Expert Review of Anticancer Therapy

ISSN: 1473-7140 (Print) 1744-8328 (Online) Journal homepage: https://www.tandfonline.com/loi/iery20

Overview and recent advances in the treatment of

neuroblastoma

Sarah B. Whittle, Valeria Smith, Erin Doherty, Sibo Zhao, Scott McCarty &
Peter E. Zage

To cite this article: Sarah B. Whittle, Valeria Smith, Erin Doherty, Sibo Zhao, Scott McCarty &
Peter E. Zage (2017) Overview and recent advances in the treatment of neuroblastoma, Expert
Review of Anticancer Therapy, 17:4, 369-386, DOI: 10.1080/14737140.2017.1285230

To link to this article: https://doi.org/10.1080/14737140.2017.1285230

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EXPERT REVIEW OF ANTICANCER THERAPY, 2017
VOL. 17, NO. 4, 369–386
http://dx.doi.org/10.1080/14737140.2017.1285230

REVIEW

Overview and recent advances in the treatment of neuroblastoma

Sarah B. Whittlea, Valeria Smitha, Erin Dohertya, Sibo Zhaoa, Scott McCartyb and Peter E. Zageb
a
Department of Pediatrics, Section of Hematology-Oncology, Texas Children‘s Cancer and Hematology Centers, Baylor College of Medicine,
Houston, TX, USA; bDepartment of Pediatrics, Division of Hematology-Oncology, University of California San Diego, La Jolla, CA and Peckham
Center for Cancer and Blood Disorders, Rady Children’s Hospital, San Diego, CA, USA

ABSTRACT ARTICLE HISTORY

Introduction: Children with neuroblastoma have widely divergent outcomes, ranging from cure in Received 14 June 2016
>90% of patients with low risk disease to <50% for those with high risk disease. Recent research has Accepted 18 January 2017
shed light on the biology of neuroblastoma, allowing for more accurate risk stratification and treatment KEYWORDS
reduction in many cases, although newer treatment strategies for children with high-risk and relapsed Neuroblastoma; INRG;
neuroblastoma are needed to improve outcomes. MYCN; ALK; immunotherapy;
Areas covered: Neuroblastoma epidemiology, diagnosis, risk stratification, and recent advances in MIBG; dinutuximab
treatment of both newly diagnosed and relapsed neuroblastoma.
Expert commentary: The identification of newer tumor targets and of novel cell-mediated immu-
notherapy agents may lead to novel therapeutic approaches, and clinical trials for regimens designed to
target individual genetic aberrations in tumors are underway. A combination of therapeutic modalities
will likely be required to improve survival and cure rates for patients with high-risk neuroblastoma.

1. Introduction classification schemes among large cooperative groups, a new

international classification system has been recently adopted
Neuroblastoma is a childhood tumor derived from primordial
that utilizes a new, more uniform staging system incorporat-
neural crest cells and is the most common extracranial solid
ing presurgical risk factors for more consistent staging of
tumor of childhood. Under normal conditions, neural crest cell
patients diagnosed and treated worldwide.
precursors migrate from the dorsal neural tube and differenti-
Using selected clinical, pathologic, and genetic factors,
ate upon reaching their appropriate locations into tissues and
patients can be subdivided into risk groups for treatment.
organs of the sympathetic nervous system, but in some cases,
Current overall survival (OS) rates for patients with non-high-
defects in neural crest cell migration, maturation, or differen-
risk neuroblastoma are greater than 90% with limited treat-
tiation, many of which remain to be defined, can lead to the
ment, and treatment regimens for patients with low- and
development of neuroblastoma. One of the interesting fea-
intermediate-risk neuroblastoma have been to designed to
tures of neuroblastoma is the unique clinical and biological
further decrease the therapeutic intensity and to reduce asso-
heterogeneity of neuroblastoma tumors, with some children
ciated toxicity. However, the long-term survival rates for
having tumors that regress completely or that spontaneously
patients with high-risk neuroblastoma are currently less than
differentiate without treatment, while other children have
50% despite aggressive, multimodal treatment and the recent
widespread metastatic tumors with poor outcomes despite
inclusion of immunotherapy with antibodies directed against
aggressive multimodal therapy. Clinical and laboratory
the GD2 antigen on neuroblastoma tumor cells. Ongoing
research has increased our understanding of the biology of
research to identify novel prognostic factors and therapeutic
neuroblastoma, and neuroblastoma risk stratification has
targets will hopefully lead to the development of improved
served as a paradigm for the use of clinical and biological
treatment strategies for these patients.
prognostic factors to develop therapies for use in the appro-
priate patient subsets.
The significant heterogeneity of neuroblastoma has fasci- 2. Epidemiology & genetics
nated investigators, and decades of research efforts have
The estimated incidence of neuroblastoma is 10.5 cases per
identified novel biomarkers for stratification and prognostica-
million children under 15 years of age in North America and
tion as well as novel cellular pathways that can be targeted by
Europe, with minimal ethnic or geographic variability [1–4].
new treatment strategies. Prognostic factors identified to date
Neuroblastoma accounts for approximately 10% of all pedia-
include clinical factors, such as the patient's age at diagnosis
tric cancers but unfortunately also accounts for up to 15% of
and the tumor stage, as well as biologic features of the tumor
deaths in children from cancer. Neuroblastoma is predomi-
itself, such as the presence or absence of amplification of the
nantly a cancer of small children, as the majority of children
MYCN oncogene. In order to address differences in disease risk

CONTACT Peter E. Zage pzage@ucsd.edu University of California San Diego School of Medicine, Moores Cancer Center, Room #5311, 3855 Health Sciences
Drive, MC 0815, La Jolla, CA, USA
© 2017 Informa UK Limited, trading as Taylor & Francis Group
370 S. B. WHITTLE ET AL.

with neuroblastoma are diagnosed before 5 years of age, with Table 1. Neuroblastoma patient symptoms.
a median age of patients at diagnosis of 19 months. Asymptomatic mass
Neck
Neuroblastoma is also the most common cancer diagnosed
Thorax
in infants and occurs slightly more often in boys than girls. Abdomen/pelvis
African American and Native American patients are more likely Horner syndrome
Spinal cord compression
to have more aggressive disease with lower survival rates,
Back pain, weakness
although the etiologies of these differences are unclear [5]. Bone pain
Large-scale national screening studies of infants via evalua- Limp
tion of urinary catecholamine levels have led to the diagnosis Refusal to walk
‘Blueberry muffin’ skin lesions
of neuroblastoma in a higher number of children, although Systemic symptoms
most of the detected tumors were low risk, with favorable Weight loss
clinical and biological features [6–9]. In multiple studies, Irritability
Fever
screening of asymptomatic infants has not resulted in Hypertension
increased detection rates for advanced disease, which usually Intractable diarrhea (VIP)
presents at an older age. Screening also did not improve OS Opsoclonus/Myoclonus/Ataxia Syndrome
Cytopenias
rates for neuroblastoma in these studies [10], suggesting that Anemia – pallor, fatigue
early detection of low-risk disease does not prevent the sub- Thrombocytopenia – bruising, bleeding
sequent progression of disease to more aggressive, high-risk Disseminated intravascular coagulation (DIC)
Tumor lysis syndrome
forms; rather, neuroblastoma tumors likely have established
features of either low- or high-risk disease at the time of
disease onset.
The underlying etiology of most neuroblastoma tumors relatively frequently in the general population and may con-
remains unknown, and although environmental factors have tribute to the development of sporadic neuroblastoma,
been suggested as possible causes, direct linkage has not although the functional roles of these germline variants and
been clearly established [11–13]. The vast majority of neuro- other somatic alterations remain to be elucidated.
blastoma tumors occur in isolation in families, suggesting that
the rates of oncogenic germline mutations in patients with
neuroblastoma are low. Approximately 1–2% of all cases of
3. Symptoms & diagnosis
neuroblastoma are associated with a positive family history
[14], with autosomal dominant inheritance with incomplete The presenting symptoms in children with neuroblastoma vary
penetrance. Cases of familial neuroblastoma often present based on the number and location of tumors and can include
with multifocal or bilateral adrenal primary tumors and are both local and systemic symptoms (Table 1). Approximately half of
diagnosed at a median of 9 months of age. Neuroblastoma patients present with localized or regional disease, with the
also occurs in patients with neurocristopathies such as remainder having distant metastases, with the bones, bone mar-
Hirschsprung disease and central hypoventilation syndrome row, and liver being common metastatic sites.
(CHS), and although the underlying etiologies of these asso- Most neuroblastoma tumors arise in the abdomen, and the
ciations are not clear, mutations in the PHOX2B gene that are most common primary site for neuroblastoma is the adrenal
associated with Hirschsprung disease and central hypoventila- gland. Abdominal masses may be asymptomatic or may result
tion have also been detected in some cases of familial neuro- in hypertension, abdominal pain, distension, or constipation
blastoma [15–17]. Furthermore, although there have been a from local effects on abdominal organs. Approximately 10–15%
number of reported cases of neuroblastoma in patients with of patients with neuroblastoma will have tumor extension into
neurofibromatosis, the role of germline NF1 mutations in the the epidural or intradural space that may lead to spinal cord
etiology of neuroblastoma remains unclear [18]. compression and paraplegia [32] (Figure 1). Neuroblastoma also
Large-scale genetic linkage studies performed in pedigrees commonly spreads to the orbital bones, resulting in periorbital
with familial neuroblastoma have identified several candidate ecchymoses (raccoon eyes) characteristic of neuroblastoma,
chromosomal predisposition regions, including 2p23-36, 12p, along with proptosis and eventual visual impairment. Tumors
and 16p, which subsequently led to the discovery of germline that originate in either the cervical or thoracic regions are more
mutations in the anaplastic lymphoma kinase (ALK) gene [19– common in infants and may be associated with Horner syn-
22]. The majority of patients with familial neuroblastoma have drome (unilateral ptosis, anhidrosis, and miosis) and respiratory
germline mutations in ALK [19], and sporadic neuroblastoma symptoms.
tumors also occasionally harbor ALK abnormalities, including Additional paraneoplastic syndromes associated with neu-
2–3% of tumors with genomic amplification and approxi- roblastoma include excess tumor production of vasoactive
mately 10% with missense mutations [19,21–25]. intestinal peptide (VIP) leading to refractory secretory diarrhea
Genome-wide association studies (GWAS) have also identi- [33] and opsoclonus-myoclonus- ataxia (OMA) syndrome. OMA
fied additional germline genetic variants in neuroblastoma syndrome occurs in approximately 2–3% of children with
patients, including single-nucleotide polymorphisms in neuroblastoma and often occurs in the setting of well-differ-
LIN28B, BARD1, and LMO1, among others [26–29] as well as entiated, low-risk tumors [34,35]. The symptoms of OMA are
other polymorphisms in other chromosomal regions yet to be generally felt to be immune mediated, and children with OMA
fully characterized [30,31]. These polymorphisms occur suffer from myoclonic jerks and random eye movements,
 EXPERT REVIEW OF ANTICANCER THERAPY 371

