes chemo-
therapy resistance and metastases through anoikis
suppression.123,124
Principal carcinoma-associated pathways seem intact
The cancer genes most commonly altered in adult
carcinogenesis (eg, TP53, CDKN2A, Ras) are rarely
aberrant in neuroblastoma. TP53 inactivating mutations
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 Seminar

are infrequent in primary tumours, although they have

been documented in cell lines at relapse.125–127 Alternative NGF TrkA–II TrkA–III TrkB
mechanisms for p53 dysfunction, including HDM2 NGF
NGF+ NGF– splice BDNF+
amplification, cytoplasmic sequestration, or TWIST1- variant
NGF NGF
mediated suppression have been proposed, but their BDNF BDNF

contribution to p53 pathway inactivation in primary

tumours remains limited.128–130 Homozygous deletion of
CDKN2A (INK4A/p16) has been identified in a subset of
neuroblastoma cell lines,131 but there is no consistent
evidence for inactivation of this locus in primary tumours.
Finally, though persuasive evidence supports RAS and BDNF

MYC gene cooperation in tumourigenesis,132–134 activation P P

P
P
BDNF
of RAS does not seem to constitute a preferred secondary P P P P P

pathway for neuroblastomas, even those with MYCN

amplification.135
Thus, major oncogenic pathways governing human
neoplasia do not seem to be deregulated in neuroblastoma
with the exception of MYCN in a subset. Indeed, the only
reliable genetically engineered mouse model of
neuroblastoma results from targeted overexpression of the
human MYCN cDNA to the murine neural crest.136,137
Alternative mechanisms of MYC or MYCN activation
could contribute to pathogenesis, though none have been
identified to date. Efforts to correlate high MYCN
expression in the absence of gene amplification with
aggressive tumour attributes have been largely
unsuccessful.138,139 These studies assessed absolute MYCN
levels but not the tumour cells ability to appropriately
regulate MYCN. Persistently activated MYCN expression
invokes potent checkpoints that act as a barrier to
tumourigenesis, such that neuroblasts with MYCN
amplification must circumvent this through selection for
loss of key checkpoint regulators, leading to a highly
malignant phenotype.140,141 By contrast, this selective
pressure might not exist in infant neuroblastomas
expressing developmentally appropriate high levels of
MYCN that can be normally regulated.
The cancer stem cell conundrum
Evidence suggests that malignancies can arise or be
maintained in a stem cell compartment with the attributes
of limitless replication and self-renewal. The Notch, Sonic
hedgehog, and Wnt/β-catenin developmental programmes
play a crucial part in stem cell determination and renewal
in diverse tissues and misappropriation of these pathways
seems to be a recurring theme in embryonal
tumourigenesis.142–144 β-catenin signalling is involved in the
maintenance and expansion of neural crest stem cells145,146
