neuroblastoma

TO STUDY THE EPIDEMIOLOGY, CLINICAL
PROFILE AND TREATMENT OUTCOME IN
NEUROBLASTOMA AT GCRI
DOCTORATE OF MEDICINE
(D. M.)
MEDICAL ONCOLOGY
JULY-2022

TO STUDY THE EPIDEMIOLOGY, CLINICAL
PROFILE AND TREATMENT OUTCOME IN
NEUROBLASTOMA AT GCRI
A Dissertation submitted to “The Gujarat University”
For the Degree of
DOCTORATE OF MEDICINE
(D. M.)
MEDICAL ONCOLOGY
Guided by
DR. APURVA A. PATEL
MBBS, MD, DM
PROFESSOR AND UNIT CHIEF OF THE DEPARTMENT OF
MEDICAL AND PAEDIATRIC ONCOLOGY, THE GUJARAT
CANCER RESEARCH INSTITUTE, AHMEDABAD, 380016,
GUJARAT.
(AFFILIATED TO BJ MEDICAL COLLEGE, GUJARAT
UNIVERSITY)
Submitted by
DR. SATISH SHARMA
JULY-2022

CERTIFICATE
This is to certify that Dr. Satish Sharma has satisfactorily completed his
dissertation work for his DM Medical oncology degree, Gujarat University, July
2022, titled “TO STUDY THE EPIDEMIOLOGY, CLINICAL PROFILE
AND TREATMENT OUTCOME IN NEUROBLASTOMA AT GCRI”
under my guidance and supervision.
DR. APURVA A. PATEL
MBBS, M.D., D.M. (medical oncology),
Professor and unit chief,
Department of medical and pediatric oncology,
The Gujarat Cancer and Research Institute,
B.J. Medical College, Ahmedabad. - 380016

DECLARATION
I, Dr Satish Sharma (MBBS, DNB) DM oncology trainee in The
Department of Medical and Pediatric oncology at The Gujarat Cancer and
Research Centre, B.J. Medical College, Ahmedabad hereby declare that the
dissertation “TO STUDY THE EPIDEMIOLOGY, CLINICAL PROFILE
AND TREATMENT OUTCOME IN NEUROBLASTOMA AT GCRI” is
the bonafide research work done by me under the guidance of Dr. Apurva A.
Patel Professor and unit chief of the Department of medical oncology, The
Gujarat Cancer and Research Institute, in fulfillment of the requirements for the
award of D.M. by Gujarat University. This dissertation has not formed the basis
for the award of any other diploma or degree to me by any university or board.
DR. SATISH SHARMA,
DM Oncology Trainee,
Department of medical and pediatric oncology,
G.C.R.I.,
Ahmedabad. - 380016

ACKNOWLEDGEMENT
The completion of my study brings me to the time to express my sense of
obligation to all those who supported me in multiple ways.
Firstly, I express my deep gratitude and sincere thanks to my esteemed
teacher and elite guide Dr. APURVA A. PATEL, Professor and Unit Chief,
Department of Medical Oncology, for his inspiring teaching, timely remarks,
untiring ceaseless efforts, and valuable suggestions, without whom this study
would not have materialized. I would also like to thank my teachers Dr Harsha
P Panchal and Dr Sonia Parikh for their constant support.
I sincerely thank Dr. Shashank Pandya, Director, of The Gujarat Cancer
Research Institute for his supportive role.
I would like to thank all my colleagues Dr Nikesh and Dr Aruj and our
beloved juniors Dr Debjyoti, Dr Yughanshu, and Dr Goutham Reddy for being
the source of constant knowledge and exchange of ideas and for all the support.
I am indebted to my wife Dr Geetanjili Sharma and my parents for their
incredible support.
Last, but not the least, a word of thanks to all the patients who kindly co-
operated with me in this study.
Dr. SATISH SHARMA

INDEX
SR. NO. TITLE PAGE NO.
1. INTRODUCTION 1
2. AIMS AND OBJECTIVES 3
3. REVIEW OF LITERATURE 4
4. MATERIALS AND METHODS 43
5. RESULTS AND OBSERVATIONS 45
6. DISCUSSION 64
7. CONCLUSIONS 74
8. LIMITATION 75
9. BIBLIOGRAPHY 76
10. ABBREVIATIONS 86
11. ANNEXURE I- CT Scan, Intra-operative Images 87
12. ANNEXURE II- INFORMED CONSENT 92
14. ANNEXURE III-PROFORMA 93
15. MASTER CHART ATTACHED

1
INTRODUCTION
Neuroblastoma is a childhood cancer with divergent courses.
Spontaneous regressions, chemotherapy-induced or spontaneous
maturation, and highly malignant tumor progressions are nowadays seen
in a predictable number of patients. Recent years have seen an improved
understanding of the molecular characteristics of neuroblastomas and
their association with clinical outcomes [1, 2]
. This has resulted in a
complex system with many subcategories [3, 4]
. However, a
comprehensive overview of a well-defined national cohort for the
complete spectrum of the disease is lacking.
Each year, about 800 children ages 0 to 14 are diagnosed with
neuroblastoma in North America. Neuroblastoma accounts for 6% of all
childhood cancers in the United States. Almost 90% of neuroblastoma is
found in children younger than 5. The average age of diagnosis is
between 1 and 2 years. Neuroblastoma is the most common cancer
diagnosed in children younger than 1. It is rare in people more than
10years of age. [5]
The 5-year survival rate tells you what percent of children live at
least 5 years after the cancer is found. The 5-year survival rate for
neuroblastoma is 81%. However, a child’s survival rate depends on many
factors, particularly the risk group of the tumor. [5]
For children with low-risk neuroblastoma, the 5-year survival rate
is higher than 95%. For children with intermediate-risk neuroblastoma,
the 5-year survival rate is between 90% and 95%. For children with high-
risk neuroblastoma, the the-5-year survival rate is around 50%. [5,6]
Neuroblastoma (NBL) is the most common extracranial solid
tumor in childhood in developed countries where it accounts for 10% of
pediatric cancers. [6]
However, in India, its precise incidence is unknown.
Approximately 2000 new cases of NBL may be expected to be diagnosed

2
per year in India. [7]
As per the ‘Indian Council of Medical Research(
ICMR) – National Council on Radiation Protection’,(NCRP) the relative
proportion of NBL in childhood (0-14 years) in seven hospital-based
cancer registries across India varied from 2.4% to 7.5% during 2007-11.
[8]
In a retrospective compilation of all childhood cancers, at PGIMER,
Chandigarh, NBL was the second most common solid tumor diagnosed
following retinoblastoma; of 3568 cases over a 14year period, 223 (6.3%)
patients were diagnosed with NBL. [9]
NBL is considered to have one of the least favorable outcomes
among pediatric cancers. At a Govt-run tertiary care center in India, NBL
would likely account for at least 20 new cases annually. The cure rate of
thigh–risk NBL in developed countries does not typically exceed 40%. [6,
10]
The outcome of the high–risk NBL in India is widely perceived to be
dismal. The factors contributing to a poor outcome of the high–risk NBL
in India, include late diagnosis, poor nutrition with resultant higher
treatment-related mortality, limited availability of an ASCT, and
treatment abandonment. With optimal risk stratification, judicious
administration of a management protocol, and good supportive care, the
outcome of children with NBL in India can hopefully be improved.
The hospital-based cancer registry in our hospital, The Gujarat
Cancer and Research Institute, Neuroblastoma is the most common
Extracranial solid tumor among children.

3
AIMS AND OBJECTIVES
AIM: To assess the epidemiological and clinical profile and treatment
outcome of Neuroblastoma patients attending Gujarat Cancer Research
Center, a tertiary care public sector hospital in western India, over a
period of two years.
PRIMARY OBJECTIVE: To study the treatment outcome of patients
undergoing any form of treatment, either chemotherapy or radiotherapy
or both, for neuroblastoma, during this period.
SECONDARY OBJECTIVES:
1. To study the demographic profile of patients.
2. To assess the most common symptoms, stage, and site of
metastasis at presentation in NBL.

4
REVIEW OF LITERATURE
Neuroblastoma is the most common childhood solid tumor. It
arises from embryonal neural crest tissue and accounts for approximately
15% of all pediatric oncology deaths. The prevalence is 10.7 cases per
1,000,000 persons aged 0-14 years and there are approximately 800 new
cases of neuroblastoma per year in the United States and occurs in 150-
200 children each year in Japan. [1, 11, 12]
The median age at presentation is
23 months and less than 10% of the cases are diagnosed after the age of 5
years. [1, 11]
This disease has a remarkable variation in clinical features,
ranging from a localized disease with spontaneous regression to
aggressive progression despite intensive treatment. [11]
Although most
tumors are sporadic, neuroblastoma rarely occurs as a familial or
syndromic disease. [1, 11]
Despite current intensive multimodality therapy,
children diagnosed at an advanced stage have a dismal prognosis with an
approximate 40% 5-year overall survival rate. [11]
Thus, to improve the
prognosis of neuroblastoma patients with intractable disease, new
therapeutic strategies are required.
SCREENING
The idea that one could detect childhood cancer preclinically by
screening has been and remains an appealing prospect. Neuroblastoma
screening for elevated urinary catecholamines led to a marked increase in
the incidence of the disease with no reduction in its mortality; hence, in
2004 using the markers studied, neuroblastoma screening has been be
abandoned throughout the world.
Points to avoid screening are: [12]
• it is not known if screening tests can tell the difference between
cancers that do and do not need to be treated

5
• screening may lead to people being treated without need
• there is a lack of quality evidence that screening children at 18 months
is beneficial
• there is no evidence that screening would reduce deaths from
neuroblastoma
ORIGIN OF NEUROBLASTOMA
Approximately half of the neuroblastomas arise in the adrenal
medulla (47%) followed by the abdominal/retroperitoneal (24%), thoracic
(15%), pelvic (3%), and neck (3%) regions. [1]
Based on these common primary tumor sites and the biological
features of neuroblastoma, it is widely accepted that the originating cell
for neuroblastoma arises from neural crest-derived sympathoadrenal
progenitor cells that differentiate to form sympathetic ganglion cells and
adrenal chromaffin cells. [13]
The neural crest, originating from the
embryonic ectoderm, develops from the neural tube after its closure and
produces diverse cell types including peripheral neurons, enteric neurons
and glia cells, melanocytes, Schwann cells, and cells of the craniofacial
skeleton and adrenal medulla. [13]
During embryogenesis, neural crest cells subsequently undergo an
epithelial-mesenchymal transition enabling the cells to delaminate,
migrate, and differentiate into various cell types that contribute to the
organism’s anatomical structures. [13]
This process is regulated by several
mechanisms, such as a complex network of external signaling, activation
of transcriptional programs, and epigenetic events. The dysregulation of
the process of neural crest cell development can alter cell specification
and deregulation of migration as well as cell differentiation, causing
hyper-neoplastic lesions that may eventually result in neuroblastoma
initiation and progression.