metastatic disease [38–40]. Either head CT or brain MRI ima-

ging should also be performed when intracranial metastases
are suspected or when otherwise clinically indicated. In addi-
tion to imaging studies, bone marrow aspirates and biopsies
from at least two independent sites are generally obtained to
determine tumor involvement [41]. The ultimate diagnosis of
neuroblastoma can be confirmed either by tumor tissue
biopsy with histopathologic diagnosis or by the combination
of either elevated urine or serum catecholamine levels or a
positive MIBG scan plus a bone marrow aspirate or biopsy
with detectable tumor cells.

4. Prognosis & risk stratification

With the biological and clinical heterogeneity of neuroblas-
toma tumors, a variety of prognostic features associated with
patient outcomes have been identified. Treatment strategies
for children with neuroblastoma have been tailored according
to the predicted response to therapy and risk of relapse for
more than 40 years [42], and treatment stratification has
become increasingly important as we obtain a better under-
standing of clinical and biological risk factors. These prognos-
tic factors range from clinical factors such as tumor stage and
Figure 1. Spinal MRI of a patient with spinal cord compression by neuroblas- the patient’s age at diagnosis, to biological features of the
toma tumor mass. A saggital T2-weighted image demonstrating the tumor mass
(white arrow) is shown. tumor such as histology and DNA ploidy, cytogenetic factors
including amplification of the MYCN oncogene and key chro-
mosomal deletions or gains, and serum tumor markers as well
occasionally associated with ataxia. Symptoms of OMA syn- as other molecular markers (Table 2).
drome often persist after resection, and can also be associated Using subsets of known clinical and biological prognostic
with dramatic long-term motor impairment, speech and lan- factors, patients with neuroblastoma can be classified into one
guage delay and significant cognitive dysfunction [36,37]. of three risk groups: low risk, intermediate risk (IR), and high
The evaluation to determine the disease stage in children risk (HR). Factors recently employed by the Children’s
with neuroblastoma commonly includes imaging of the pri- Oncology Group (COG) for risk stratification included patient
mary tumor site with CT or MRI to determine primary tumor age at diagnosis, tumor stage, tumor histopathology using the
size and regional invasion and spread as well as additional International Neuroblastoma Pathology Classification (INPC)
imaging of the chest, abdomen, and pelvis to identify spread system [43–45], DNA index (ploidy), and the presence of
to other distant sites (Figure 2). Meta-iodobenzylguanidine absence of MYCN amplification (Table 3). For many children
(MIBG) scans can be used to detect primary tumors and meta- with neuroblastoma, this risk stratification and subsequent
static sites, with approximately 90% of patients having MIBG- treatment determination has been successful (Figure 3), as
avid tumors. For those patients who do not have MIBG-avid the outcomes for children with low- and intermediate-risk
disease, [18 F]-fluorodeoxyglucose positron emission tomogra- neuroblastoma have been excellent, with approximately 90%
phy (FDG-PET) scans are recommended for detecting long-term event-free survival rates. However, children with

a b

Figure 2. Image of a patient with meta-iodobenzylguanidine (MIBG) avid neuroblastoma. (a). Abnormal MIBG activity within the head, chest, abdomen and multiple
osseous structures in the cervical, thoracic and lumbar spine. Additional abnormal increased uptake is seen within the sternum, bilateral proximal humeri, scapulae,
bilateral iliac bones, sacrum, bilateral ischii, bilateral femora and proximal tibiae. (b). Fused MIBG/CT axial imaging demonstrating MIBG uptake in the sternum,
vertebral body, and posterior rib (top), and in the bilateral humeri, vertebral body, and sternum (bottom).
372 S. B. WHITTLE ET AL.

Table 2. Neuroblastoma prognostic factors and features.

Favorable Unfavorable
Clinical factors
Stage 1, 2, 4S 3, 4
Age at diagnosis <547 days >547 days
Biologic tumor features
Shimada histology Favorable Unfavorable
Ganglioneuroblastoma Intermixed Nodular
DNA index Hyperdiploid, near triploid Near diploid, near tetraploid
Vascularity Low High
Cytogenetics
MYCN oncogene Non-amplified Amplified (>10 copies)
Chromosome 1p36 Normal Deletion/LOH
Chromosome 11q Normal Deletion/LOH
Chromosome 17q Normal Gain
Serum tumor markers
Ferritin Low High
LDH Low High
NSE Low High
Other miscellaneous markers
Caspase 8 Expressed Repressed (methylation)
RASSF1A Expressed Repressed (methylation)
Trk A High Low
Trk B High
CD44 High Low
MRP Low High

Table 3. Children's Oncology Group risk stratification.

Tumor stage Patient age at diagnosis MYCN amplification Histology Ploidy Risk group
1 Any Any Any Any Low
2 Any Not Amplified Any Any Low
4S <365 days Not Amplified Favorable DI > 1 Low
2a Any Not Amplified Any Any Intermediate
3 <547 days Not Amplified Any Any Intermediate
3 >547 days Not Amplified Favorable Any Intermediate
4 <365 days Not Amplified Any Any Intermediate
4 365–547 days Any Favorable DI > 1 Intermediate
4S <365 days Not Amplified Any DI = 1 Intermediate
4S <365 days Not Amplified Unfavorable Any Intermediate
2 Any Amplified Any Any High
3 >547 days Not Amplified Unfavorable Any High
3 Any Amplified Any Any High
4 <365 days Amplified Any Any High
4 365–547 days Any Any DI = 1 High
4 365–547 days Any Unfavorable Any High
4 365–547 days Amplified Any Any High
4 >547 days Any Any Any High
4S <365 days Amplified Any Any High
a
Patients with >50% residual tumor and/or symptomatic disease. DI: DNA index

high-risk neuroblastoma remain a challenge, with long-term studies have not been uniform, making it difficult to com-
survival rates for children with high-risk neuroblastoma cur- pare the results of clinical trials.
rently under 50%. To address this concern, investigators and members of
Until recently, the International Neuroblastoma Staging major national and international cooperative groups from
System (INSS) was used for neuroblastoma tumor staging North America, Europe, and Asia developed the International
[46,47]. INSS guidelines were initially developed in 1986 Neuroblastoma Risk Group (INRG) classification and staging
and subsequently revised in 1993, and these guidelines systems using data from over 8000 international patients
employed the combination of imaging and bone marrow with neuroblastoma to update and replace the INSS
evaluation for appropriate staging of neuroblastoma (Table 5). The INRG staging system (INRGSS) uses preoperative
tumors (Table 4). The development and use of interna- radiological features and image-defined risk factors (IDRFs) to
tional staging systems such as the INSS has provided con- distinguish low-risk tumors from high-risk tumors [48,49]. The
sistency in the staging of patients with neuroblastoma INRG classification system was developed based on analysis of
worldwide. However, cooperative groups from different 13 clinical and biological variables for effects on event-free
regions of the world have not consistently used the same survival rates, including patient age at diagnosis, INRGSS
markers to classify patient risk and treatment strategies, tumor stage, serum lactate dehydrogenase and ferritin levels,
and therefore the patient cohorts treated on risk-based tumor histological category, grade of differentiation, tumor
 EXPERT REVIEW OF ANTICANCER THERAPY 373

abnormalities, DNA ploidy, the primary tumor site (adrenal or

nonadrenal) and the presence or absence of metastatic dis-
ease. Seven clinically relevant and statistically significant fac-
tors (tumor stage, patient age, tumor histological category and
grade of differentiation, MYCN gene amplification status, chro-
mosome 11q aberration, and DNA ploidy) were incorporated
into the INRG classification system (Table 6) [48]. This INRG risk
stratification is currently in use in multiple national and inter-
national clinical trials, with validation of its efficacy and rele-
vance for patient outcomes pending results of these
studies [50].