and neural progenitors.147 An engineered gain-of-function
β-catenin allele targeted to neural tissues causes marked
neural progenitor expansion by promoting cell cycle entry
at the expense of differentiation.146 This pathway might
contribute to maintenance of neuroblastoma stem cells as
well, raising the question of whether emerging inhibitors
of this pathway might have therapeutic usefulness. Indeed,
our own unpublished data suggest that aberrant β-catenin
signalling could play a part in promoting a high-risk
MAPK+ Apoptosis Constitutively active PI3K+
Differentiation PI3K+ Metastasis
Survival Chemoresistance
Oncogenesis Angiogenesis
Angiogenesis
Figure 5: Schematic representation of neurotrophin tyrosine kinase receptor signalling in neuroblastoma
Neural TrkA (TrkA-II) is the preferred receptor for nerve growth factor (NGF) and is critical in development of the
peripheral nervous system. Favourable neuroblastomas frequently express high levels of TrkA but do not express
NGF. In the presence of locally-derived NGF, TrkA expressing neuroblasts terminally differentiate into mature
neural cells, whereas in the absence of NGF apoptosis (programmed cell death) ensues. Biologically unfavourable
neuroblastomas can express a constitutively active TrkA isoform (TrkA-III) that is oncogenic and promotes
angiogenesis independent of NGF. More commonly, unfavourable neuroblastomas express the TrkB receptor and
its ligand, brain-derived neurotrophic factor (BDNF), constituting an autocrine survival loop that sustains
proliferation and promotes chemoresistance and metastasis. P=phosphorylation events. MAPK+=mitogen-
associated protein kinase pathway signalling. PI3K=inositol-phosphate-3-kinase signalling.
phenotype through induction of MYC and additional
advantageous target genes in neuroblastomas without
MYCN amplification.
Stem cells also seem programmed to subvert many death
stressors, sparing them from local insults to allow for
tissue regeneration and repair. Neural stem cells promote
resistance to cytokine and death receptor mediated
apoptosis at least partly through developmentally down-
regulated caspase-8 and up-regulated PEA15.148 A
substantial proportion of primary neuroblastomas lack
caspase-8 expression,149 yet ectopic restoration of caspase-8
in deficient cells does not fully restore apoptosis. These
data suggest that inhibition of apoptosis occurs at multiple
levels, and that absent caspase-8 expression might indicate
a developmental programme or sustained stem cell feature
of neuroblasts rather than a tumour-specific somatic
mutation.140 This distinction has important implications
for experimental therapeutics targeting these latent death
signalling pathways.
Genome-wide profiling applied to neuroblastoma
Despite extensive published work correlating common
genomic alterations with disease outcome, no bonafide
target genes have been identified in neuroblastoma with
the exception of MYCN. Hemizygous deletions are typically