6
Neuroblastoma derives only from precursor cells or stem cells of
the sympathoadrenal lineage but never from the other lineages derived
from neural crest cells. Hence, the oncogenic events that cause
neuroblastoma may occur after the point in which migrating cells choose
to differentiate into sympathetic neurons. The super enhancer properties
of neuroblastoma cell lines have revealed two neuroblastoma subtypes: a
Noradrenergic (ADRN) type and a Mesenchymal (MES) type. These
subtypes exhibit distinct expression patterns in core regulatory circuitry-
related genes. [14, 15]
MES-type neuroblastoma cells and neural crest-
derived precursor cells share common features, whereas ADRN-type cells
are committed to the adrenergic lineage (Figure 1). Both cell types can
spontaneously interconvert to generate neuroblastoma with high
transcriptional plasticity.
Fig. 1

7
CHROMOSOMAL COPY NUMBER ALTERATIONS
Chromosomal copy number changes are the most common genetic
event in neuroblastoma (Figure 2). The best-characterized copy number
alteration associated with poor prognosis is the amplification of the
MYCN oncogene. [16]
It was also reported that the loss of heterozygosity
(LOH) on chromosome 1p correlates with poor prognosis of
neuroblastoma and several candidate tumor suppressor genes have been
identified in the common LOH regions of 1p including TP73, CHD5,
CAMTA1, KIF1B, CASZ1, and mir-34A. [17]
However, because MYCN
amplification and 1p LOH are not observed in approximately half of all
high-risk neuroblastoma patients, it has been suggested that genetic
aberrations other than MYCN amplification and 1p LOH are involved in
the development and progression of the disease.
Chromosomal deletion of 11q can be detected in 35%-40% of
primary neuroblastomas. [18]
Recently, several candidate genes
responsible for 11q LOH, such as CADM1[19]
, TSLC1[20]
, H2AFX[21]
, and
ATM[22]
, were reported by different groups. Because there is no
mechanism ensuring their complete inactivation, our group and others
revealed that synthetic lethality could be induced by treatment with a
PARP inhibitor in neuroblastoma cells with ATM haploinsufficiency. [22]
Reportedly, ATM silencing promotes neuroblastoma progression
independently of MYCN amplification. [22]
Notably, although the 11q
deletion is predominantly detected in tumors without MYCN
amplification and 1p LOH, it remains highly correlated with the dismal
prognosis of neuroblastoma patients. [18, 23]
In a study comprising a large
cohort of neuroblastoma cases registered with the Children’s Oncology
Group study, 11q LOH and 1p LOH or MYCN amplification were
independent poor prognostic markers as determined by multivariable
analysis. [18]

8
ALK is an orphan receptor tyrosine kinase normally expressed only
in the developing embryonic and neonatal central nervous system.
Because of chromosomal translocation, ALK fusion proteins are
constitutively active and have been characterized in various human
malignancies. [24]
Previously, various genome-wide studies have revealed
that ALK amplification and somatic mutations occur in <10% of primary
neuroblastoma cases (Figure 3). [25, 26, 27, 28]
Since ALK is located
proximal to the MYCN locus, it can be coamplified with MYCN; however,
solitary ALK amplification has rarely occurred. Additionally, mutations
are found in almost all cases of familial neuroblastoma. [25]
Besides the 17q gain, unbalanced translocations of 17q with 1p or
11q are found relatively frequent in neuroblastoma. [25, 31]
Previous studies
have reported that high expression of BIRC5, NME1, PPMID, and ncRAN
occurs in a subset of tumors with 17q gain, but the candidate genes
responsible for this remain elusive. [30, 31, 32, 33]
Several other recurrent
partial chromosomal imbalances have been detected by metaphase
comparative genomic hybridization and single nucleotide polymorphism
arrays including losses of 3p, 4p, 9p, and 19q and gains of 1q, 2p, 7q, and
11p [23, 26, 34]
Furthermore, hemizygous deletions and sequence alterations
of the chromatin remodeling genes, ARID1A (1p36) and ARID1B (6q25),
were identified in a subset of cases and associated with early treatment
failure and decreased survival. [35]
Subsequent functional analyses
suggested that ARID1A and ARID1B were haploinsufficient tumor
suppressor genes in MYCN-driven neuroblastoma. [36]

10
CHROMOSOMAL REARRANGEMENTS AND INSTABILITY
Recent genomic studies of neuroblastoma tumors using whole-
genome sequencing have identified loss-of-function genetic alterations
(somatic mutations, small indels, and single nucleotide variations) of
ATRX, which encodes chromatin remodeling proteins in the telomeric
region, in approximately 10% of patients with neuroblastoma. [36, 37]
Patients whose tumors had ATRX alterations were typically older than 5
years, had an indolent disease course, and a dismal prognosis. Moreover,
the rearrangements of the promoter region of TERT encoding the catalytic
subunit of telomerase were detected in approximately 25% of
neuroblastoma cases. [37, 38]
In support of an oncogenic role for TERT,
neuroblastoma cell lines having rearrangements or MYCN amplification
exhibited both upregulated TERT expression and enzymatic telomerase
activity. ATRX and TERT alterations are involved in telomere
maintenance through telomerase activity. Usually, they are not present in
cases with MYCN amplification, suggesting that telomere lengthening
represents a central mechanism that defines the high-risk group without
MYCN amplification. [39, 40]
Chromothripsis, a chromosomal instability phenomenon, describes
a new oncogenic mechanism caused by many sudden rearrangements in
the same cell in one or more chromosomes. This is in contrast to the
conventionally described mechanism in which the accumulation of
mutations over time causes cancer. [41]
Chromothripsis was observed in at
least 2%-3% of all cancers, with the highest frequencies detected in soft
tissue tumors. [42]
Recently, whole-genome sequencing also identified
chromothripsis in advanced stages of neuroblastoma. [43]
Chromothripsis-
related structural abnormalities are correlated with amplification of the
MYCN or CDK4 genes and 1p LOH [43]
, indicating that chromothripsis
suppresses neuroblastoma cell differentiation through allelic loss of

11
potential tumor suppressor genes on 1p36 involved in the induction of
differentiation. Chromothripsis is also associated with chromosomal
rearrangements of TERT resulting in a significant increase in telomere
length. [39, 40]
GENETIC RISK FACTORS
Genome-wide association studies (GWAS) further disclosed that
neuroblastoma is a complex genetic disease related to common
polymorphic alleles that can influence neuroblastoma development. At
least 12 highly significant polymorphic alleles have been identified that
can influence the development of neuroblastoma. [44]
Fig. 4
Although each association has a modest individual effect on
disease initiation, multiple perturbations can cooperate in an individual
patient to promote malignant transformation during neurodevelopment.
Many GWAS-defined neuroblastoma susceptibility genes have been

12
identified including CASC15, BRCA1-associated RING domain protein 1
(BARD1), LMO1, DUSP12, DDX4, IL31RA, HSD17B12, HACE1,
LIN28B, LINC00340, LOC729177 (FLJ44180), and NEFL. These genes
display oncogenic or tumor-suppressive functions related to the disease.
[45, 46]
The discovery of these susceptibility loci demonstrates the utility of
analyzing GWAS signals for clues into the underlying biology driving
neuroblastoma genesis.
Conversely, unlike retinoblastoma, familial neuroblastoma is
extremely rare (1%-2% of cases). [47]
Familial neuroblastoma is typically
consistent with an autosomal dominant pattern of inheritance with
incomplete penetrance. A remarkable heterogeneity of clinical behavior is
observed within pedigrees in terms of age at diagnosis, histology, and
aggressiveness. Although familial neuroblastoma is very rare, these
pedigrees provide a unique opportunity to identify the genetic drivers of
neuroblastoma.
The first predisposition gene identified in neuroblastoma was
PHOX2B, a gene encoding a paired homeodomain transcription factor
that promotes cell cycle exit and neuronal differentiation that plays a
critical role in the development of neural crest-derived autonomic
neurons. Germline mutations of PHOX2B occur in <10% of hereditary
cases of neuroblastoma, whereas somatic PHOX2B mutations are rarely
found in sporadic cases. [48]
PHOX2B mutations usually occur in neural
crest-derived disorders, such as congenital central hypoventilation
syndrome and Hirschsprung’s disease. The families with nonpolyalanine
repeat expansion mutations typically experience the most severe
phenotype, neuroblastoma-Hirschsprung’s disease-hypoventilation
syndrome association.
Thus, these observations suggest that perturbations in the
PHOX2B-regulated differentiation pathway may be a common genetic

13
factor responsible for these diseases derived from the neural crest. A
more common lesion associated with familial neuroblastoma is found in
the ALK locus. Approximately 80% of families with neuroblastoma
harbor mutations in ALK and they have also been rarely found in
germline and tumor cells of sporadic neuroblastoma. [25, 26]
Most of these mutations are located in the tyrosine kinase domain
and lead to constitutive phosphorylation, indicating that ALK mutations
found in neuroblastoma may be oncogenic drivers. Pleiotrophin are
known as natural ligand of ALK. [49]
ALK expression in the developing
sympathoadrenal progenitor of the neural crest is high and it may regulate
the balance between proliferation and differentiation through multiple
signal pathways including the MAPK and RAS-related protein 1 signal
transduction pathways. Heritable mutations in ALK are the first example
of a familial pediatric cancer arising from mutations in an oncogene.
HEREDITARY NEUROBLASTOMA [26]
Heritable neuroblastoma is a rare phenomenon, and the pediatric
oncologist should reassure parents of any newly diagnosed patient that
the risk to siblings (particularly in the absence of high-risk features such
as multifocal primary tumors) is very low. Analyses of the published
pedigrees in the past three decades strongly support the original
conclusion of an autosomal-dominant mode of inheritance with
incomplete penetrance.
SPONTANEOUS REGRESSION
Spontaneous regression of neuroblastoma has been well
documented in infants with stage 4S disease (e.g., patients <1 year of age
with metastasis limited to the skin, liver, or bone marrow). [50]
The actual
prevalence of neuroblastoma regression is known; nevertheless, previous
studies have provided evidence that regression may be at least as common
as clinically detected neuroblastoma and probably about 200-fold higher

14
than the clinically detected disease. [50]
Segmental chromosomal aberrations have characterized the tumors
from patients with stage 4 disease, whereas the majority of stage 4S
tumors are near triploid with the whole chromosomal gains. [51]
Importantly, patients with 3p and 11q abnormalities in stage 4S tumors
showed an inferior outcome compared with those without these
alterations, particularly in MYCN single-copy tumors. [51]
The accurate
mechanisms responsible for spontaneous regression are not fully known,
but several plausible mechanisms have been proposed to date. [52, 53, 54]
One of the candidate key mechanisms underlying tumor regression
is the nerve growth factor (NGF) dependency of neuroblastoma cells. [55]
NGF binds to one of the neurotrophin receptors, TRKA, and high
expression of TRKA has been observed in localized neuroblastoma and 4S
tumors. [56, 57]
When cells derived from these tumors were cultured with
endogenous NGF, they underwent neuronal differentiation and survived
for months. By contrast, cell death by apoptosis can occur within a week
in the absence of NGF. [54]
Hence, these in vitro culture conditions appear to recapitulate the
behavior of TRKA-expressing neuroblastomas in patients with neuronal
differentiation or spontaneous regression (apoptosis), depending on the
presence or absence, respectively, of NGF in the microenvironment.
Conversely, most high-risk neuroblastomas exhibited high telomerase
activity and a poor prognosis, whereas the majority of the 4S tumors had
low telomerase activity or short telomeres. [58]
These findings suggest that
telomere crisis has a role in spontaneous tumor regression. Interestingly,
when a neuroblastoma cell line was transfected with a dominant- negative
form of telomerase, the cells displayed increased apoptosis. [59]
Furthermore, neuroblastoma cells with a dominant-negative form

15
of telomerase exhibited reduced tumorigenicity in a mouse xenograft
model. Thus, these data indicate that the loss of telomerase activity and
telomere shortening are candidate mechanisms that lead to spontaneous
regression of neuroblastoma. Another potential explanation of
spontaneous regression is tumor destruction mediated by an antitumor
immune response. Approximately 50% of patients with paraneoplastic
opsomyoclonus syndrome, which is correlated with antineuronal
antibodies, differentiated tumors, and a favorable outcome in patients
with neuroblastoma, present with neuroblastoma.
This suggests that the other 50% either had neuroblastoma that
regressed or a de novo autoimmune disease. [60, 61, 62, 63]
However, whether
a humoral or cellular immune response mediates spontaneous regression
remains unclear.
EPIGENETIC REGULATION
Epigenetic alterations affecting the expression of genes relevant to
neuroblastoma development were initially reported over a decade ago and
several studies have suggested that aberrations in gene DNA methylation
or histone modification are related to clinical outcome. [64, 65, 66, 67]
Based
on the analysis of promoter hypermethylation in 45 candidate genes in 10
neuroblastoma cell lines and 10 selected genes in 118 primary
neuroblastomas through methylation-specific PCR, Alaminos et al.
reported that the CpG island hypermethylation portrait showed distinct
patterns for MYCN-amplified versus nonamplified tumors. [68]
They also
discovered that promoter hypermethylation of the HOXA9 was related to
mortality in older patients compared with infants and tumors lacking
MYCN amplification. By contrast, hypermethylation of the proapoptotic
gene, TMS1, and the cell cycle-related gene, CCND2, was correlated
with advanced-stage tumors. [68]
Additionally, it was reported that specific chromosomal regions