5. Treatment – low- and intermediate-risk

neuroblastoma

Figure 3. Event-free survival (EFS) based on Children’s Oncology Group (COG)

Non-high-risk neuroblastoma is a heterogeneous group com-
risk stratification. Kaplan-Meier survival curves were calculated from the time of prised of patients with low- and intermediate-risk disease,
diagnosis for children enrolled onto COG, Children’s Cancer Group and Pediatric representing slightly more than half of newly diagnosed
Oncology Group Neuroblastoma trials who were classified as low, intermediate,
or high risk at the time of diagnosis. (From Park JR, Bagatell R, London WB, et al.
patients. The group includes patients with non-MYCN ampli-
Children’s Oncology Group 2013 blueprint for research: neuroblastoma. Pediatr fied localized tumors as well as most infants with metastatic
Blood Cancer 2013;60(6):986, with permission. © 2012 Wiley Periodicals, Inc.). disease. Outcomes are generally excellent for these children,
with variable treatment strategies including observation alone,
surgical resection, or moderate doses of chemotherapy with
Table 4. International Neuroblastoma Staging System (INSS). surgical resection [51,52] (Figure 3).
Stage Definition For those patients with localized, resectable disease, surgi-
1 Localized tumor with complete gross excision, with or without cal resection alone is generally curative, and chemotherapy is
microscopic residual disease; representative ipsilateral lymph nodes effective salvage for those with relapses after resection [53–
negative for tumor microscopically (nodes attached to and 55]. Low-stage tumors with favorable biological features often
removed with the primary tumor may be positive)
2A Localized tumor with incomplete gross excision; representative do not metastasize even after incomplete resection, and there-
ipsilateral nonadherent lymph nodes negative for tumor fore, for these patients, chemotherapy is reserved for those
microscopically with life or organ-threatening symptoms, such as spinal cord
2B Localized tumor with or without complete gross excision; with
ipsilateral nonadherent lymph nodes positive for tumor. Enlarged compression or respiratory compromise. The COG P9641 study
contralateral lymph nodes must be negative microscopically demonstrated excellent OS for these patients with surgical
3 Unresectable unilateral tumor infiltrating across the midline, with or resection alone, with a 5-year OS rate of 99% for those with
without regional lymph node involvement; or localized unilateral
tumor with contralateral regional lymph node involvement; or INSS stage 1 and 96% for patients with asymptomatic INSS 2A
midline tumor with bilateral extension by infiltration (unresectable) or 2B tumors [52]. The European International Society of
or by lymph node involvement Pediatric Oncology Neuroblastoma Group trial LNESG1 also
4 Any primary tumor with dissemination to distant lymph nodes, bone,
bone marrow, liver, skin and/or other organs (except as defined for demonstrated that surgical resection alone is curative in the
stage 4S) vast majority of patients with localized disease, with an OS
4S Localized primary tumor (as defined for stage 1, 2A or 2B), with rate of 99% for stage 1 patients and 93% for stage 2
dissemination limited to skin, liver, and/or bone marrow (limited to
infants <1 year of age and bone marrow with <10% tumor cell patients [55].
involvement) In recent years, clinical trials for infants with localized adre-
nal masses have demonstrated excellent outcomes with
observation alone. These masses tend to spontaneously
regress, and can be observed without surgical resection or
Table 5. International Neuroblastoma Risk Group Staging System (INRGSS). chemotherapy. In a COG prospective trial observing infants
Stage Definition less than 6-months old with small adrenal masses, 81% of
L1 Localized tumor not involving vital structures as defined by the list of subjects were effectively managed with expectant observation
image-defined risk factors and confined to one body compartment alone, while the remaining 19% eventually underwent resec-
L2 Localized tumor with the presence of one or more image-defined risk
factors (IDRFs)
tion. None required chemotherapy. 3-year event-free survival
M Metastatic disease (except stage MS) (EFS) rates were 97% with OS rates of 100% [56]. This strategy
MS Metastatic disease in children younger than 18 months of age at has become standard of practice, and the active COG
diagnosis with metastases limited to the skin, liver, and/or bone
marrow
ANBL1232 study (NCT02176967) has extended the age of
Adapted from [49].
observation for a subset of localized tumors up to 12 months
to determine whether observation alone is appropriate in this
age group as well.
mitosis-karyorrhexis index (MKI), MYCN gene amplification sta- Infants with stage 4S/MS neuroblastoma may demon-
tus, the presence or absence of chromosome 1p or 11q strate spontaneous regression, likely due to the unique
374 S. B. WHITTLE ET AL.

Table 6. International Neuroblastoma Risk Group (INRG) Risk Stratification.

INRG Patient age MYCN Gene 11q Pre-treatment risk
stage (months) Tumor histology Tumor differentiation amplification Aberration DNA ploidy group
L1/L2 Any GN maturing, Any Any Any Any Very Low
GNB intermixed
L1 Any Any* Any No Any Any Very Low
MS <18 Any Any No No Any Very Low
L2 <18 Any* Any No No Any Low
L2 ≥18 GNB nodular, Differentiating No No Any Low
neuroblastoma
M <18 Any Any No Any Hyperdiploid Low
L2 <18 Any* Any No Yes Any Intermediate
L2 ≥18 GNB nodular, Differentiating No Yes Any Intermediate
neuroblastoma
L2 ≥18 GNB nodular, Poorly Differentiated No Any Any Intermediate
neuroblastoma or Undifferentiated
M <18 Any Any No Any Diploid Intermediate
L1 Any Any* Any Yes Any Any High
L2 Any Any Any Yes Any Any High
M <18 Any Any Yes Any Any High
M ≥18 Any Any Any Any Any High
MS <18 Any Any Yes Any Any High
MS <18 Any Any Any Yes Any High
*
Except GN maturing, GNB intermixed.
GN: ganglioneuroblastoma; GNB: ganglioneuroblastoma.

biological features of neuroblastoma in infants, including In a recent European study, children with localized, unre-
near-triploid DNA content and increases in expression of sectable neuroblastoma lacking MYCN amplification received a
genes from the chromosome 1p36 region [57]. However, a reduction in chemotherapy dose intensity before an attempt
subset of these patients require immediate treatment due at surgical resection. Although this strategy demonstrated
to an increased risk for complications and death due to encouraging results in children older than 12 months, children
disease. This group includes those with massive hepato- with favorable clinical features but with tumors with unfavor-
megaly or very large tumors causing mechanical able genomics or histology had worse outcomes than those
obstruction, respiratory distress, or liver dysfunction. The with favorable biologic features [59]. Therapy has generally
youngest of the stage 4S/MS patients (those less than 3- not been reduced for those children with neuroblastoma
months old) have the highest rates of mortality and between 12 and 18 months with stage 4 disease and diploid
require careful observation and immediate treatment for tumors or for those with localized unresectable disease with
any of the above symptoms [58]. A scoring system evalu- unfavorable histology due to worse outcomes for these
ating the above symptoms of clinical deterioration is being patients [51,59,60].
prospectively evaluated in a current COG study Overall, outcomes for patients with low- or intermediate-
(NCT02176967). risk neuroblastoma continue to remain excellent despite sig-
Treatment for intermediate-risk neuroblastoma is com- nificant reductions in therapy in recent years. Efforts to further
prised of moderate doses of multiagent chemotherapy and reduce chemotherapy exposure and surgical morbidity are
surgical resection. Using clinical and genetic data, recent trials ongoing in certain subsets of this group. The current COG
have successfully reduced treatment for these patients while study ANBL1232 (NCT02176967) is investigating further reduc-
maintaining excellent survival. Risk stratification in these stu- tion of therapy for children <18 months of age with localized
dies was performed based on not only the patient’s age and tumors and favorable biology as well as expanding the obser-
tumor stage, but also on biologic features of the tumor, vation only strategy to infants <12 months old with small
including DNA content, segmental chromosomal aberrations tumors. This study is also investigating a clinical scoring sys-
and histologic features. The COG A3961 study subdivided tem to guide therapy in non-high-risk patients with the high-
patients with IR neuroblastoma into those with favorable biol- est mortality rates, namely infants with stage 4S disease with
ogy (having both favorable histology and a DNA index of evolving hepatomegaly.
more than 1) and those with unfavorable biology (having
unfavorable histology, a DNA index of 1 or less, or both),
where patients with favorable biology received 4 cycles of 6. Treatment – high-risk neuroblastoma
chemotherapy and those with unfavorable biology received Children with high-risk neuroblastoma account for approxi-
8 cycles. The final results demonstrated an 88% EFS rate and a mately half of all patients diagnosed with neuroblastoma.
96% OS rate at 3 years using this stratified treatment Despite a variety of changes in the treatment strategy for
approach, suggesting that subgroups of patients could main- patients with high-risk neuroblastoma over the years, this
tain excellent outcomes with reduced treatment [51]. Further group continues to have poor outcomes and remains one of
reduction in number of cycles given for subsets of patients is the most challenging to treat. Long-term survival rates for
being evaluated in subsequent trials, for which follow-up is children with high-risk neuroblastoma are currently around
ongoing. 40–50% in large cooperative group studies [61–64]. Although
 EXPERT REVIEW OF ANTICANCER THERAPY 375