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 Seminar

dozens of megabases without demonstrable biallelic efforts.154–156 Pathway discovery might be further facilitated
inactivation of regional candidate genes, suggesting that by integrating genomic DNA alterations with mRNA
complex multiple gene repression or haploinsufficiency expression profiles. Transcriptional profiles have been
could be involved. Whole genome copy number alterations used to define prognostic signatures157 and regional CNAs
(CNAs) detected with array-based comparative genomic have been correlated with transcriptional alterations
hybridisation have confirmed the true complexity of (figure 6).152 Biological pathways engaged or suppressed as
chromosomal aberrations in neuroblastoma,150 identified a result of recurring CNAs are being sought. To date, these
distinct genetic subgroups,151,152 and inferred models for include upregulation of genes involved in host immune
progression.153 Refined platforms are likely to replace response and antigen processing in high-risk
region-specific methods to detect crucial CNAs used in neuroblastomas without MYCN amplification, as well as
risk-stratification schemas, and enhance gene discovery enrichment for sympathoadrenal developmental genes in
low-risk neuroblastomas.152 Ultimately, improvements in
survival will probably result from innovative treatment
approaches based on a better understanding of the crucial
biological pathways responsible for neuroblastoma
initiation and progression. A systems biology approach to
the neuroblast could be a prerequisite to such improved
understanding.
Age
INSS stage
1p36 New approaches to relapsed disease
MYCN
11q23 Although there are highly effective salvage therapies for
11q23 UNB
17q patients with low-risk and intermediate-risk disease who
PSMC5 (17)
ATP5G1 (17)
have local relapses, recurrent disease in patients with
RPS7 (17)
NME1 (17)
high-risk neuroblastoma remains a clinical challenge.
NME1 (17)
NME1 (17)
During the past several years, an expanding portfolio of
NME2 (17)
NME2 (17) new agents and combinations has been developed for
MCM7
DDX48 (17) use in the high-risk relapse setting.
PPM1D (17)
ODC1 (2)
ODC1 (2)
MYCN (2)
MYCN (2)
Cytotoxic agents
SMURF (17)
DDX1 (2)
The topoisomerase 1 inhibitors topotecan and irinotecan
NAG (2)
ALK are often used early in the relapse setting because of
ALK
NCAM1 (11)
HLA
their efficacy and their acceptable toxicity profile.158–161
HLA
HLA The efficacy of topotecan is enhanced when it is
ID2 (2)
combined with low dose cyclophosphamide.162 The
ID2 (2)
combination of irinotecan and temozolomide is well
CLSTN1 (1)
GNB1 (1)
tolerated, and efficacy is being studied. A promising
STX12 (1) novel cytoxic agent is ABT-751, an oral tubulin-binding
CAMTA1 (1)
agent that is not a classic multidrug resistance pump
CDC42 (1)
SLC35E2 (1)
substrate. Early clinical trials suggest activity against
PUM1 (1)
FYN refractory neuroblastoma,163 and a phase II trial of this
FYN
RERE (1) agent is in progress.
RERE (1)
CD44 (1)
DBH
TH
NTRK1 Targeted delivery of radionuclides
BA12 (1)
NCAM1
IGSF4 (11)
Because recurrent neuroblastoma is often a radiation
IGSF4 (11)
AASDHPPT (11)
sensitive systemic disease, there has been interest in the
PTP4A2 (1) use of radioactive molecules that are selectively
MEIS1
GATA3 concentrated in neuroblastoma cells. Approaches have
PHOX2B
included attachment of radionuclide to MIBG,164–166
somatostatin analogues,167–169 and anti-GD2 antibodies.170–172
Figure 6: Neuroblastomas classified into clinically meaningful subsets based on gene expression profiling Low dose ¹³¹I-MIBG has been shown to be effective for
Heat map representation of unsupervised two-way agglomerative hierarchical clustering of 101 primary
neuroblastomas and human fetal brain147 show five main sample groups S1 (red), S2 (yellow), S3 (blue), S4 (pink),
disease palliation.173 Dose escalation of ¹³¹I-MIBG was
and S5 (green) in addition to fetal brain control (light blue). Clinical and genomic co-variates labelled below with associated with hematopoietic toxicity.165 However, doses
dark boxes indicating: Age=older than 1 year at diagnosis; Stage=4; MYCN=Amplified; 1p36=LOH (loss of up to 18 mCi/kg are tolerable with stem cell support. A
heterozygosity); 11q23=LOH; 11q23 UNB=LOH; 17q=unbalanced gain. Gray denotes that the condition is not recent study showed a response rate of around 40% in
known for that sample. Three main gene clusters are noted as G1 (blue), G2 (yellow), and G3 (red). Genes of interest
are noted (with chromosome number in parentheses for those mapping to regions showing CNAs commonly
heavily pretreated patients.174,175 Current clinical in-
detected in neuroblastoma), and those shown more than once indicate different probe sets for the same gene on vestigation is focusing on further dose intensification of
the microarray chip. MIBG and the combination of ¹³¹I-MIBG with myelo-