16
could be identified as uniquely hypermethylated or hypomethylated in
stage 4S tumors compared with other stages. They comprised
transcription factors genes associated with neural crest development
sympathetic neural differentiation. [68]
Notably, E2F1 binds to the TERT
promoter which is hypermethylated in stage 4S compared with stage 4
tumors. Lower expression of TERT was observed in stage 4S compared
with stage 4 tumors [68]
indicating that TERT DNA methylation also
regulates telomerase activity in stage 4S neuroblastoma.
More recently, genome-wide methylation analysis using Infinium
Human 450 K Bead Chips resulted in more comprehensive studies.
Henrich et al. applied an integrative approach to analyze the global
methylation patterns, transcriptomes, and copy number aberrations of 105
neuroblastoma cases, complemented by primary tumor- and cell line-
derived global histone modification analyses and epigenetic drug
treatment in vitro. [69]
IMMUNOLOGY AND IMMUNOTHERAPY
Initial evidence suggesting the existence of an immune response to
neuroblastoma was provided in 1968 when blood leukocytes from
neuroblastoma patients, including 50%-70% lymphocytes, inhibited
colony formation and exhibited cytotoxicity against their neuroblastoma
cells and allogeneic neuroblastoma cells [70]
in vitro. Also, tumors from
infant cases contained high numbers of leukocytes [68, 71]
, and
neuroblastoma in infants often showed spontaneous regression. [72, 73]
Together, these findings suggest that neuroblastoma has a characteristic
immune mechanism and the development of an antineuroblastoma
therapy based on the immune system is warranted. Several studies
support the importance of T cells and natural killer (NK) cells in the
immune response to cancer, including neuroblastoma. [74, 75]
Normally,
cytotoxic T cells (CTLs) exhibit cytotoxic activity upon presentation of

17
HLA class 1, but most neuroblastoma cells do not express HLA class I
and II molecules and thus could represent better targets for NK cells than
for CTLs. [75]
Recent studies revealed an antitumor role for NK cells in
high-risk neuroblastoma patients.
Venstrom et al. reported that killer immunoglobulin-like receptor
(KIR) and HLA gene polymorphisms interact to govern NK cell function
associated with disease progression and survival in high-risk
neuroblastoma cases treated with autologous hematopoietic stem cell
transplant (AHSCT). [76]
Those with a “missing ligand” KIR-HLA
compound genotype had a 46% lower risk of death at 3 years after
AHSCT compared with patients who possessed all ligands for inhibitory
KIR. [76]
Among all KIR-HLA combinations, 16 patients lacking the HLA-
C1 ligand for KIR2DL2/KIR2DL3 exhibited the highest 3year survival
rate (81%). In this study, the survival rate was more strongly associated
with a “missing ligand” than with tumor MYCN amplification. [76]
Thus,
NK cells have a promising role in immunotherapy in high-risk
neuroblastoma. [77]
The most obvious contribution of monoclonal antibodies (mAbs) to
high-risk neuroblastoma treatment came from the discovery of a high-
level expression of disialoganglioside (GD2) in neuroblastoma cells and
from the generation of mAbs to this surface molecule. A phase I/IB study
assessed the combination of IL-2 and murine anti-GD2 antibody 14G2A
in patients with recurrent neuroblastoma. [78]
Phase I studies were also
done to evaluate the chimeric anti-GD2 mAb ch14.18 in refractory or
relapsed patients [79]
and in patients who responded to high-dose therapy
and AHSCT. [80]
A subsequent phase I study tested the combination of
antibody dependent cellular cytotoxicity-enhancing cytokines (GMCSF
and IL-2) and ch14.18 therapy combined with cis retinoic acid (RA)

18
following high-dose therapy and AHSCT and found the regimen to be
tolerable. [81]
This clinical trial progression culminated in a recently
completed phase III randomized study of cis-RA together with ch14.18,
IL-2, and GMCSF vs. cis-RA only for patients with high-risk
neuroblastoma who had a clinical response to induction therapy and
myeloablative consolidation therapy/AHSCT.
Immunotherapy after consolidation significantly improved event-
free survival and overall survival. Hence, these findings indicate that anti-
GD2 therapy combined with GM-CSF and IL-2 will improve the survival
of patients with high-risk neuroblastoma. Other immunologic approaches
for high-risk patients with neuroblastoma include the development of
chimeric antigen receptor-modified T cells (CAR-T) and Bi-specific T-
cell Engager. [82, 83]
Although these approaches primarily use GD2 as a
tumor antigen at present, their clinical effectiveness remains unknown.
CLINICAL FEATURES
NBL can originate from anywhere along the sympathetic chain. It
is considered in the differential diagnosis of a mass arising in neck,
mediastinum, abdomen or pelvis. Nearly two-thirds of primary tumors
occur within the abdomen. [6, 10]
Adrenal NBL is more frequent in older
children than infants (40% vs. 25%). [6]
Thoracic and cervical primary
tumors are more common in infants1. Metastasis can occur to regional
lymph nodes, and by hematogenous spread to distant sites; predominantly
BM, cortical bone, liver and skin. Overall, metastatic disease is observed
in 50% patients1. It is frequent in older children as compared to infants
(60% vs. 40%). [6]
Paraspinal NBL can result in compression of nerve roots and spinal
cord. Occasionally NBL may be associated with paraneoplastic
syndromes such as opsoclonus-myoclonus-ataxia syndrome and
intractable watery diarrhea. [6, 10]
Hypertension is common; it can be

19
managed with long-acting ACE inhibitor (e.g. enalapril) or calcium
channel blocker (e.g. amlodipine)1. It typically resolves with surgical
resection/chemo-reduction of the tumor following which the anti-
hypertensive drugs can be stopped.
DIAGNOSIS
• TUMOUR MARKERS
• Catecholamine Metabolites [84]
Catecholamine metabolites represent the most sensitive and
specific tumor markers. While the determination of vanillylmandelic acid
(VMA) and homovanillic acid (HVA) in a clean void urine sample is
considered essential, the additional value of dopamine is less clear. The
simultaneous measurement of urinary creatinine permits reliable VMA
and HVA estimates in spot urine samples avoiding the uncomfortable
24hrs urine collection. The determination of VMA, HVA, and dopamine
in serum samples may be useful in some instances, but is 10–15% less
sensitive. The usefulness of catecholamine metabolites as early markers
of recurrence may be limited.
• Neuron-Specific Enolase
Neuron-specific enolase (NSE) is synthesized by neuroblastoma
cells and used as an immunohistochemical marker. Elevated serum levels
have been reported in other neuroectodermal tumors such as Ewing’s
sarcoma, small cell lung cancer, and pheochromocytoma, as well as in
acute lymphoblastic leukemia and non-Hodgkin’s lymphoma. [84]
High
levels at diagnosis were associated with poor outcome in several studies
(cutoff levels 30–100 ng/ml) when corrected for stage. [85]
Neuron-
specific enolase is less specific for neuroblastoma than the catecholamine
metabolites, but is more prognostic, and similarly valuable for monitoring
recurrent disease.
• Ferritin

20
Neuroblastoma cell lines and tumors produce and secrete ferritin
which is biochemically different (glycosylated, electrophoretic
characteristics) from that secreted by normal cells. Elevated serum ferritin
levels have been observed not only in neuroblastoma but also in
Hodgkin’s disease, leukemia, and breast cancer. While tumor cells from
infants with stage-4 and stage-4S tumors contained equivalent amounts of
ferritin, the highest serum levels were only observed in children with
stage-4 disease with poor prognosis. Although ferritin is a robust
prognostic marker at diagnosis [86]
, it is unsuitable for monitoring the
disease, because it becomes elevated from frequent blood transfusions
during chemotherapy; thus, ferritin appears helpful for estimating the
prognosis, but not for diagnosis and monitoring.
• Lactate Dehydrogenase
Several multivariate analyses demonstrated that elevated serum
lactate dehydrogenase (LDH) levels provide additional prognostic
information that is independent of stage, age, and other factors. [87]
LDH
is elevated in most children with stage-4 disease.
Table: 1 Sensitivity of abnormal homovanillic (HVA) or
vanillylmandelic acid (VMA) in relation to stage of neuroblastoma [6]
Stage of
disease
Sensitivity of abnormal HVA and/or VMA
1 78 – 85%
2 – 3 80 – 100%
4 92 – 100%
4S 100%
• BIOPSY
IHC aids in distinguishing from other small round blue cell tumors.
IHC markers for NBL classically include CD56, synaptophysin, tyrosine
hydroxylase and neuron-specific enolase8. In India, biopsy is often
performed with a Tru-Cut® needle under image guidance. An open

21
biopsy in operation-theatre under general anesthesia may often be done in
developed countries to obtain adequate material for experimental
biological studies. [80]
Surgical procedures performed at presentation are
highlighted in Table 2.
Table: 2 Surgical procedures at presentation [6]
Surgical procedures at
presentation
Tumor
Resection INRG: L1 (Localized tumor; IDRF
negative)
Biopsy only •INRG: L2 (Localized tumor; IDRF
positive)
•INRG: M and MS (metastatic disease)*
Observation only
(No resection or biopsy)
Adrenal mass in selected infants <3 months
old
*A biopsy may not be required if BM biopsy is infiltrated. Pl read
text. INRG, The International Neuroblastoma Risk Group staging system.
IDRF: image defined risk factors.
• ROLE OF BONE MARROW IN PRIMARY DIAGNOSIS
A biopsy of the tumor, besides confirming diagnosis is essential, as
well as desirable for histopathological grading and requesting for
prognostic markers (e.g. MYCN). However, a difficulty in obtaining
biopsy from the primary tumor may be observed for several reasons,
including, clinical instability, risk of bleeding secondary to
thrombocytopenia/coagulopathy, paraspinal mass with spinal cord
compression, or adverse logistics, such as an unduly late date for image-
guided biopsy in a busy center or other resource limitations. [86]
Given the
high rate of metastatic disease and BM infiltration (up to 60%) observed
in patients> 18 months age, an upfront bilateral BMA with trephine
biopsy may be considered for primary diagnosis in patients with
suggestive clinical/radiological profile. A diagnosis of NBL can be made
with observation of unequivocal tumor cells (e.g., syncytia or

22
immunocytologically positive clumps of cells) in the BM along with
raised urine/serum VMA/HVA1. [84]
In younger children, there is a lower
incidence of marrow infiltration, and a greater need for risk stratification
based on tumor pathology and biology. Accordingly, a tumor biopsy is
more desirable in the younger age-group. Patients >2 years with extensive
marrow involvement may not require a tumor biopsy as genetic studies
can be done in a clearly involved marrow specimen.
• FINE-NEEDLE ASPIRATION CYTOLOGY
FNAC is an easy, rapid and accessible investigation for diagnosis
of solid tumors. With the availability of immunocytochemical markers
applied to the cell block, the diagnosis of NBL can be made with
experienced hands. However, a biopsy is encouraged, particularly in
patients in whom MYCN amplification (MYCN-A) status will make a
critical difference in treatment approach [84]
(Pl read the later section titled
MYCN).
STAGING
Radiological staging of the primary tumor is commonly performed
with a contrast-enhanced CT scan in tumors, which primarily arise in the
chest, abdomen or pelvis. [6, 10, 88]
MRI is a superior modality for
paraspinal lesions, particularly when associated with nerve root/cord
compression. [6, 88]
Either CT or MRI may be used for a cervical mass. [88]
Metastatic evaluation classically includes bilateral BMA and trephine
biopsy and an MIBG (meta-iodobenzyl guanidine) scan. [6, 10, 88]
The
SIOP-PODC guidelines for NBL recommend obtaining a CT of the neck,
chest, abdomen and pelvis in all patients. [88]
We suggest that in patients
who undergo adequate metastatic work-up with MIBG or FDG-PET and
a BM examination, CT of the primary tumor site alone should suffice.
• MIBG /Bone scan/ FDG-PET: Which one to choose?
MIBG is the most sensitive metastatic investigation for skeletal and