the treatment regimens used for children with high-risk neu- novel agents prior to attempting consolidation in hopes of
roblastoma have evolved somewhat over the past decade, the improving clinical outcomes [69].
standard regimens continue to have 4 main components: (1)
Induction chemotherapy, (2) Local Control, (3) Consolidation,
6.2. Local control
and (4) Maintenance therapy.
Local control is a critical component of high-risk neuroblas-
toma therapy to prevent local recurrence of disease. Local
control treatment modalities include surgical resection, gen-
6.1. Induction erally after 4–6 cycles of induction therapy, and external beam
radiation to the primary site and other sites of active, residual
Induction is critical for children with high-risk neuroblastoma, disease. There are conflicting reports regarding the necessity
as the response to induction as measured by semi-quantitative of gross total resection in achieving the most optimal local
MIBG scoring systems is a key prognostic indicator [65]. control rates. A study from Germany using the NB97 protocol
Although a majority of children with high-risk neuroblastoma reported that the extent of surgical resection following induc-
do achieve remission after induction chemotherapy, many tion had no impact on survival rates for patients with stage 4
patients relapse even after additional consolidation therapy. neuroblastoma who were diagnosed at 18 months of age or
Furthermore, as many as 20% of patients with high-risk neu- older [70]. Additionally, a recent study evaluated the utility of
roblastoma experience disease progression during or have an surgical resection in a subgroup of patients who were poor
inadequate response to induction therapy [4]. Tumor response responders to induction therapy (tumor volume >50% of
rates are also lower in adolescents and adults, who often have initial volume after 6 cycles of induction therapy). Results
indolent, chemoresistant tumors compared to tumors in showed that patients who were poor responders to induction
younger children that tend to be more responsive to che- and underwent tumor resection had a statistically significant
motherapy [66,67]. improvement in 3-year OS over those where no surgery was
Although the specific induction regimens currently in use attempted. However, there was no significant difference in 3-
differ between different cooperative groups, most currently year EFS, and gross total resection versus subtotal resection
employed induction regimens for high-risk neuroblastoma had no effect on these outcomes [71]. Castel and colleagues
utilize a combination of anthracyclines, platinum-containing reported the results of an analysis of 98 stage IV patients, and
compounds, alkylating agents and topoisomerase II inhibitors. determined that the final outcome for patients was deter-
The most recently completed protocol for high-risk neuroblas- mined more by metastatic relapses than by the degree of
toma treatment employed by the COG utilized 6 cycles of upfront surgical resection [72]. In addition to the results of
induction chemotherapy, including the combination of topo- these individual studies, the roles of surgeon experience and
tecan and cyclophosphamide for the first 2 induction cycles, surgical techniques used in the outcomes of patients have not
cisplatin and etoposide for cycles 3 and 5 and cyclophospha- been adequately addressed, making further analyses of the
mide, vincristine and doxorubicin for cycles 4 and 6. Data role of surgical resection in the management of patients with
reflecting the efficacy and long-term outcomes in patients high-risk neuroblastoma difficult.
treated with this regimen are currently being collected [68]. In addition to surgical resection, another key component of
In contrast to the COG approach, European protocols have local control is radiotherapy, given the exquisite radio-sensi-
utilized OPEC/COJEC regimens, which include vincristine, cis- tivity of neuroblastoma tumors. Radiation therapy is typically
platin, etoposide, and cyclophosphamide in OPEC, with addi- administered after the completion of consolidation therapy
tional carboplatin for COJEC. In a recent study, a regimen (see below), but until recently there was little published data
using ‘rapid’ COJEC aiming to increase treatment dose inten- about its efficacy. A 2014 study where 30 patients received
sity was evaluated. Rapid COJEC was administered in 8 cycles, 24–30 Gy to the primary site and 24 Gy to MIBG-avid meta-
separated by 10 day intervals, allowing for completion of static lesions showed a 5-year progression-free survival rate of
induction within 70 days from administration of the first 48%, an OS rate of 59% and a 5-year primary site local control
drug. A randomized trial showed no difference in outcomes rate of 84%. The 5-year local control rate for metastatic sites
between patients treated with the rapid COJEC regimen com- was 74%. The 5-year progression-free survival rates for
pared to those treated with standard OPEC/OJEC induction patients with 0, 1, 2, and >3 post-induction MIBG-avid sites
therapies [62], and due to the ability to deliver induction over were 66%, 57%, 20%, and 0%, respectively, further suggesting
a shorter timeframe, rapid COJEC has been incorporated into that the number of MIBG-avid sites present after induction
the standard treatment regimen for these children with high- therapy is predictive of patient outcomes [73]. The most
risk neuroblastoma. recently completed COG protocol utilized 21.6 Gy external
Patients with high-risk neuroblastoma who do not respond beam radiation to the presurgical tumor volume and an addi-
to induction therapy are an even more difficult subgroup of tional 14.4 Gy boost to any gross residual tumor, with hopes
patients to treat, with long-term survival rates less than 20%. that this intervention would reduce both side effects and rates
Further genomic and proteomic analyses of these patients and of local recurrence [68].
their tumors are critical for both understanding the underlying Late effects in children with cancer are an unfortunate
mechanisms of treatment resistance as well as identifying consequence of radiation therapy, likely due to the sensitivity
novel targets for therapy. Some investigators have proposed of growing and developing normal tissues and the longer life
considering these patients for phase I and II clinical trials with expectancy for survivors [74]. Many techniques, such as
376 S. B. WHITTLE ET AL.