2114 www.thelancet.com Vol 369 June 23, 2007


ablative therapies.176 Future trials will likely combine
¹³¹I-MIBG with radiosensitising agents.
Immunotherapy
GD2 is a disialoganglioside that is highly expressed in
most neuroblastomas, and GD2-targeted therapies using
various monoclonal antibodies have been assessed in the
phase I and II setting, and now are being studied, with or
without cytokines, in phase III trials for neuroblastoma
patients in first response.172,177,178 Other immunotherapeutic
strategies in early development include DNA,179 cellular180
and anti-idiotypic vaccination strategies181 as well as use
of engineered cytolytic T lymphocytes for cellular
immunotherapy.182
Retinoids
A randomised trial of 13-cis-retinoic acid following
myeloablative chemotherapy established the importance
of retinoids in therapy for high-risk patients.4 The
present goal is to identify the retinoid compound that
gives optimum systemic exposure without excess
toxicity183 Fenretinide can produce multi-log cell kill in
multiple neuroblastoma cell lines, even those resistant
to other retinoids.184,185 Phase I studies have shown that
the drug is generally well-tolerated, and a phase II trial
has been completed within the Children’s Oncology
Group.186,187 Newer formulations of this drug are
currently in development to facilitate oral administration
to young children.
Angiogenesis inhibitors
Tumour vascularity is correlated with an aggressive
phenotype in neuroblastoma,188 making angiogenesis
inhibitors an attractive therapeutic option. Additionally,
pro-angiogenic molecules seem to be differentially
expressed in high-risk tumours,189–191 whereas lower-risk
tumours are characterised by a stromal compartment
that provides anti-angiogenic molecules in the micro-
environment.192 Pre-clinical studies of anti-angiogenic
agents in neuroblastoma models have met with varying
degrees of success.193–199 Several strategies focused on
neutralisation of circulating vascular endothelial growth
factor are currently being studied.
Tyrosine kinase inhibitors
CEP-701, a small molecule inhibitor of Trk tyrosine
kinase, has been shown to have a substantial growth
inhibitory effect on neuroblastoma in vivo,200,201 and a
phase I clinical trial of this compound is ongoing.
Other tyrosine kinase inhibitors, including inhibitors
of the epidermal growth factor receptor, might have
activity against neuroblastoma,202 and are entering
clinical trials. Imantanib mesylate has also been
studied in neuroblastoma because some tumours
seem to express c-kit, PDGFR,203,204 or both, but
activating mutations in these receptors have not
been reported. Screens of the neuroblastoma kinome
www.thelancet.com Vol 369 June 23, 2007
Seminar
for other potential drug targets, especially those in late
stage clinical development for adult malignancies, are
continuing.
Other strategies
Epigenetic silencing of genes that are crucial for induction
of programmed cell death, such as caspase-8, seems
to occur frequently in neuroblastoma.149 Therefore,
demethylating agents such as decitabine are currently
being studied. Histone deacetylase inhibitors have also
shown preclinical activity against neuroblastoma.205,206 At
least three histone deacetylase inhibitors are now in clinical
trials for patients with refractory solid tumours. Heat shock
protein 90 inhibitors are also of interest because these
agents alter the function of molecules associated with
neuroblastoma cell growth and proliferation, including the
type I insulin-like growth factor receptor, AKT, and
TrkB.207,208 With an expanding portfolio of potential drugs to
be tested in paediatric phase I clinical trials, it is becoming
increasingly important to have a firm biological rationale
and evidence of efficacy in preclinical models to help
prioritise drug development.
Future directions
Advances in our understanding of the fundamental
genomic alterations that are associated with varying
tumour behaviour and patient outcome have moved us
closer to the goal of precise prognostication based on
molecular analysis of a panel of patient-specific and
tumour-specific variables. Array-based methods to be used
in the diagnostic setting seem plausible in the reasonably
near future.209 Although we have made some progress in
identifying neuroblastoma-specific molecular targets for
novel therapeutics, much work still needs to be done in
this area. Improved understanding of normal neuro-
development of the sympathicoadrenal system will help
us identify the key mutational events that initiate neuro-
blastoma tumourigenesis. Defining these events, as well
as those that reliably predict for the acquisition of a
high-risk phenotype, might ultimately direct us to the key
pathways that can be exploited therapeutically.
Acknowledgments
This work was supported in part by NIH grants R01-CA78545,
R01-CA87847, P01-CA97323, R01-NS049814, P30CA60553, the
Abramson Family Cancer Research Institute, the Children’s Oncology
Group (U10-CA98543), the Caitlin Robb Foundation, PANDA
Foundation, The Neuroblastoma Children’s Cancer Society, Friends
for Steven Paediatric Cancer Research Fund, Neuroblastoma Kids,
Alex’s Lemonade Stand, and the Elise Anderson Neuroblastoma
Research Fund.
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