23
soft tissue. [6]
MIBG can be labeled with either 131
I or 123
I11
. 123
I is
considered the radiopharmaceutical of choice as it has a more favorable
dosimetry and provides better image quality, allowing accurate
anatomical localization. [89]
Nevertheless, 131
I is what is commonly
available in major centers of India. Moreover, the iodine tracer is sourced
from elsewhere by most Indian centers, making it available intermittently,
often on a periodic (say, monthly) basis. When unavailable, or non-MIBG
avid (in up to 10% patients), a TC-99-diphosphonate scintigraphy (bone
scans) is performed1,8. 18FDG-PET is another alternative in this
situation8. The advantage of 18FDG-PET over a bone scan is that FDG-
PET can be used for re-evaluation as well. [90]
A recent study was conducted at AIIMS, New Delhi to compare the
diagnostic value of FDG-PET/CT with 131
I-MIBG scintigraphy in 40
pediatric neuroblastoma patients. [91]
On a patient-based comparison,
there was no significant difference between FDG-PET/CT and I-MIBG
(p=1. 00), however, FDG-PET/CT was superior to I-MIBG on a lesion-
based comparison (p<0.0001). Although no difference was noted for
primary lesions (p=1. 00), PET/CT was superior to I-MIBG scintigraphy
for the detection of lymph nodal (p=0. 001) and bone/BM lesions (p=0.
007). [91]
Response evaluation following chemotherapy is recommended
with MIBG. FDG-PET is a suitable alternative for response evaluation, in
the absence of MIBG. [88]
A bone scan is not reliable for re-evaluation. [90]
Any detectable response on bone scan may be evident for up to 6-8
months after therapy. On the contrary, response to therapy in the first 4-
12 weeks may result in “flare phenomenon” and increased uptake related
to the process of healing. [90]
A BM examination is an essential staging investigation in the NBL
and cannot be replaced by an imaging modality. The essential and

24
desirable investigations to be obtained in a patient with NBL prior to
treatment are listed in Table 3. [90]
Table: 3 Essential and desirable investigations in the diagnosis and
staging of neuroblastoma
Essential investigations Desirable investigations
•Complete blood count
•Serum electrolytes, uric acid, bilirubin,
liver transaminases, creatinine
• PT, Aptt (prior to invasive procedures
such as biopsy)
•CT of primary tumor with IV contrast
•Tumor biopsy with IHC markers
•Bilateral BMA and trephine
•MIBG (FDG-PET / TC-99-
diphosphonate scintigraphyalternatives
when MIBG negative or unavailable)
•MYCN gene amplification in stage 4
tumor in patients <18 months of age, and
for all stage 3 tumors.
• Serum LDH and ferritin when MYCN
unavailable in stage 3 and <18 months
old patients with stage 4
•MRI in paraspinal
tumors
•MYCN gene
amplification in all
patients
•Urinary VMA and
HVA
•DNA ploidy in tumor
tissue
•Segmental
chromosomal
aberrations (deletion of
1p, 3p, 4p or 11q or
gain of 1q, 2p or 17q)
• Staging systems: International Neuroblastoma Staging System
(INSS) vs. International Risk Group Staging System (INRGSS)
The INSS has traditionally been used by the major cooperative
groups. It carries certain inherent limitations. Firstly, the expertise and
opinion of an individual surgeon can decide whether a tumor is stage 1
(complete gross excision) or stage 3 (unresectable). [92]
Using the same
reasoning, a tumor can be “down staged” or “upstaged” simply by ability
or inability to do surgery, when the outcome is determined by several
other salient factors such as age, histopathology and tumor biology.
Further, the system is heavily dependent on lymph node sampling during
surgery, which cannot be performed in all patients with uniformity and
consistency. [92]
Subsequently, a need to classify patients based on more

25
robust criteria was perceived which led to the INRGSS based on pre-
surgical radiology and metastatic status. Such a classification offers the
advantages of permitting central review based on radiology, as well as
removing the confounding effect of surgical treatment on the stage of the
patient. [92]
The staging systems are illustrated in following Tables.
Table: 4 International Neuroblastoma Staging System [93]
1 Localized tumor with complete gross excision, with or without
microscopic residual disease; representative ipsilateral lymph
nodes negative for tumor microscopically (nodes adherent to and
removed with the primary tumor may be positive)
2A Localized tumor with incomplete gross excision; representative
ipsilateral non-adherent lymph nodes negative for tumor
microscopically
2B Localized tumor with or without complete gross excision, with
ipsilateral non-adherent lymph nodes positive for tumor. Enlarged
contralateral lymph nodes must be negative microscopically
3 Unresectable unilateral tumor infiltrating across the midlinea
, with
or without regional lymph node involvement; or localized
unilateral tumor with contralateral regional lymph node
involvement; or midline tumor with bilateral extension by
infiltration (unresectable) or by lymph node involvement
4 Any primary tumor to dissemination to distant lymph nodes,
bone, bone marrow, liver, skin and/or other organs (except as
defined for stage 4S)
4S Localized primary tumor (as defined for stage 1, 2A, or 2B), with
dissemination limited to skin, liver, and/or bone marrowb
(limited
to infants < 1 year of age)
1. The midline is defined as the vertebral column. Tumors must
originate on one side and infiltrate beyond the opposite of the
vertebral column. b. Marrow involvement in stage 4S must be <
10% of total nucleated cells on aspirate/trephine. More
extensive involvement would be considered as stage 4. MIBG
(if done) scan must be negative in marrow.
Table: 5 International Risk Group Staging System [92]
L1 Localized tumor without IDRF*. The tumor must be confined
within one body compartment, neck, chest, abdomen, or pelvis.
The isolated finding of intraspinal tumor extension that does not

26
fulfill the criteria for an IDRF* is consistent with stage L1.
L2 Localized tumor with image defined risk factors. The tumor may
be ipsilaterally continuous within body compartments (i.e., a left-
sided abdominal tumor with left-sided chest involvement should
be considered stage L2). However, a clearly left-sided abdominal
tumor with right-sided chest (or vice versa) involvement is
defined as metastatic disease.
M Distant Metastasis (i.e., not contiguous with the primary tumor)
except as defined for MS. Non-regional (distant) lymph node
involvement is metastatic disease. However, an upper abdominal
tumor with enlarged lower mediastinal nodes or a pelvic tumor
with inguinal lymph node involvement is considered loco-
regional disease. Ascites and a pleural effusion, even with
malignant cells, do not constitute metastatic disease unless they
are remote from the body compartment of the primary tumor.
MS Distant Metastasis in children younger than 18 months (547
days) with sites of metastasis limited to skin, liver, and/or bone
marrow. Bone marrow involvement should be limited to <10% of
total nucleated cells on smears or biopsy. MIBG scintigraphy
must be negative in bone and bone marrow. Provided there is
MIBG uptake in the primary tumor, bone scans are not required.
The primary tumor can be L1 or L2 and there is no restriction
regarding crossing or infiltration of the midline.
* Image defined risk factors are predefined specific radiology-
based criteria which would render the tumor difficult to resect upfront.
Image defined risk factors (IDRF) [92]
• Ipsilateral tumor extension within two body compartments
✓ Neck-chest, chest-abdomen, abdomen-pelvis
• Neck
✓ Tumor encasing carotid and/or vertebral artery and/or internal
jugular vein
✓ Tumor extending to base of skull
✓ Tumor compressing the trachea
• Cervico-thoracic junction
✓ Tumor encasing brachial plexus roots

27
✓ Tumor encasing subclavian vessels and/or vertebral and/or
carotid artery
✓ Tumor compressing the trachea
• Thorax
✓ Tumor encasing the aorta and/or major branches
✓ Tumor compressing the trachea and/or principal bronchi
✓ Lower mediastinal tumor, infiltrating the costo-vertebral junction
between T9 and T12
• Thoraco-abdominal
✓ Tumor encasing the aorta and/or vena cava
• Abdomen/pelvis
✓ Tumor infiltrating the porta hepatis and/or the hepatoduodenal
ligament
✓ Tumor encasing branches of the superior mesenteric artery at the
mesenteric root
✓ Tumor encasing the origin of the celiac axis, and/or of the
superior mesenteric artery
✓ Tumor invading one or both renal pedicles
✓ Tumor encasing the aorta and/or vena cava
✓ Tumor encasing the iliac vessels
✓ Pelvic tumor crossing the sciatic notch
• Intraspinal tumor extension whatever the location provided that:
✓ More than one third of the spinal canal in the axial plane is
invaded and/or the perimedullary leptomeningeal spaces are not
visible and/or the spinal cord signal is abnormal
• Infiltration of adjacent organs/structures

28
✓ Pericardium, diaphragm, kidney, liver, duodeno-pancreatic
block, and mesentery
• Conditions to be recorded, but not considered IDRFs
✓ Multifocal primary tumors
✓ Pleural effusion, with or without malignant cells
✓ Ascites, with or without malignant cells
• Risk stratification
Conventional factors which are utilized for risk stratification
include; age, stage, histopathological grading, serum lactate
dehydrogenase (LDH), serum ferritin, DNA ploidy, MYCN gene
amplification status, and segmental chromosomal aberrations. [90]
• Age
Age <18 months confers a superior prognosis, when there is no
MYCN gene amplification. A metastatic disease has a good outcome in
this age group as well.
• Histopathological classification
The original Shimada pathological classification of the NBL, has
been replaced by the more comprehensive International Neuroblastoma
Pathological Classification (INPC) system (Table below)16. The system
incorporates age, differentiation, maturation, stroma, and mitosis-
karyorrhexis index. Tumors are classified as favorable or unfavorable.
The INRG pre-treatment classification schema includes INPC as one of
the criteria. [92]
INPC has been underutilized in developing countries due
to a lack of sub-specialization in Pediatric pathology in majority of the
institutions. The multi-tasking pathologist is often unaware of INPC. The
SIOP-PODC risk stratification has not included it in the main schema.
However, it encourages INPC whenever expertise is available. [88]
Patients with stage 4 disease in the age group of 12-18 months are

29
considered high-risk, if the tumor is classified unfavorable by INPC
and/or diploidy/hypodiploidy (even if MYCN not amplified). [88]
Table: 6 International Neuroblastoma Pathology Classification [94]
Category and subtype Prognostic group
Neuroblastoma (Schwannian
stroma-poor)
Undifferentiated
Poorly differentiated
Differentiating
All UH
< 1.5 years with MKI < 4% FH,
rest UH
< 1.5 years with MKI < 4% and
1.5-5 years with MKI < 2% FH,
rest UH
Ganglioneuroblastoma,
intermixed (Schwannian stroma-
rich)
FH
Ganglioneuroma
(Schwannian stroma-dominant)
Maturing
Mature
FH
Ganglioneuroblastoma,
nodular (Schwannian
stroma-rich/stroma-dominant and
stroma poor)
UH
FH, Favorable histology; UH, Unfavorable histology; MKI, Mitosis-
karyorrhexis index.
• Correlation of Histopathology with MYCN Amplification and
trkA Expression
There is a reproducible correlation between the molecular event of
MYCN amplification and the morphological manifestations in pNTs. [95]
Those tumors with amplified MYCN typically are of the undifferentiated
or poorly differentiated subtype of neuroblastoma (Schwannian stroma-
poor) with markedly increased mitotic (proliferating) and karyorrhectic
(apoptotic) activities [95]
, an unfavorable histology group according to the
International Neuroblastoma Pathology Classification. The presence of
prominent nucleoli in neuroblastic cells of undifferentiated or poorly
differentiated neuroblastoma, often associated with unfavorable prognosis