intensity-modulated radiation therapy (IMRT), have been shown this combination to be well tolerated. The primary
adopted in an attempt to reduce the exposure of normal reported side effect was mucositis, but all patients were able
tissues to the toxicities of radiation therapy. Proton beam to receive ASCR as scheduled and all were reported to have no
radiation therapy has been shown to reduce the side effects evidence of disease at time of follow up (median 409 days)
of radiation therapy by minimizing doses of radiation deliv- [82]. Further trials to further evaluate the efficacy of the Bu-
ered to normal tissues surrounding treated tumors [75]. Mel combination are underway.
Although the use of proton beam radiation in children with An additional critical therapeutic question for patients with
neuroblastoma has been hampered by both cost and limited high-risk neuroblastoma is the utility of repeated cycles of mye-
access, early studies have suggested that proton beam radia- loablative therapy with ASCR for patients with high-risk neuro-
tion may provide some benefit to children with advanced blastoma. The COG pilot study ANBL00P1 was developed to
neuroblastoma [76,77], and further studies are underway to assess the feasibility and toxicity of a repeated cycle of myeloa-
better evaluate the safety and efficacy of proton beam radia- blative therapy with ASCR during treatment for high-risk neuro-
tion for children with neuroblastoma (NCT02112617). blastoma. 41 patients were enrolled in this study and received
standard COG induction therapy. Those that were allowed to
proceed to transplant received myeloablative therapy with thio-
tepa and cyclophosphamide prior to the first ASCR and then CEM
6.3. Consolidation
prior to the second. The reported 3-year EFS rate for these
Consolidation therapy in most neuroblastoma treatment regi- patients was 44.8%, with an OS rate of 59.2%, and only 2 patients
mens includes myeloablative chemotherapy and autologous experienced treatment-related mortality. From these results, it
stem cell rescue (ASCR). The benefit of myeloablative therapy was concluded that the repeated course is feasible and should be
with ASCR in high-risk neuroblastoma treatment regimens has assessed further with a larger, randomized trial [83]. The most
been demonstrated in a number of studies, and a meta-ana- recently completed COG protocol randomized patients to
lysis from 2015 revealed a statistically significant improvement receive either a single course of myeloablative therapy with
in EFS when myeloablative therapy combined with ASCR were ASCR or two courses repeated consecutively. Preliminary data
used for patients [78]. However, the source of stem cells used from this trial demonstrate an improvement in outcomes with
for stem cell rescue for children with high-risk neuroblastoma tandem transplant compared to single transplant, with similar
has been the subject of some debate. Stem cells to be used for toxicity profiles, and data collection from patients treated on this
ASCR are typically harvested after 2–3 cycles of induction trial is ongoing [68].
chemotherapy. Peripheral blood has become the preferred European trials have looked at the utility of repeated
source of these stem cells because of easier collection, higher courses of myeloablative therapy with stem cell rescues as
yield, lower incidence of tumor cell contamination, and faster well, although the myeloablative therapy used included thio-
blood count recovery [79,80]. Continued concerns regarding tepa prior to the initial ASCR and then Bu-Mel prior to the
the potential for circulating neuroblastoma tumor cell contam- second. This regimen was employed in patients classified with
ination led to studies evaluating the efficacy of stem cell very high-risk neuroblastoma due to poor response of meta-
purging prior to reinfusion for children with high-risk neuro- static sites to induction therapy. Results of the initial 26
blastoma. In a recently completed trial for children with high- patients using this regimen have been reported, with patients
risk neuroblastoma through COG, 468 patients were rando- having an EFS rate of 37.3% and an OS rate of 69% [84]. Based
mized to receive purged vs. non-purged stem cells after 6 on these promising results, investigators are likely to incorpo-
cycles of induction and myeloablative consolidation therapy. rate this tandem regimen as part of the next SIOPEN protocol
Stem cells to be purged were mixed with immunomagnetic for children with high-risk neuroblastoma.
beads with a panel of 5 monoclonal antibodies targeting However, despite these recent results, additional studies
neuroblastoma tumor cell surface markers. Results of this have also suggested that the benefit of myeloablative therapy
study showed no statistically significant difference in survival with ASCR is minimal in the setting of current treatment regi-
rates between the groups, and the authors concluded that mens. In a retrospective non-randomized study of historical
purging does not alter patient outcomes [64]. results at a single institution with extended follow-up, patients
The optimal myeloablative regimen for patients with high- with high-risk neuroblastoma treated with or without myeloa-
risk neuroblastoma has also been the subject of numerous blative therapy had similar survival rates. Therefore, the
studies. The current myeloablative regimen employed in authors concluded that courses of myeloablative therapy
many protocols includes 3 days of melphalan with 4 days of may not be needed to improve outcome when anti-GD2
carboplatin and etoposide (CEM). However, recent European immunotherapy is used for consolidation after dose-intensive
data suggests that consolidation with busulfan and melphalan conventional chemotherapy [85]. These mixed results suggest
(Bu-Mel) in patients following induction with the rapid COJEC that the optimal consolidation regimen for patients with high-
regimen provides superior outcomes with no increased toxi- risk neuroblastoma remains to be determined, likely through
city concerns [81]. This SIOPEN study reported 3-year event- carefully controlled, large-scale international clinical trials.
free survival rates of 49% for those who underwent condition-
ing with Bu-Mel, compared to 33% reported for those with
6.4. Maintenance
CEM. Early data from a single institution pilot study where
patients were treated using the COG induction regimen, fol- Patients with high-risk neuroblastoma typically enter the
lowed by consolidation with the Bu-Mel combination, has maintenance phase of therapy after the completion of
 EXPERT REVIEW OF ANTICANCER THERAPY 377

induction chemotherapy, surgical resection, myeloablative + subcutaneous GM-CSF + isotretinoin) showed an overall
therapy with ASCR, and radiation therapy. While many improvement in OS in patients treated with the combination
patients achieve complete clinical remission with the above regimens [89]. Infusion times for 3F8 are significantly shorter
multimodal approach, relapse remains common, suggesting than those for ch14.18, and, although side effects associated
that minimal residual disease is an important factor in neuro- with 3F8 infusions are similar to those with ch14.18, a huma-
blastoma relapse. Over the years, several post-consolidation nized version of the 3F8 antibody is currently under investiga-
treatment options have been studied in an attempt to eradi- tion and may represent a better tolerated form of
cate residual disease and further improve event-free survival maintenance immunotherapy that can be administered on
rates. an outpatient basis.
The CCG-3891 study demonstrated the efficacy of isotreti- Another potential approach to reduce the relapse rates for
noin (13-cis-retinoic acid), a synthetic retinoid that decreases children with neuroblastoma involves re-purposing the anti-
proliferation and induces differentiation in neuroblastoma protozoal drug difluoromethylornithine (DFMO) for use in
cells, in patients with minimal residual disease when it was maintenance therapy for high-risk neuroblastoma. DFMO is
administered after chemotherapy or stem cell rescue. These an irreversible inhibitor of ornithine decarboxylase, the rate
results established a standard for the use of non-cytotoxic limiting enzyme in polyamine synthesis and whose gene a
differentiation therapy for maintenance therapy for high-risk known target of MYCN [90,91]. An open-label, single-agent,
neuroblastoma treatment [63,86]. In this study, patients were multicenter phase II study through the Neuroblastoma and
randomly assigned to receive either six cycles of isotretinoin or Medulloblastoma Translational Research Consortium (NMTRC)
no further therapy after consolidation. The 3-year EFS rate was was developed to evaluate the efficacy of DFMO as mainte-
significantly better among the 130 patients who were nance therapy in high-risk neuroblastoma patients that had
assigned to receive isotretinoin compared to the rate among achieved remission after standard therapy. Subjects received
the 128 patients assigned to receive no further therapy [86]. 27 cycles of oral DFMO at doses of 500–1000 mg/m2/day daily
Similar trends toward improved survival rates, although not for 28 day cycles. DFMO was well tolerated, with the most
reaching statistical significance, were found in the same common significant toxicity being grade 3 transaminitis
patient cohorts with extended follow-up [63]. reported in 4% of patients. The EFS and OS rates for these
A number of clinical trials testing antibodies directed patients were 92% and 98% at 2 years, significantly higher
against the GD2 ganglioside – a cell-surface marker expressed than historical controls. For the group of patients (n = 73) who
on the surface of neuroblastoma tumor cells – were performed were previously enrolled on the COG ANBL0032 study incor-
in the 1980s and 1990s and demonstrated promising results in porating immunotherapy into high-risk neuroblastoma main-
children with neuroblastoma. A recently completed rando- tenance, the 2-year event-free and OS rates were 95% and
mized-controlled phase III trial demonstrated that the addition 98%, respectively, suggesting increased survival rates for
of the anti-GD2 chimeric monoclonal antibody ch14.18 (dinu- patients who received both anti-GD2 immunotherapy and
tuximab) with cytokines granulocyte-macrophage colony-sti- DFMO [92–93]. These initial promising results will need to be
mulating factor (GM-CSF) and interleukin-2 (IL-2) to standard confirmed, however, in larger randomized trials, and the safety
isotretinoin maintenance therapy significantly improved short- and efficacy of extended maintenance therapy for children
term survival in high-risk patients, with both higher EFS and with high-risk neuroblastoma in first remission is being further
OS rates compared to standard therapy after two years of explored in an international clinical trial (NCT02395666).
follow-up [87], leading to FDA approval of dinutuximab for Overall, outcomes for patients with high-risk neuroblastoma
use in children with high-risk neuroblastoma. Data collection have been improving over the past several years with the
for this study is ongoing to determine whether this difference incorporation of immunotherapy into maintenance therapy
in survival rates is sustained over longer time periods. regimens, and recent results offer hope that extended main-
However, immunotherapy with ch14.18 is associated with a tenance therapy for patients with high-risk neuroblastoma in
number of significant side effects, including allergic reactions, first remission will result in further improvement in outcomes.
fever, hypotension, capillary leak syndrome, and pain related Efforts to improve outcomes using molecularly targeted thera-
to the cytokine effects, and ch14.18 also requires extended pies are ongoing as well, with studies to utilize the ALK inhibitor
infusion times, triggering searches for alternate forms of crizotinib in patients with known ALK mutations being devel-
immunotherapy that may be better tolerated or require oped and studies to utilize targeted therapies determined from
shorter infusion times that can be completed on an outpatient genomic analysis of initial diagnostic tumor samples already
basis. ongoing (NCT02559778). These studies offer the promise of
3F8, another murine monoclonal antibody against the GD2 further exciting results contributing to our understanding of
ganglioside, has also shown efficacy as consolidation therapy neuroblastoma pathogenesis and to our ability to determine
for patients in first remission. 3F8 treatment used for main- the most effective treatment regimens for individual patients.
tenance therapy in children with stage IV neuroblastoma
resulted in significant improvement in EFS rates compared to
historical controls in a cohort of 34 patients [88]. A larger 7. Treatment – relapsed and refractory
retrospective analysis of 166 patients with high-risk neuroblas- neuroblastoma
toma in first remission who received 3 different treatment Despite recent advances, many patients with high-risk neuro-
regimens (3F8 alone, 3F8 + IV GM-CSF + isotretinoin, or 3F8 blastoma will have either refractory disease that responds
378 S. B. WHITTLE ET AL.