30
[96]
, can be an additional hallmark of MYCN amplification. Favorable
histology neuroblastoma tumors include both poorly differentiated and
differentiating subtypes: although there is no difference in the level of
trkA expression between these two histological subtypes, tumors of
differentiating subtype are diagnosed in significantly older children
(usually between 1 and 5 years of age) than those of poorly differentiated
subtype (newborn to 1.5 years of age).
Table: 7 Very low and low-risk NBL: Risk stratification and
management [97, 98]
Risk
group
Criteria Management
Very low-
risk
Small adrenal mass
detected in infants < 3-6
months age or antenatally
Observation
Low-risk* L1, any age, MYCN non-
amplified
Surgery/observation only
Infants aged ≤18 months
with localized
unresectable (INRG: L2),
MYCN non-amplified
tumors
Can observe with three
monthly imaging if no LTS
and lacking SCA. If status of
SCA is not known, or if LTS
present: treat with 2-4 courses
of carbo/etop. If LTS still
persist, administer: CADO X
2. Surgery after chemo only if
IDRF negative, otherwise
observation.
Infants aged ≤ 12 months
with INRG stage MS
(INSS: 4S), MYCN non-
amplified
<3 months; even if
asymptomatic: treat with 2-4
courses of carbo/etop. If
Philadelphia score (see Table
12) ≥ 2: treat with 2-4 courses
of carbo/etop. If still
symptomatic, proceed with
CADO X 2. If Philadelphia
score is <2: observation.
*If NMYC unavailable in patients otherwise classified as low-risk,
assume to be low-risk.

31
SCA: segmental chromosomal aberrations. LTS: life-threatening
symptoms. IDRF: image defined risk factors; INRG, The International
Neuroblastoma Risk Group staging system.
Table: 8 Intermediate-risk NBL: Risk stratification and management
[97, 98]
Criteria Management
Age >18 months with
localized unresectable
(INRG: L2) non-MYCN
amplified tumors(or) low
serum ferritin and/or
LDH when MYCN
unknown^
Histology: INPC differentiating
Rx 4 courses of chemotherapy
VP/Carbo x 2 – if tumor responds continue
with further VP/Carbo x 2, if no response
continue with CADO x 2 then: If IDRF
negative, proceed for surgical resection
Histology: INPC undifferentiated or poorly
differentiated <5 yrs of age
Rx 6 courses of chemotherapy, surgery,
radiotherapy & cis-retinoic acid
VP/Carbo x 2, CADO x 2;
Followed by:
•If tumor response & IDRF negative then:
surgical resection followed by VP/Carbo
x 1, CADO x 1 if there was response to
initial VP/Carbo, or CADO x 2 if no
initial response to VP/Carbo
•If no tumor response & IDRF persist:
surgical resection followed by CADO x 2
•If tumor response but IDRF persist:
VP/Carbo x 1, CADO x 1 if there was
response to initial VP/Carbo, or CADO x
2 if no initial response to VP/Carbo then
surgical resection
•Surgery should be followed by
radiotherapy + 6 courses of 13 cis-
retinoic acid
If > 5 years with undifferentiated or poorly
differentiated tumor histology, consider
treatment according to high-risk protocol.
Stage INRG: L1, MYCN
amplified age ≤10 years
Rx 6 courses of chemotherapy:
VP/Carbo x 2, CADO x 2, VP/Carbo x 1,
CADO x 1
Surgery
Radiotherapy + 6 courses of 13 cis-retinoic

32
acid.
Age ≤12 months with
stage 4(INRG: M)
disease, MYCN not
amplified (or) low serum
ferritin &/or LDH when
MYCN unknown^
VP/Carbo x 2 then
•If response shown, proceed for further
VP/Carbo x 2,& if metastatic remis••sion
achieved proceed for surgical resection of
primary, if metastatic remission not
achieved continue with CADO x 2-4
courses to achieve metastatic remission
•If no response after initial VP/Carbo
proceed for CADO x 2-4 to achieve
••metastatic remission.
•Note: metastatic remission is all sites
other than the liver. Surgical resection
••when metastatic remission achieved &
no further chemotherapy
INPC, International Neuroblastoma Pathological Classification system.
SIOP-PODC, International Society of Pediatric Oncology- Committee on
Developing Countries; INRG, The International Neuroblastoma Risk
Group staging system, LDH, Lactate dehydrogenase. IDRF: image
defined risk factors.
^If NMYC unavailable in INSS3 and infants with INSS4, serum LDH
≥750 IU/L and/or serum ferritin ≥ ≥120 ng/ml can be used as surrogate
markers to classify the patient as high-risk.

33
Table: 9 High-risk NBL: Risk stratification and management [97, 98]
Criteria Management
•Stage 2 with MYCN-
amplified
•Stage 3 with MYCN-
amplified (or) high
high LDH when MYCN
unknown^
•Stage 4 with age < 18
months with MYCN-
amplified (or) high
high LDH when MYCN
unknown^
•Stage 4S with MYCN-
amplified
•Stage 4, 12-18 months,
MYCN-non amplified
with segmental
chromosomal
aberrations
•Stage 4 ≥18 months
age#
High-risk protocol (induction
chemotherapy, surgery, autologous
hematopoietic stem cell transplant,
radiotherapy, differentiation therapy)
Stage 4, age: 12-18
months, MYCN non-
amplified with numerical
chromosomal aberrations
High-risk protocol, but receive only
COJEC and surgery
#Irrespective of MYCN status, all INSS 4 ≥18 months of age will be
classified as high-risk.
^If NMYC unavailable in INSS 3 and infants with INSS 4, serum LDH
≥≥750 IU/L and/or serum ferritin ≥120 ng/ml can be used as surrogate
markers to classify the patient as high-risk.
Table: 10
Life-threatening symptoms: The presence of any of these symptoms is
an indication for chemotherapy [97, 98]
Intraspinal neuroblastoma
Patients who either have symptoms of spinal cord compression or have
a spinal tumor component that occupies more than 1/3rdof the spinal

34
canal on the axial plane and/or the perimedullary leptomeningeal
spaces are not visible and/or the spinal cord signal is abnormal.
Systemic upset
•Pain requiring opiate treatment
•Gastrointestinal
•Vomiting needing nasogastric/IV support
•Weight loss >10% body weight (NB: diarrhea with VIP does not
respond to chemotherapy and is a definite indication for surgery)
•Respiratory
•Respiratory distress without evidence of infection
•Tachypnoea >60
•Oxygen need
•Ventilatory support
•Cardiovascular System
•Hypertension
•IVC compression +/- leg oedema
•Renal
•Impaired renal function, creatinine increased x2 ULN
•Poor urine output, less than 2Ml/kg/hour
•Hydroureter/hydronephrosis
•Hepatic
•Abnormal liver function >2 ULN
•Evidence of DIC
•Platelets <5 x 109/L
•Bladder/Bowel dysfunction secondary to a mass effect.
•A very large tumor volume causing concern of possible tumor
rupture and/or the possible rapid development of systemic upset.
TREATMENT
• Low-Risk Neuroblastoma [92]
Low-risk neuroblastoma is defined as disease that is curable with
no or minimal cytotoxic therapy and is strongly associated with
spontaneous regression. Approximately 40% of neuroblastoma patients
have low-risk disease. According to the COG this category includes
patients with INSS stage – 1, 2, & 4s. Currently, stage 1 in all age groups
is considered low risk regardless of biologic markers and the same holds
for infants with stage-2 disease. In older patients, lack of MYCN

35
amplification is the only biologic finding needed for classifying stage 2 as
low risk. Most patients with low-risk neuroblastoma are cured with
surgery alone, while a subset of low-risk infants with small adrenal
tumors can be safely observed without surgery or other treatment. The
excellent outcome is due, in part, to a high incidence of spontaneous
tumor regression observed with this group of tumors. The identification
of biologic markers associated with favorable prognosis has facilitated
treatment reduction for ever greater numbers of neuroblastoma patients.
While gross total resection of a localized neuroblastoma remains the
current treatment recommendation for most patients, it is now well
recognized that such a procedure is not justified if it entails acute risks
such as loss of a major organ (e.g., kidney) or damage to important nerves
(e.g.,brachial or sacral plexus) as the residual biologically favorable
disease will likely remain stable or even regress spontaneously.
Alternatively, a partial resection followed by chemotherapy or
observation alone may be reasonable, given the very small risk that
residual biologically favorable tumor might evolve into lethal metastatic
disease.
Table: 11 Chemotherapy for low and Intermediate-risk disease
VP/Carbo (Etoposide [VP16] and carboplatin) [98]
Dose (mg/Kg)
(For patients
weighing ≤ 10
kg)
Dose (mg/m2)
(For patients
weighing > 10
kg)
Day 1 Day
2
Day
3
Carboplatin 6.6 200 ✓ ✓ ✓
Etoposide 5.0 150 ✓ ✓ ✓
Carboplatin: In 5% dextrose (5 ml/kg) over 1 hr daily x 3 days.
Etoposide: 0.9% saline (12.5 ml/kg) over 2 hrs daily x 3 days
Courses of VP/Carbo are given at 21 day intervals
CADO (Cyclophosphamide, doxorubicin and vincristine)
Dose Dose Day Day Day Day Day

36
(mg/Kg)
(For patients
weighing ≤
10 kg)
(mg/m2)
(For patients
weighing >
10 kg)
1 2 3 4 5
Cyclophosp
hamide
10 300 ✓ ✓ ✓ ✓ ✓
Doxorubici
n
1 30 ✓ ✓
Vincristine 0.05 1.5 ✓ ✓
Cyclophosphamide: 5% dextrose (5 ml/kg) over 1 hr, daily x 5 days
Doxorubicin: 0.9% saline over 1-6 hours on days 4 and 5
Vincristine: Bolus injection on days 1 and 5
• Intermediate-Risk Neuroblastoma [92]
According to the current Children’s Oncology Group (COG)
Neuroblastoma Risk Stratification System, this grouping includes infants
with INSS stages 3 or 4 tumors that lack MYCN amplification, infants
with stage-4S disease with normal MYCN copy number and either
unfavorable histology or diploidy, and children >1 year of age with
favorable histology stage-3 tumors that lack MYCN amplification. Based
on these clinical and biologic criteria, approximately 15% of all patients
diagnosed with neuroblastoma are classified as intermediate risk
• Treatment for Stage-3 Neuroblastoma
In the current COG study, infants with stage 3 continue to receive
chemotherapy, but only four cycles if disease is hyperdiploid vs eight
cycles if disease is diploid, and carboplatin is used, rather than cisplatin,
in an attempt to reduce toxicity (combinations of platinum compounds,
etoposide, cyclophosphamide, doxorubicin, and/or vincristine). Older
patients with intermediate-risk stage-3 neuroblastoma). In the current
COG study, these patients receive only four cycles of chemotherapy. The
French Society of Pediatric Oncology achieved their result using
alternating cycles of carboplatin/ etoposide and

37
cyclophosphamide/doxorubicin/ vincristine (maximum of three cycles of
each combination), in moderate doses. [99]
In a large CCG study,
treatment included 9 months of combined usage of cisplatin, etoposide,
cyclophosphamide, and doxorubicin. [100]
In one large POG study, cycles
of high-dose cisplatin/etoposide alternated with low-dose
cyclophosphamide/ doxorubicin, and in a successor POG study, patients
received cycles of cyclophosphamide, etoposide, vincristine, plus either
cisplatin or carboplatin.
• Treatment of Infant Stage-4 Neuroblastoma
In the current COG study, infants with non-MYCN-amplified stage
4 are treated with modest-dose chemotherapy, four cycles if tumor is
hyperdiploid and has favorable histopathology, and eight cycles if tumor
is diploid and/or has unfavorable histopathology.
• Treatment for Stage-4S Neuroblastoma
COG uses the presence of diploidy and unfavorable histopathology
to confer intermediate risk status on stage 4S with the implication of a
need for treatment with chemotherapy (up to eight cycles in the current
COG study) For symptomatic hepatomegaly low dose chemotherapy
and/or radiotherapy (e.g., 150 cGy/fraction) have been used with variable
success.
Table: 12
Philadelphia score for infants with stage 4S disease [101]
Clinical
parameter
Score 0 Score 1 Score 2
Emesis Absent >10% intake Severe requiring
intravenous fluids
Respiratory
involvement
Absent Respiratory rate
>60, O2support
requirement
CPAP, Ventilation
Edema
(obstruction of
venous return)
Absent Pedal Sacral and scrotal
Renal Absent Oliguria < urea/creatinine above

38
involvement 2ml/kg/hr age appropriate level
Liver
involvement
Absent - Evidence of DIC/
Platelet count <
50,000/Μl
CPAP, Continuous positive airway pressure; DIC, Disseminated
intravascular coagulation. A total score ≥ ≥1 in newborns and ≥2 in older
infants will warrant treatment in 4S disease.
• High-Risk Neuroblastoma [92]
The high-risk group in neuroblastoma is comprised primarily of
children (>1 year of age at diagnosis) with stage-4 disease and stage 3
with tumor MYCN amplification or unfavorable histopathology. Therapy
for high-risk neuroblastoma is currently divided into four phases
1. Induction Therapy combinations of vincristine, cisplatin, etoposide,
cyclophosphamide, and carboplatin.
2. Local Control Surgical resection of the tumor during or at
completion of chemotherapy induction, in order to remove residual
viable tumor.
3. High-dose marrow ablative therapy Myeloablative therapy while
prolonging progression-free survival may only have a small effect
on the long-term cure rate.
4. Therapy of Minimal Residual Disease New approaches to eliminate
minimal residual disease tried with agents such as 13-cis-retinoic
acid, fenretinide, anti-GD2 monoclonal antibodies,
immunocytokines, genetically engineered vaccines, anti-angiogenic
therapy, small molecule inhibitors of tyrosine kinase genes, or
histone deacetylase inhibitors.
• Surgical Technique
It is almost never possible to obtain a clear microscopic margin;
thus, dissection generally proceeds along the pseudocapsule of the tumor.
Sectioning of the tumor overlying vital structures or its partial removal

39
allows better visualization in order to achieve a gross total resection. The
use of titanium surgical clips can improve hemostasis and lymphostasis
while marking involved areas for subsequent radiotherapy.
• Surgical complications
• Vascular Arterial or venous laceration:
primary repair
Arterial laceration: graft
Renovascular hypertension
Lymphatic ascites
• Genitourinary Nephrectomy
Renal infarction (arterial or venous
occlusion or thrombosis)
Ureteral transection or fibrosis
Neurogenic bladder
Bladder perforation
Urinary tract infection
• Gastrointestinal Intussusception
Chronic diarrhea
Gastric atony
Motility disorders
• Nervous Spinal cord injury with paralysis
Horner’s syndrome
Recurrent nerve injury
Brachial or lumbosacral plexus injury
Sensory loss
• RADIOTHERAPY
• Low Risk Disease [94]
The current standard of care is to reserve radiation therapy only for

40
those low-risk patients whose disease is not adequately controlled with
surgery and chemotherapy. In the current COG protocol for low risk
neuroblastoma (#P9641), a dose of 21 Gy is recommended for stage-1
and stage-2 patients who require radiotherapy.
• Intermediate-Risk Disease [94]
Radiation therapy is indicated for patients with clinical
deterioration despite chemotherapy and surgery or those with persistent
tumor after chemotherapy and second-look surgery.
• Stage-4S Disease
A unique use of radiation therapy is for infants with stage-4S
disease who have respiratory distress or compression of abdominal
viscera from massive liver involvement. [102]
A very low dose of
radiation, three fractions of 1.5 Gy each, is extremely effective in
reversing these life-threatening problems without a significant risk of
long-term complications.
• High-Risk Disease
The majority of patients with high-risk disease do benefit from the
addition of radiation therapy to the combined modality treatment
paradigm. This group of patients have excellent local control when the
primary site is managed with complete surgical resection followed by
approximately 21 Gy; however, this dose of radiation does not appear to
be adequate if complete resection is not achieved. Radiation therapy is an
indispensable tool in the management of neuroblastoma metastases, either
as part of initial therapy or as palliation.
• TREATMENT OF RELAPSED &
• REFRACTORY NEUROBLASTOMA [95]
status Treatment
approach
• Primary refractory – Novel chemotherapy, MIBG +
myeloablative therapy, MRD therapy (Minimal residual disease

41
therapy).
• Early relapse – Novel chemotherapy, then targeted therapy,
myeloablative therapy, MRD therapy.
• Late relapse – Standard combination chemotherapy, surgery,
radiotherapy or MIBG, and novel MRD therapy.
• Multiple relapse – Low-toxicity oral chemotherapy or outpatient
targeted therapy
• Novel therapies:
Prolongation of induction therapy with intensification of dose if
there has been some response to treatment, or else adding
chemotherapeutic agents that differ in their mechanism of action from
those used in induction, biologic response modifiers, or targeted
radiotherapy
• New agents with potential in Neuroblastoma
Table: 13 HDAC histone deacetylase inhibitors
Cytotoxi
c
Apoptoti
c
Pathway
Immunolog
ic
Anti-
angiogenic
Retinoids Targeted
radiotherap
y
Topoiso
merase
inhibitor
s
HDAC
inhibitor
s
Antibodies Thalidomid
e
Fenretidin
e
131
I-MIBG
Alkylato
rs
Demethy
lating
agents
Cytokines Antibodies Other
retinoids
131
I-anti-
GD2
Cross-
linkers
Tyrosine
kinase
inhibitor
s
Vaccines Small
molecules
• Stem cell apheresis
G-CSF10 μg/kg/day single dose is administered for mobilizing the
stem cells. The apheresis is typically done on day 4-5. In the rare case of
poor mobilization, Injection Plerixa forcan be considered (Dose: 0.24
mg/kg subcutaneous, 10 hrs prior to apheresis, expensive – Rs ~ 62,000

42
for a vial of 1.2 ml = 24 mg). [103]
A femoral dialysis catheter size eight Fr
(available as double lumen: Arrow/Wygon/Medcomp) is typically used
for apheresis in patients below 5yrs of age.
• Autologous Transplant in Neuroblastoma
The use of the patient’s own stem cells to support recovery from
high-dose chemotherapy is more properly referred to as high-dose chemo
with stem cell rescue (HDC/SCR). The HDC regimen used to prepare the
patient is usually myeloablative, meaning that no bone marrow recovery
is possible without SCR. There are also submyeloablative HDC regimens,
in which the SCR is used to speed recovery.

43
MATERIALS AND METHODS
PLACE OF STUDY: Department of Medical Oncology, The Gujarat
Cancer andResearch Institute, BJ Medical College, Ahmedabad,
Gujarat, India.
STUDY DESIGN: Prospective observational Study.
STUDY DURATION: Two years (September 2019 to August 2021)
SAMPLE SIZE: 26 patients
INCLUSION CRITERIA:
• Histo-pathologically proven patients of neuroblastoma.
• New cases of neuroblastoma who have never received chemotherapy
before.
• Those who has given informed consent.
EXCLUSION CRITERIA:
• Patients with co – existing second cancers / history of prior cancer.
METHODOLOGY:
The work has been undertaken to study the epidemiological,
clinical profile and treatment outcome of neuroblastoma in children who
presented in the outpatient department of Medical Oncology, The Gujarat
Cancer and Research Institute, BJ Medical College, Ahmedabad from
September 2019 to August 2021.
After obtaining informed consent from the patient’s/parents,
relevant history was taken and examination was done. Routine
investigations done included hematological, blood bio-chemistry, urine
analysis and chest and abdominal radiographs. Specific investigations
included, USG (ultrasonography) abdomen, FNAC (fine needle aspiration
cytology) of the swelling, CECT (contrast enhanced computed

44
tomography) abdomen and chest when required, MRI (magnetic
resonance imaging), tumor markers, bone marrow biopsy, bone scan.
Tissue samples were taken at the time of open biopsy for
histopathological diagnosis and were stored in special preservative
solution. Neuroblastoma was confirmed by histopathology or
immunohistochemistry tissues.
Neuroblastoma was staged according to the International
Neuroblastoma Staging System. Follow-up of the patients was done in all
patients. N-myc sample was sent for affordable patients.
The treatment was planned according to the stage of disease,
clinical examination, and investigative workup.
Investigations like USG or CECT were occasionally done
whenever needed in which the presence or absence of residual disease or
metastasis was noted. Follow-up was carried out till the date of
compilation of this work.
DATA ANALYSIS AND INTERPRETATION:
Data were entered into Microsoft Excel (Windows 7; Version
2007) and analyses were done using the same.
Descriptive statistics such as mean and standard deviation (SD) for
continuous variables, frequencies, and percentages were calculated for
categorical variables were determined.
Bar charts and pie charts were used for a visual representation of
the analyzed data.

45
OBSERVATIONS AND RESULTS
This study conducted at The Gujarat Cancer andResearch Institute,
B. J Medical College, Ahmedabad, is a prospective observational study
done between September 2019 and August 2021 and it encompasses a
total of 26 children who were diagnosed to have Neuroblastoma. All
patients had positive Synaptophysin and Chromogranin on
immunohistochemistry. All the patients who were not histologically
proven were excluded from the study. Most of the patients had advanced
disease at presentation.
The most common presenting complaint was an abdominal
lump, followed by abdominal pain. Patients also presented with non-
specific complaints such as decreased urine output and fever. During
study-period two major Covid peak waves came, which drastically
reduced the inflow of patients.

46
TABLE 1: AGE DISTRIBUTION
No. %
≤1 year 5 19%
>1 year 21 81%
As per the above table 1, patients of 1 year and less than 1 year are
5 (19%), while 21 (81%) patients are more than 1 year of age. The
minimum age of presentation was 3 months, and the oldest child was 7
years of age.
The majority of the patient in this study were in the age group 1-5
years.
5
21
0
5
10
15
20
25
≤1 year >1 year
Age Distribution

47
TABLE 2: SEX DISTRIBUTION
In this study group, the total number of boys was 18 (69%) and
the total number of girls was 8 (31%).
18
8
0
2
4
6
8
10
12
14
16
18
20
Male Female
Sex Distribution
No. %
Male 18 69%
Female 8 31%

48
TABLE 3(a): TUMOUR SITES DISTRIBUTION
Location No. %
Suprarenal 16 62%
Renal 1 4%
Pelvic 3 12%
Mediastinal 2 8%
Abdominal 2 8%
Bone 2 8%
Table 3(a) - shows the location of the tumor, the majority of the
patients 16 (62%) were found it to be suprarenal in location, the most
common location. Followed by Pelvis 3 (12%), Mediastinal, abdominal
and Bone all for 2 (8%), Renal 1 (4%) location. The patient was
radiologically scanned with CT scan, USG abdomen pelvis.

49
3(b)- Bar Graph
3(b). This bar graph shows the most common location of
neuroblastoma in this study was the suprarenal region. Patients with
metastatic disease presented with nonspecific complaints. Common sites
for metastasis were bone, mediastinum.
16
1
3
2 2 2
0
2
4
6
8
10
12
14
16
18
Location of Tumour

50
TABLE 4: SYMPTOMS DISTRIBUTION
Symptoms No. %
Abdominal swelling 15 58%
Abdominal Pain 7 27%
Decrease Urine 2 8%
cough 2 7%
Table (4a) - Of the 26 children in the study, the most common
presenting symptom was abdominal swelling (58%) noted by the
parents or referring physician. Abdominal pain (27%) was the next
common complaint. Nonspecific complaints like decreased urine and
cough were noted in 15%.