poorly to therapy or will experience disease recurrence, and to regimen of 50 mg/m2/day irinotecan with 150 mg/m2/day
date there are no well-established, curative treatment regi- oral temozolomide over 5 days in a single-institution study
mens for the majority of these patients. The reported 5-year reported 2 complete responses among 19 patients with refrac-
OS rate for patients after the first relapse of neuroblastoma is tory disease, with 7 mixed responses and 10 with stable dis-
20% [94], with outcomes dependent on the time of relapse ease (SD) [109]. A subsequent multi-institutional study used
and the initial patient tumor stage [94–96]. A recently pub- lower doses but still found an overall response rate of 15%,
lished meta-analysis of three phase II clinical trials run through with an additional 53% having stable disease [110].
the SIOPEN group in Europe reported median progression free Additional commonly used chemotherapy regimens for
survival rates of 12.5% and 5.7% for patients with refractory children with relapsed neuroblastoma include combinations
and relapsed disease, respectively, while median OS rates were of ifosfamide, carboplatin, and etoposide (ICE). A single-insti-
27.9 months for patients with refractory disease versus tution study demonstrated treatment comprised of ifosfamide
11.0 months for patients with relapsed disease, confirming (2000 mg/m2 daily for 5 days), carboplatin (500 mg/m2 daily
the poor outcomes in both cohorts of patients [97]. for 2 days), and etoposide (100 mg/m2 daily for 5 days) was
Recent data reported by Modak and colleagues [98] well tolerated. Disease regression was achieved in 14 of 17
demonstrate that patients with an isolated relapse can be patients (82%) with a new relapse, 13 of 26 patients (50%)
salvaged successfully, often with surgery and/or focal radiation with refractory neuroblastoma, and 12 of 34 patients (35%)
therapy. When additional chemotherapy is needed for these who were treated for progressive disease during chemother-
patients, these authors recommend using agents with known apy [111]. The ICE regimen also demonstrated responses in
anti-neuroblastoma activity rather than experimental therapy. 37% of patients with relapsed or refractory high-risk neuro-
However, relapsed patients most commonly present with blastoma, with an additional 17% of patients having stable
metastatic, chemoresistant disease whose biology is likely sig- disease [112], while 15 of 16 patients receiving ICE as front-line
nificantly different from the initial disease due to mutations treatment for patients older than one year of age with stage 4
and other adaptations induced by prior chemotherapy. neuroblastoma had major responses, with a 37% disease-free
For those patients with relapsed neuroblastoma who are survival rate [113], suggesting a role for ifosfamide-based
treated with additional chemotherapy, initial regimens are chemotherapy regimens for both frontline and relapsed neu-
typically based on chemotherapy regimens with mechanisms roblastoma treatment.
of action different from those of the agents previously used. Additional forms of therapy have focused on cell surface
Topotecan and irinotecan are topoisomerase inhibitors that markers specific for neuroblastoma tumor cells. Because
are commonly used for treatment of relapsed neuroblastoma, neuroblastoma tumors arise from neural crest progenitors,
and both have shown single agent activity in preclinical and nearly 85% of tumors express the norepinephrine transpor-
clinical studies [99–104]. Topotecan is primarily used in com- ter on their cell surface, suggesting that the use of benzyl-
bination with cyclophosphamide, and the initial phase II study guanidine analogs that bind to this transporter would
using cycles of this combination (250 mg/m2/day cyclopho- effectively and selectively target these cells. Approximately
sphamide and 0.75 mg/m2/day topotecan for 5 days each) 90% of neuroblastoma tumors are MIBG-avid, and infusions
achieved objective responses in 6 of 13 neuroblastoma of 131I-MIBG allow for the targeted delivery of radiation
patients [105]. Further studies have shown increased response therapy directly to any sites of active disease. Initial studies
rates in patients treated with the cyclophosphamide/topote- showed overall response rates of 21–47% in patients with
can combination compared to topotecan alone (2 mg/m2/day neuroblastoma [114–116], and subsequent studies have
for 5 days), although no difference in OS rates were observed used ASCR after MIBG treatment, allowing for the adminis-
[106]. Higher doses of cyclophosphamide and topotecan com- tration of higher radiation doses. In a large phase II study,
bined with vincristine have also shown impressive results, 164 patients with relapsed or refractory neuroblastoma
including an overall response rate of 19% for patients with were treated with 131I-MIBG. Approximately one-third of
primary refractory neuroblastoma and 52% for those with first patients (36%) had evidence of clinical response; with
relapse [107]. An additional topotecan-containing regimen in approximately one-third (34%) having stable disease for a
widespread use combines topotecan (1.5 mg/m2/day for median of 6 months [117], leading to efforts to include 131I-
5 days) with a 48-hour infusion of doxorubicin (45 mg/m2) MIBG therapy as a part of upfront consolidation treatment
and vincristine (2 mg/m2), repeated every 21 days (TVD). The for children with high-risk neuroblastoma. Additional clinical
initial phase II study of TVD in patients with relapsed neuro- trials are ongoing through the New Agents in
blastoma reported an overall response rate of 64% in 25 Neuroblastoma Therapy (NANT) consortium to identify the
patients, with 4 complete responses [108]. The TVD regimen best of anticancer agents to combine with MIBG therapy
has since been incorporated into the SIOPEN HR-NBL-1 treat- (NCT02035137). However, despite the demonstrated efficacy
ment protocol for high-risk neuroblastoma (NCT01704716) as of MIBG therapy, major challenges continue to include the
salvage therapy for patients who do not have sufficient limited number of institutions capable of administration of
responses in metastatic sites of disease after induction. radioactive iodine to pediatric patients, and the accompany-
Irinotecan is primarily used in combination with the alkylat- ing need for available stem cells for autologous stem cell
ing agent temozolomide, and irinotecan/temozolomide com- rescue.
binations have been reported to have significant response While these chemotherapy and MIBG-based regimens have
rates in patients with neuroblastoma. Investigators using a demonstrated some success in the treatment of children with
 EXPERT REVIEW OF ANTICANCER THERAPY 379