51
Bar graph ( 4b) – most common symptom in patients with the localized
disease was abdominal swelling.Patient with advanced and metastatic
disease patients presented with non-specific complaints such as decreased
urine output, cough.
15
7
2 2
0
2
4
6
8
10
12
14
16
Abdominal
Swelling
Abdominal Pain Decrease Urine cough
Symptoms Distribution

52
TABLE 5(a): BONE MARROW BIOPSY
BM No. %
Yes 16 62%
No 10 38%
Table 5(a) shows Bone marrow aspiration and Biopsy - Among 26
patients 16 (62%), underwent bone marrow biopsy. Out of 10 patients, 2
patients didn’t give consent for biopsy. 3 patient biopsy reports came
inconclusive. Rest were metastatic at presentation.
16
10
Bone Marrow
Yes
No

53
TABLE 5(b): BONE MARROW BIOPSY POSITIVITY
BM No. %
POSITIVE 10 62%
NEGATIVE 6 38%
5(b) – Pie chart showing bone marrow biopsy was done in 16
patients, in which it was positive in 10 (62%) patients, leading to up-
gradation of the stage to stage 4. Out of 10 marrow which came positive 6
patients had localized disease on radiological investigation. Bone marrow
positivity upgraded stage, and course of action.

54
6: TUMOUR STAGING
Staging No. %
1 0 0%
2A 0 0%
2B 0 0%
3 6 23%
4 20 77%
4S 0 0%
Table 6(a)- shows that among 26 patients studied, 20 (77%) were
of stage 4 of INSS while 6 (23%) were of stage 3.

55
Bar graph- shows the majority of the patient presented in stage 4 or
advanced stage. There were no patients in stage 1 or 2, although 6 patients
were in stage 3, rest were in stage 4.
0 0 0
6
20
0
0
5
10
15
20
25
1 2A 2B 3 4 4S
Tumour Staging

56
TABLE 7: TREATMENT
Treatment No. %
POG Protocol 10 38%
Cyclophosphamide + Topotecan 5 19%
Carboplatin + Etoposide 6 23%
OPEC 5 19%
As Table 7 shows, the majority of the patients 10 (38%) were
treated with POG protocol, followed by Cyclophosphamide plus
Topotecan 5 (19%), Carboplatin plus Etoposide 6 (23%), and OPEC
protocol in 5 (19%).

57
TABLE 8: Metastatic vs Non-metastatic
Table 8 shows- Out of 26 patients, 3 were metastatic at
Presentation, whereas 23 were non-metastatic.
Metastatic Non-metastatic
Number of patients 3 23
Percentage 12 88

58
TABLE 9: EFFECT OF LOCKDOWN DUE TO COVID.
Without lockdown With lockdown
Table (9) - This study was conducted during covid peak waves, as
we can see in this table as the number of patients was significantly higher
without lockdown than with lockdown due to many logistics issues
0
2
4
6
8
10
12
14
16
18
20
With lockdown Without Lockdown
No of Patient
With lockdown Without Lockdown

59
Table 10- N-MYC Status
N-myc amplified N-myc nonamplified
Total number of patients (4) (1/4) (3/4)
Percentage 25% 75%
Table 10 - Out of 26 patients, N-MYC was sent for 4 patients, in
which N-MYC was amplified for 1 patient, while it was not amplified for
one patient.
Percentage
N-myc amplified N-myc nonamplified

60
Table 11 - RESPONSE AND EVALUATION
Response
and
Evaluation
Complete
response
(CR)
Stable
Disease
(SD)
Progressive
disease (
PD)
Number
of patients 4
14 8
Table 11(a)- shows out of 26 patients who had taken chemotherapy, 4
patients had a complete radiological response (CR) in 4 patients, Stable
Disease in 14 patients, and 8 patients had progressive disease as per recist
criteria
No of Patients
Complete Response(CR) Stable Disease(SD) Progression(PR)

61
Table 12 COMPLIANCE
Regular
follow up
Irregular
follow up
Lost
to
follow
up
Number of
patients
12 11 3
Table (12) – shows out of 26 patients 12 patients were on regular follow up,
11 were on irregular follow up , while 3 patients were lost to follow up.
No of Patients
Regular Follow up Irregular Follow up Lost to Follow Up

62
TABLE 13: OUTCOME
Table 13(a)- According to the above table for the final outcome of
the patients, 8 (31%) of patients were on the treatment with palliative
chemotherapy. 5 (19%) with a residual calcified mass of various sizes, 5
(19%) with the progression of the disease, and 1 (4%) with post-surgical
remission. 7 (27%) patients abandoned the treatment after initiation.
Outcome No. %
Residual calcified mass 5 19%
Post-surgery remission 1 4%
Progression 5 19%
Palliative chemo 8 31%
Lost follow up 7 27%

63
Bar graph - The majority of patients after having
the stable disease were eventually shifted on
palliative chemotherapy ( 8 ), only one patient had
post-surgery remission, while the rest of the
patients had stable disease.
0
5
10
15
20
25
30
35
Residual Calcified
mass
Post-surgery
remission
Progression Palliative Chemo Lost follow Up
Outcome
Number Percentage

64
DISCUSSION
The Gujarat Cancer and Research Institute, BJ Medical College,
Ahmedabad is a government-run institution and caters to children
belonging to the lower socio-economic strata. The majority of parents of
children with this disease are illiterate and ignorant of even the common
pediatric problems. This is one of the reasons why a good number of our
patients in the study presented to us in the advanced stage of the disease.
Most of our children presented with a mass in the abdomen or with
abdominal distension, which was either noticed by the parents or by the
referring physician.
In this study a total of 26 children were diagnosed to have
Neuroblastoma.
1.The age & sex distribution:
In this study group of 26 cases(n) total number of boys was 18
(69%) and the total number of girls were 8 (31%). The male to female
ratio is 2.25:1, as compared to the study done by Dr Puligundla Krishna
Chaitanya[104]
in which sex ratio is 1.14:1.
The youngest age at presentation was 1 year and the oldest child
was 7 years. The median age of presentation is 3.5 years, as compared
to the study done by Dr. Puligundla Krishna Chaitanya[104]
in which is 4
years, which is similar to our study.

65
Distribution - AGE AND SEX
Parameters
Present
Study
Dr Puligundla
Krishna
Chaitanya[104]
PGIMER(102)
Median Age of
Presentation
3.5 year 4 year 3.6
Sex Ratio 2.25:1 1.14:1 1.6;1
2. LOCATION
In my study location of the tumor found in the adrenal is 62%
which is similar to be 50% in the study done by Grosfeld JL[106]
. In this
study, mediastinal and pelvic tumors are 4% and 12% respectively, as
compared to the study done by Grossfeld JL[106]
which is found to be 20%
and 5%. According to literature and previous study, the most common
location is an abdominal mass, which is also the case in this study, but
the proportion is less as compared to international studies as there are
many patients present in the early stage.

66
Location of
Tumour
Current
Study
Grosfeld JL[106]
ICMR (93)
Adrenal 62% 50% 66 %
Pelvic 12% 5% 12 %
3. SYMPTOMS
In this study of 26 children, the most common presenting symptom
was abdominal swelling (58%), while in a study done by Zahida Akhter
et al.[105]
9 (100%) patients had the same symptoms. Abdominal pain
(12%) and suprarenal pain (12%) were the next common complaint in this
study while in a study done by Zahida Akhter et al. [105]
5 (55%) had the
same symptoms. Patients in this study have presented in an advanced
stage. Lack of health awareness may be a reason for that.
In this study, 77% of patients were found with stage 4, while in a
study done by Zahida Akhter et al. [105]
55% of patients were found with
stage 4S but no patients with stage 4.

67
PRESENTING SYMPTOM
Parameters
Current
Study
Zahida Akhter et
al. [105]
ICMR
study(93)
Abdominal
swelling as a
most common
symptom
58% 100% 70%
Stage 4 disease 77% - 60%
4. TREATMENT
In a study done by Gaurav Kharya et al[107]
. There were 33 cases of
NB. Fifteen cases either did not opt for therapy or were abandoned after
initial therapy. Of the remaining 18 cases, 7 had low risk, 6 had
intermediate risk and 5 had high-risk NB. Five low-risk patients
underwent surgery alone whereas watchful waiting was observed in two-
stage IV‐S patients. Intermediate risk patient was treated as per
COGA3961 protocol. High-risk patients received 3–5 courses of OPEC
as induction chemotherapy followed by surgical resection/debulking,
another 2–4 cycles of chemotherapy, autologous peripheral blood stem
cell transplant, and finally 6 cycles of 13‐cis‐retinoic acid. Whereas in my
study 27% of patients abandoned the treatment after initial therapy, 38%
on POG protocol, and others with the chemotherapy.

68
Due to covid lockdown, there was a logistics issue related to
transportation, inter and intrastate travel leading to irregular follow-
up. So, many patients couldn’t come hospital for the due date of
chemotherapy, which may be the reason why there was no optimal
response on induction chemotherapy, and many patients were put on
palliative chemotherapy.
Chemotherapy
Current
Study
Gaurav
Kharya et
al[107]
PGIMER
( 102 )
Cyclophosphamide +
Topotecan
5 7 4
Carboplatin + Etoposide 6 6 10
OPEC 5 5 2

69
5. BONE MARROW BIOPSY
In this study out of 26 patients, 16 patients consented for bone
marrow biopsy underwent, out of that 10 patients bone
marrow came positive
Marrow Involvement
Present
study
Pulkit
Rastogi
(87)
Marrow
positivity
62 % 54.5 %
Marrow Positivity
Present Study Pulkit Rastogi

70
6. Response and Evaluation
Out of 26 patients, 18 patients had stable /partial response (69
%), while 8 patients had progressive disease.
Present
study
Berthold
(89)
Response
rate
69 % 63 %
60
61
62
63
64
65
66
67
68
69
70
Present Study Berthold
Response Rate
Present Study Berthold

71
7. EFFECT Of LOCKDOWN DUE TO COVID
During this study, there was a COVID-19 pandemic, which put an unrivaled
burden on the health care system. There was a nationwide lockdown that
began on March 24, 2020. Many cancer centers were converted into covid
facilities. There was a sharp noticeable decline in all cancer patients,
including that of neuroblastoma. During the lockdown, there was a follow-up
of only 7 patients, while without lockdown we had a follow-up of 19 patients.
Without lockdown With lockdown

72
8. HISTOPATHOLOGY
In this study, all cases were of Neuroblastoma. There was no case
of ganglioneuroblastoma.
N-myc Amplification detected using PCR
The method for N-myc amplification used is the southern blot
which is quantitative but requires a large amount of DNA that can be only
obtained by surgical excision or open biopsy.
In this study, N-myc was sent for 4 patients, out of which N-myc
came positive for one patient.
COMPARISON OF MYCN AMPLIFICATION OF OUR STUDY
WITH OTHER INDIAN and FOREIGN STUDY
Present
study
AIIMS
STUDY
AMERICAN
SOCIETY OF
ONCOLOGY
MYCN
Amplification
25% 30% 20-25%

73
0
10
20
30
40
50
60
70
80
MYCN
Amplified(n=3)
MYCN
Amplified(n=7)
N-myc Amplification
MYCN Amplified(n=3)

74
CONCLUSION
This study was conducted on 26 patients with neuroblastoma at the Department of
medical oncology, Gujarat Cancer and Research Institute(GCRI), Ahmedabad, Gujarat,
India from 1
The key findings are summarized below: -
1. Majority of patients were of more than 1 year of age.
2. Median age was 3.5 years.
3. Majority of patients were in age group 1-5 years of age.
4. Out of males and females, males were involved in majority.
5. Most common presentation was abdominal mass.
6. Most common location of the tumor was the adrenal.
7. Only 3 patients presented as denovo metastatic.
8. Bone marrow is involved in 60 % of patients
9. Significant reduction in the number of patients follow due to
COVID Lockdown.
10.Response rate with chemotherapy 60 %.
11.N-MYC was positive in 25 % of patients
12.Almost 2/3rd
of patients were of stage 4 INSS at the time of
diagnosis.
13.Majority of patients were treated with POG protocol for
induction, OPEC as palliative chemotherapy regimen

75
LIMITATIONS
1. Most of the studies in the literature have not done a comprehensive
analysis of different modalities of treatment like surgery, induction
chemotherapy, Radiation, and their impact, so the data from this study
cannot be generalized.
2. Sample size of this study was 26, and the period of the study was 2
years. Hence the complete follow up of the cohort could not be done.
3. Further studies with a large sample size and in collaboration with
surgeons and radiation oncologists might help to improve outcomes.
4. Despite of these limitations, much effort have been made to represent
this data.
5. Due to the unprecedented COVID-19 crisis, patients were lost to
follow up or had irregular follow up.