relapsed neuroblastoma, subsequent disease relapses and pro- neuroblastoma. SF1126, a pan PI-3 kinase inhibitor, was
gression often leave these children with limited further treat- shown to have potent antitumor activity in neuroblastoma
ment options. Over the past decade, however, extensive preclinical models [131], suggesting the PI3K/mTOR/Akt path-
investigations into the pathogenesis of neuroblastoma have way as a therapeutic target in neuroblastoma, and SF1126 is
resulted in a number of novel targets for new therapies, and also currently being tested in a phase I clinical trial through
several agents have been identified that are highly active in the NANT consortium for children with neuroblastoma
preclinical models. Furthermore, clinical trials focused on (NCT02337309). Nifurtimox is a nitrofuran compound that
therapies guided by genomic alterations have shown early has been used since the 1970s as a primary form of therapy
promise, with the potential for individualized treatment lead- for Chagas’ disease, a parasitic infection caused by
ing to increased rates of response for patients with relapsed Trypanosoma cruzi [132,133]. Preclinical studies have shown
disease. that nifurtimox inhibits neuroblastoma cell growth in vitro and
One of the most exciting recent success stories in neuro- in vivo [134,135], and in early-phase clinical trials tumor
blastoma treatment is the recent discovery of a key role for responses were seen in patients treated with nifurtimox at a
the anaplastic lymphoma kinase (ALK) in the pathogenesis of dose of 30 mg/kg/day both as a single agent and in combina-
familial neuroblastoma [19–22]. Activating mutations in ALK tion with chemotherapy [136], leading to an ongoing national
are found in a large majority of familial cases of neuroblas- phase II trial (NCT00601003).
toma, which account for approximately 2% of all cases of More recent clinical trials have focused on the potential of
neuroblastoma, and ALK gene mutations or gene amplifica- personalized therapy for children with relapsed neuroblas-
tions have been identified in up to 15% of sporadic high-risk toma, using genomic information to direct molecularly tar-
neuroblastoma cases [19,118]. Furthermore, wild-type ALK geted therapies against the tumors in cases most likely to
expression is elevated in high-risk compared to low-risk neu- derive clinical benefit. Pilot studies have demonstrated the
roblastoma tumors [119]. A subsequent phase I trial using the feasibility of generating therapeutic treatment plans based
ALK inhibitor crizotinib in children with relapsed and refrac- on genomic profiling in less than 12 days [137], and a fol-
tory neuroblastoma has been completed [120], and further low-up national, multi-institutional phase I trial showed clinical
studies have identified synergistic combinations of ALK and benefit in 64% of patients (defined as disease stabilization for
mTOR inhibitors [121], suggesting a potential role for ALK at least one cycle of therapy or partial response), with an
inhibitors in the treatment of children with tumors with overall response rate of 7% and progression free survival
mutant ALK. Novel second-generation ALK inhibitors, such as time of 59 days [138]. A subsequent separate single-institution
ceritinib (LDK378), that are effective against the ALKF1174L study demonstrated that incorporation of tumor DNA sequen-
mutant that is resistant to crizotinib [122,123] are currently cing data into clinical management of patients was feasible,
being evaluated in clinical trials (NCT01742286). revealed potentially actionable findings in nearly half of
Other recent studies have identified the Aurora A kinase as patients, and directly led to changes in treatment and family
a potential therapeutic target in neuroblastoma tumors. The genetic counseling for some patients [139]. Although the
Aurora A kinase has a critical role regulating the mitotic results of these studies demonstrate the feasibility and safety
checkpoint complex and is essential for appropriate comple- of performing real-time genomic profiling to guide treatment
tion of mitosis [124]. However, when aberrantly overex- decision making for pediatric neuroblastoma patients, the lack
pressed, Aurora A leads to genomic instability, suppression of control groups has limited the assessments as to whether
of p53 function and resistance to apoptosis [125]. In neuro- better clinical outcomes resulted from this targeted therapy
blastoma tumors, expression of Aurora A kinase correlates approach compared to outcomes that would have occurred
with advanced stage and high-risk disease [126,127]. Small- with standard care. Ongoing national studies are underway to
molecule inhibitors of this kinase block proliferation and soft better determine the efficacy of molecularly guided therapy in
agar colony formation of neuroblastoma tumor cells and these patient populations (NCT02162732, NCT02520713).
increase sensitivity to chemotherapy [126]. Subsequent Although the efficacy of immunotherapy has been demon-
phase I trials in children with relapsed tumors demonstrated strated in patients with neuroblastoma who have minimal
some evidence of efficacy of the Aurora A kinase inhibitor residual disease, the role of immunotherapy in patients with
MLN8237 (alisertib) both alone and in combination with irino- relapsed or refractory disease is the focus of several recent and
tecan and temozolomide in children with relapsed neuroblas- ongoing studies. A recent trial combining chemotherapy with
toma [128,129], suggesting a potential role for Aurora A kinase ch14.18 (dinutuximab) demonstrated promising results. In a
inhibitors in future neuroblastoma therapy. small cohort of 6 patients with relapsed or refractory neuro-
Numerous additional ongoing clinical trials are exploring blastoma resistant to multiple prior therapies, 5 of 6 achieved
the safety and tolerability of novel agents in children with either complete or partial response with the combination
relapsed neuroblastoma. In addition to the ongoing studies therapy. This cohort included 2 patients with bulky soft tissue
of DFMO for extended maintenance therapy (see above), masses, both with complete resolution, as well as complete
DFMO has been evaluated as a single agent in a phase I response of all subjects with bone marrow disease [140]. These
clinical trial [130] and is also under investigation in combina- results led to a current COG study combining ch14.18 (dinu-
tion with other anticancer agents, including celecoxib tuximab) with irinotecan and temozolomide (NCT01767194).
(NCT02030964) and the proteasome inhibitor bortezomib In a separate study of the Hu14.18-IL-2 immunocytokine, there
(NCT02139397) in clinical trials for children with relapsed were no responses observed in 13 patients with measurable
380 S. B. WHITTLE ET AL.

soft tissue neuroblastoma tumors, whereas in those with only while a screening study using an siRNA library identified the
MIBG-avid or bone marrow disease, there were 5 complete checkpoint kinase 1 (CHK1) as a potential target [161].
responses out of 23 patients [141]. Further analyses have Transcriptome analysis of neuroblastoma tumor formation in
revealed that mismatches for natural killer (NK) cell KIR/KIR- the MYCN transgenic mouse model identified the centromere-
ligand genotypes and polymorphisms in the Fcγ receptor have associated protein E (CENP-E) as an additional potential thera-
also been associated with better responses to anti-GD2 immu- peutic target [162]. Further studies to both identify novel agents
notherapy [142,143]. With the significant side effects and targeting these pathways and identify novel critical biological
known limitations of anti-GD2 antibody immunotherapy, pathways are underway, potentially leading to the development
many other immunologic approaches have been evaluated of a number of novel therapeutic approaches to recurrent neu-
recently, including therapy with immunomodulatory CTLA4 roblastoma in the near future.
checkpoint inhibitors [144], antitumor vaccines [145,146], and
cell-based immunotherapy using either NK cells [147] or anti-
GD2 targeted autologous T cells [148], which have been 8. Late effects
shown to have antitumor activity, including activity in cases As outcomes for neuroblastoma have improved over the last
with measurable disease [149,150]. Next-generation chimeric 20 years, so too have the number of long-term survivors.
antigen receptor T cells, in which the constructs will include Survivors incur significant late effects directly related to the
costimulatory domains to activate the T cell, are currently intensity of their treatment, and as neuroblastoma treatment
being developed [151]. has become more aggressive, both the incidence and severity
In addition to the numerous recently completed and of late effects has increased as well. Patients with high-risk
ongoing clinical trials of novel agents in children with relapsed disease receive intensive multimodal therapy, with a wide
neuroblastoma, a number of additional targets have been variety of potential late effects. While treatment is less inten-
identified and are the subject of ongoing studies to identify sive for low- and intermediate-risk patients, potential disease
appropriate targeted therapeutic agents. Because of the asso- site-specific late effects as well as effects from chemotherapy
ciation of MYCN amplification with high-risk cases of neuro- exposure are still a reality.
blastoma, it represents an attractive potential therapeutic Hearing loss is one of the most common late effects for
target [152], and numerous efforts to develop MYCN inhibitors survivors of high-risk neuroblastoma, mainly due to exposure
have been made in the past. However, as a nuclear transcrip- to platinum compounds. High-frequency hearing loss may
tion factor, MYCN is difficult to target therapeutically. A recent affect up to 73% of individuals treated [163]. A study of
screening study of cancer cell lines, however, has identified European survivors found that nearly half of patients devel-
JQ1, an inhibitor of the bromodomain and extra-terminal (BET) oped hearing loss, and 9% had severe hearing loss (Brock
class of proteins, as a potent inhibitor of MYCN [153]. JQ1 grade 3 and 4), often requiring the use of hearing aids [164].
displaces BRD4 from the MYCN promoter, leading to inhibition Platinum agents are also responsible for the approximately
of MYCN transcription, cell cycle arrest and apoptosis, and 10–40% of survivors that experience long-term renal toxicities
therefore BRD4 inhibitors represent an exciting new class of including tubular dysfunction, hypertension, proteinuria/
therapeutic agents for children with relapsed neuroblastoma. hematuria, and chronic renal failure [165,166]. Survivors of
The role of signaling through the RAS-MAPK pathway in high-risk neuroblastoma treatment also have a relative risk
neuroblastoma tumor cells is poorly understood, but activat- as high as 16.2 for developing cataracts, including a 13.6-fold
ing mutations in the genes of members of this pathway have increase in the risk of blindness if cranial radiation was a
been identified in subsets of neuroblastoma tumors at diag- component of therapy [167]. Dental disease is a common
nosis [154] and at relapse [155]. Furthermore, expression of late effect and may be severe in some cases [168]. Varied
the gene for the Ras GTPase-activating protein (RasGAP) NF1 is neurological conditions, ranging from sensory deficits to
also associated with neuroblastoma patient outcomes, and overt epilepsy are also potential effects of high-risk treatment.
recent studies have identified a potential role for NF1 as a These conditions are directly associated with surgical interven-
mediator of retinoid resistance in neuroblastoma cells [156]. tions or radiotherapy but may also be secondary to receiving
Additional studies have demonstrated efficacy of the novel therapy with vinca-alkaloids.
MEK inhibitor binimetinib in preclinical models of neuroblas- The subsequent linear growth of neuroblastoma survivors
toma [155,157], suggesting that RAS-MAPK pathway inhibitors is impacted, both from endocrine and non-endocrine etiolo-
may be effective in patients with relapsed neuroblastoma. gies. Musculoskeletal changes as a result of therapy, primarily
Other potential targets in neuroblastoma tumors that have scoliosis and osteoporosis, can decrease linear growth
been identified recently include the RET tyrosine kinase, which is [163,167]. Effects on the endocrine system also create growth
expressed primarily on neural crest-derived cells and is required hormone deficiency, hypothyroidism, insulin resistance, pre-
for peripheral nervous system maturation. Studies have demon- mature ovarian failure and delayed puberty [163]. Most of
strated that RET is required for retinoic acid-induced neuroblas- these endocrinopathies are seen in the high-risk neuroblas-
toma differentiation [158], and that RET inhibition is effective in toma group and are increased with use of total body irradia-
neuroblastoma preclinical models [159]. Other recent studies tion (TBI) [163,167].
have identified the polo-like kinase 1 (PLK1) as a potential target Other organ systems such as the pulmonary and cardiac
for neuroblastoma therapy, based on screens of a library of systems may also be affected by treatment with chemother-
kinase inhibitors in neuroblastoma preclinical models [160], apy agents and radiation therapy. Cardiac toxicity may be
 EXPERT REVIEW OF ANTICANCER THERAPY 381

more related to effects of radiation, since the cumulative neuroblastoma. The critical genetic and proteomic aberrations
doses of anthracyclines used in treating neuroblastoma are that either directly cause neuroblastoma or influence its
not typically high. Late effects may also be related to the response to standard therapy are rapidly being identified,
primary site of disease. For example, patients with spinal providing the key molecular targets for future drug develop-
cord compression may have neurologic sequelae including ment and for individualized treatment strategies. A wide range
paraplegia or sensory deficits, or alternatively may have sig- of novel therapies are currently undergoing preclinical and
nificant scoliosis or kyphosis as a result of radiation therapy or clinical evaluation, and the extensive national and interna-
laminectomy to treat the cord compression [169]. Learning tional collaborations currently focused on studying this dis-
disabilities are also a potential secondary complication from ease will provide opportunities to test these new approaches
neuroblastoma treatment, in addition to psychosocial late in carefully controlled clinical trials that should result in more
effects such as low income and lower rates of employ- precise and effective therapeutic regimens. In the meantime,
ment [165]. improved international strategies to stratify patients based on
While rare, second malignant neoplasms are a potentially established clinical and biological criteria will serve to ensure
devastating late effect of neuroblastoma treatment. Between that patients receive appropriate therapeutic intensity.
2% and 7% of neuroblastoma survivors develop a second
malignancy as a result of their therapy, with a cumulative
30-year incidence for high-risk patients of approximately 10%
10. Five-year view
[170–172]. High-risk therapy, in particular, contains several
known risk factors for secondary malignancies, including alky- Neuroblastoma is a heterogeneous tumor whose molecular
lating agents, topoisomerase II inhibitors, platinum com- and genetic features dramatically affect clinical behavior.
pounds, and radiotherapy. The types of second malignancies Although a number of genetic aberrations are strongly asso-
experienced by patients after therapy for high-risk neuroblas- ciated with neuroblastoma patient outcomes, only genetic
toma are broad, with the most common diagnoses including factors that were routinely evaluated by the large cooperative
renal cell carcinoma, thyroid carcinoma, acute myeloid leuke- groups before 2002 were included in the analysis of prognos-
mia, sarcomas, and lymphoma. Hematologic malignancies tic criteria for the recently developed INRG classification sys-
tend to develop more rapidly than solid tumors after comple- tem. New technologies are now available for genomic,
tion of neuroblastoma treatment [170]. Despite these risks, epigenomic, and proteomic analyses, and numerous studies
recurrence of disease still remains more likely than secondary suggest that these analyses will lead to a further improvement
malignancies, particularly in cases of high-risk neuroblastoma. in neuroblastoma patient risk stratification. Analyses of chro-
Further complicating the ability to predict and prevent mosomal structure and gene expression profiles have been
future side effects is the more widespread use of many types shown to add critical prognostic information to individual
of novel therapies for children with high-risk neuroblastoma, genetic aberrations, and the power of these analyses is clearly
such as MIBG therapy. MIBG therapy in particular has been shown by the identification of known neuroblastoma risk
linked to new-onset thyroid dysfunction, secondary leukemias, factors (such as amplification of the MYCN gene) in addition
and myelodysplastic syndrome in early studies [173,174]. to several consistently identified novel genes and pathways
Pharmacogenomics is an evolving field that may assist in the whose role in neuroblastoma pathogenesis remains to be
understanding of genetic variations that may allow for predic- elucidated. The results of these studies will need to be vali-
tion of risks for specific chemotherapy toxicities. Continued dated in larger multinational patient cohorts to establish
studies with long-term monitoring will be crucial in helping whether these changes are independent of other genetic risk
identify those who are at highest risk, and persistent follow-up factors.
with these patients is important in helping address the myriad Clearly, risk classification will continue to be refined with
possible late effects they may experience. advances in technology and in our understanding of the
fundamental alterations that are associated with tumor
behavior and patient outcomes. For patients with non-
9. Expert commentary
high-risk cases of neuroblastoma, these factors will help to
Modern treatment for children with neuroblastoma is based identify rare patients who still require therapy as treatment
on accurate diagnosis and prognostication, with treatment intensity continues to be reduced in this population. For
strategies based on known risk factors. Recent international those patients with high-risk disease, identification of mar-
efforts to develop uniformly defined risk groups will greatly kers for those patients at highest risk of treatment failure
facilitate the comparison of risk-based clinical trials conducted and ultimately of disease recurrence is needed to signifi-
worldwide and the future development of international colla- cantly improve long-term survival rates. The optimal system
borative studies. The treatment of patients with high-risk and for risk stratification is likely to require a comprehensive
relapsed neuroblastoma remains a challenge, however, and analysis that includes both mRNA and microRNA expression,
identification of novel agents for treatments targeted at bio- epigenetic modifications, and genetic and proteomic ana-
logically relevant pathways and of novel therapies harnessing lyses, which will each require technologies capable of gen-
the innate immune system may provide new opportunities for erating rapid and reproducible results. To remain clinically
improved outcomes for these patients. relevant, the INRG classification system will need to undergo
The future holds promise for making considerable continued modification as new information is identified in
advances in our understanding and treatment of the clinical setting and confirmed to be prognostic in
382 S. B. WHITTLE ET AL.

prospective studies. With the identification of smaller 7. Hiyama E, Iehara T, Sugimoto T, et al. Effectiveness of screening for
cohorts of biologically distinct neuroblastoma patients, the neuroblastoma at 6 months of age: a retrospective population-
need for international consensus will become even more based cohort study. Lancet. 2008;371:1173–1180.
8. Schilling FH, Spix C, Berthold F, et al. Neuroblastoma screening at
critical. Future studies will likely include more refined risk one year of age. N Engl J Med. 2002;346:1047–1053.
classifications and more individualized treatments based on 9. Woods WG, Gao RN, Shuster JJ, et al. Screening of infants and mor-
specific tumor molecular and genetic aberrations. tality due to neuroblastoma. N Engl J Med. 2002;346:1041–1046.
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risk disease to <50% for those with high risk disease.
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● Treatment strategies designed to target biologically rele- chromium and nickel exposure during pregnancy and impact on
vant targets and pathways represent the future of neuro- child outcomes. J Toxicol Environ Health A. 2015;78:1348–1368.
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● Overall, outcomes for patients with low or intermediate risk ogy of neuroblastoma: a study of 426 cases at the Institut Gustave-
Roussy in France. Pediatr Blood Cancer. 2004;42:99–105.
neuroblastoma continue to remain excellent despite signif-
15. Trochet D, Bourdeaut F, Janoueix-Lerosey I, et al. Germline muta-
icant reductions in therapy. tions of the paired-like homeobox 2B (PHOX2B) gene in neuroblas-
● Despite a variety of changes in the treatment strategy for toma. Am J Hum Genet. 2004;74:761–764.
patients with high-risk neuroblastoma over the years, this 16. Rohrer T, Trachsel D, Engelcke G, et al. Congenital central hypo-
group continues to have poor outcomes. ventilation syndrome associated with Hirschsprung’s disease and
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● Molecularly guided therapy for children with neuroblas- neuroblastoma, von Recklinghausen’s neurofibromatosis,
Hirschsprung’s agangliosis and jaw-winking syndrome. Acta
toma represents a safe and potentially effective treatment
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strategy in children with relapsed neuroblastoma. 19. Mosse YP, Laudenslager M, Longo L, et al. Identification of ALK as a
major familial neuroblastoma predisposition gene. Nature.
2008;455:930–935.
Funding • This series of manuscripts (#’s 19-22) represent the initial
reports detailing the role of ALK mutations in neuroblastoma
This paper was not funded.
and identifying ALK as the first molecular therapeutic target in
neuroblastoma detected by large-scale DNA sequencing
efforts.
Declaration of interest 20. Janoueix-Lerosey I, Lequin D, Brugieres L, et al. Somatic and germ-
line activating mutations of the ALK kinase receptor in neuroblas-
The authors have no relevant affiliations or financial involvement with any
toma. Nature. 2008;455:967–970.
organization or entity with a financial interest in or financial conflict with
21. George RE, Sanda T, Hanna M, et al. Activating mutations in ALK
the subject matter or materials discussed in the manuscript. This includes
provide a therapeutic target in neuroblastoma. Nature.
employment, consultancies, honoraria, stock ownership or options, expert
2008;455:975–978.
testimony, grants or patents received or pending, or royalties.
22. Chen Y, Takita J, Choi YL, et al. Oncogenic mutations of ALK kinase
in neuroblastoma. Nature. 2008;455:971–974.
23. Schulte JH, Bachmann HS, Brockmeyer B, et al. High ALK receptor
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