76
BIBLIOGRAPHY
1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet.
2007;369:2106–20.
2. Cheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer
genomics and immunotherapy. Nat Rev Cancer. 2013;13:397–411.
3. Maris JM. Recent advances in neuroblastoma. N Engl J Med. 2010;362:2202–
11.
4. Louis CU, Shohet JM. Neuroblastoma: molecular pathogenesis and therapy.
Ann Rev Med. 2015;66:49–63.
5. Seigel R, et al.: Cancer Statistics 2021. CA: A Cancer Journal for Clinicians.
2021 Jan; 71(1):7-33.
6. Gurney JG, Severson RK, Davis S, Robison et al. Incidence of cancer in
children in the United States. Sex-, race-, and 1-year age- specific rates by
histological type. Cancer. 1995;75 (8):2186-95.
7. Linet MS, Ries LA, Smith MA et al. Cancer surveillance series: recent
trends in childhood cancer incidence and mortality in the United States.. J
Natl Cancer Inst. 1999 ;91(12):1051-8.
8. Ognjanovic S, Linabery AM, Charbonneau B et al.Trends in childhood
rhabdomyosarcoma incidence and survival in the United States, 1975-2005.
Cancer. 2009;115(18):4218-26.
9. Smith MA, Altekruse SF, Adamson PC et al. Declining childhood and
adolescent cancer mortality.Cancer. 2014 ;120(16):2497-506.
10. Cancers in childhood. Consolidated report of hospital based cancer
registries, 2007-2011. NCDIR-NCRP, Bangalore. India. 2013: 19-25.
11. Matthay KK, Maris JM, Schleiermacher G, et al. Neuroblastoma. Nat Rev Dis
Primers. 2016;10(2):16078.
12. Nakagawara A, Li Y, Izumi H, et al. Neuroblastoma. Jpn J Clin Oncol.
2018;48(3):214-41.
13. Cheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer
genomics and immunotherapy. Nat Rev Cancer. 2013;13(6):397-411.
14. van Groningen T, Koster J, Valentijn LJ, et al. Neuroblastoma is composed of

77
two super-enhancer-associated differentiation states. Nat Genet.
2017;49(8):1261-6.
15. Kimura S, Sekiguchi M, Watanabe K, et al. Association of highrisk
neuroblastoma classification based on expression profiles with differentiation
and metabolism. PloS One. 2021;16(1):e0245526.
16. Brodeur GM, Seeger RC, Schwab M, et al. Amplification of Nmyc in untreated
human neuroblastomas correlates with advanced disease stage. Science.
1984;224(4653):1121-4.
17. Bown N. Neuroblastoma tumour genetics: clinical and biological aspects. J Clin
Pathol. 2001;54(12):897-910.
18. Attiyeh EF, London WB, Mosse YP, et al. Chromosome 1p and 11q deletions
and outcome in neuroblastoma. N Engl J Med. 2005;353(21):2243-53.
19. Michels E, Hoebeeck J, De Preter K, et al. CADM1 is a strong neuroblastoma
candidate gene that maps within a 3.72 Mb critical region of loss on 11q23.
BMC Cancer. 2008;8(1):173.
20. Ochiai H, Takenobu H, Nakagawa A, et al. Bmi1 is a MYCN target gene that
regulates tumorigenesis through repression of KIF1Bbeta and TSLC1 in
neuroblastoma. Oncogene. 2010;29(18):2681-90.
21. Takagi M, Yoshida M, Nemoto Y, et al. Loss of DNA damage response in
neuroblastoma and utility of a PARP inhibitor. J Natl Cancer Inst.
2017;109(11):djx062.
22. Mandriota SJ, Valentijn LJ, Lesne L, et al. Ataxia-telangiectasia mutated
(ATM) silencing promotes neuroblastoma progression through a MYCN
independent mechanism. Oncotarget. 2015;6(21):18558-76.
23. Uryu K, Nishimura R, Kataoka K, et al. Identification of the genetic and clinical
characteristics of neuroblastomas using genome-wide analysis. Oncotarget.
2017;8(64):107513-29.
24. Takita J. The role of anaplastic lymphoma kinase in pediatric cancers. Cancer
Sci. 2017;108(10):1913-20.
25. Mosse YP, Laudenslager M, Longo L, et al. Identification of ALK as a major
familial neuroblastoma predisposition gene. Nature. 2008;455(7215):930-5.
26. Chen Y, Takita J, Choi YL, et al. Oncogenic mutations of ALK kinase in
neuroblastoma. Nature. 2008;455(7215):971-4.

78
27. Janoueix-Lerosey I, Lequin D, Brugieres L, et al. Somatic and germline
activating mutations of the ALK kinase receptor in neuroblastoma. Nature.
2008;455(7215):967-70.
28. George RE, Sanda T, Hanna M, et al. Activating mutations in ALK provide a
therapeutic target in neuroblastoma. Nature. 2008;455(7215):975-8.
29. Lastowska M, Cotterill S, Pearson AD, et al. Gain of chromosome arm 17q
predicts unfavourable outcome in neuroblastoma patients. U.K. Children’s
Cancer Study Group and the U.K. Cancer Cytogenetics Group. Eur J Cancer.
1997;33(10):1627-33.
30. Islam A, Kageyama H, Takada N, et al. High expression of 78urviving, mapped
to 17q25, is significantly associated with poor prognostic factors and promotes
cell survival in human neuroblastoma. Oncogene. 2000;19(5):617-23.
31. Godfried MB, Veenstra M, v Sluis P, et al. The N-myc and cmyc downstream
pathways include the chromosome 17q genes nm23-H1 and nm23-H2.
Oncogene. 2002;21(13):2097-101.
32. Saito-Ohara F, Imoto I, Inoue J, et al. PPM1D is a potential target for 17q gain
in neuroblastoma. Cancer Res. 2003;63(8):1876-83.
33. Yu M, Ohira M, Li Y, et al. High expression of ncRAN, a novel non-coding
RNA mapped to chromosome 17q25.1, is associated with poor prognosis in
neuroblastoma. Int J Oncol. 2009;34(4):931-8.
34. Scaruffi P, Coco S, Cifuentes F, et al. Identification and characterization of
DNA imbalances in neuroblastoma by highresolution oligonucleotide array
comparative genomic hybridization. Cancer Genet Cytogenet. 2007;177(1):20-
9.
35. Sausen M, Leary RJ, Jones S, et al. Integrated genomic analyses identify
ARID1A and ARID1B alterations in the childhood cancer neuroblastoma. Nat
Genet. 2013;45(1):12-7.
36. Shi H, Tao T, Abraham BJ, et al. ARID1A loss in neuroblastoma promotes the
adrenergic-to-mesenchymal transition by regulating enhancer-mediated gene
expression. Sci Adv. 2020;6(29):eaaz3440.
37. Peifer M, Hertwig F, Roels F, et al. Telomerase activation by genomic
rearrangements in high-risk neuroblastoma. Nature. 2015;526(7575):700-4.
38. Valentijn LJ, Koster J, Zwijnenburg DA, et al. TERT rearrangements are

79
frequent in neuroblastoma and identify aggressive tumors. Nat Genet.
2015;47(12):1411-4.
39. Cheung NK, Zhang J, Lu C, et al. Association of age at diagnosis and genetic
mutations in patients with neuroblastoma. JAMA. 2012;307(10):1062-71.
40. Pugh TJ, Morozova O, Attiyeh EF, et al. The genetic landscape of high-risk
neuroblastoma. Nat Genet. 2013;45(3):279-84.
41. Kloosterman WP, Koster J, Molenaar JJ. Prevalence and clinical implications of
chromothripsis in cancer genomes. Curr Opin Oncol. 2014;26(1):64-72.
42. Gröbner SN, Worst BC, Weischenfeldt J, et al. The landscape of genomic
alterations across childhood cancers. Nature. 2018;555(7696):321-7.
43. Molenaar JJ, Koster J, Zwijnenburg DA, et al. Sequencing of neuroblastoma
identifies chromothripsis and defects in neuritogenesis genes. Nature.
2012;483(7391):589-93.
44. Bosse KR, Maris JM. Advances in the translational genomics of neuroblastoma:
from improving risk stratification and revealing novel biology to identifying
actionable genomic alterations. Cancer. 2016;122(1):20-33.
45. Decock A, Ongenaert M, Vandesompele J, et al. Neuroblastoma epigenetics:
from candidate gene approaches to genome-wide screenings. Epigenetics.
2011;6(8):962-70.
46. Fetahu IS, Taschner-Mandl S. Neuroblastoma and the epigenome. Cancer
Metastasis Rev. 2021;40(1):173-89.
47. Ritenour LE, Randall MP, Bosse KR, et al. Genetic susceptibility to
neuroblastoma: current knowledge and future directions. Cell Tissue Res.
2018;372(2):287-307.
48. Raabe EH, Laudenslager M, Winter C, et al. Prevalence and functional
consequence of PHOX2B mutations in neuroblastoma. Oncogene.
2008;27(4):469-76.
49. Stoica GE, Kuo A, Powers C, et al. Midkine binds to anaplastic lymphoma
kinase (ALK) and acts as a growth factor for different cell types. J Biol Chem.
2002;277(39):35990-8.
50. Brodeur GM. Spontaneous regression of neuroblastoma. Cell Tissue Res.
2018;372(2):277-86.

80
51. Lavarino C, Cheung NK, Garcia I, et al. Specific gene expression profiles and
chromosomal abnormalities are associated with infant disseminated
neuroblastoma. BMC Cancer. 2009;9(1):44.
52. Spitz R, Hero B, Ernestus K, et al. Deletions in chromosome arms 3p and 11q
are new prognostic markers in localized and 4s neuroblastoma. Clin Cancer
Res. 2003;9(1):52-8.
53. Brodeur GM, Nakagawara A, Yamashiro DJ, et al. Expression of TrkA, TrkB
and TrkC in human neuroblastomas. J Neurooncol. 1997;31(1-2):49-55.
54. Nakagawara A, Brodeur GM. Role of neurotrophins and their receptors in
human neuroblastomas: a primary culture study. Eur J Cancer.
1997;33(12):2050-3.
55. Nakagawara A. Molecular basis of spontaneous regression of neuroblastoma:
role of neurotrophic signals and genetic abnormalities. Hum Cell.
1998;11(3):115-24.
56. Nakagawara A, Arima-Nakagawara M, Scavarda NJ, et al. Association between
high levels of expression of the TRK gene and favorable outcome in human
neuroblastoma. N Engl J Med. 1993;328(12):847-54.
57. Kogner P, Barbany G, Dominici C, et al. Coexpression of messenger RNA for
TRK protooncogene and low affinity nerve growth factor receptor in
neuroblastoma with favorable prognosis. Cancer Res. 1993;53(9):2044-50.
58. Hiyama E, Hiyama K, Yokoyama T, et al. Correlating telomerase activity levels
with human neuroblastoma outcomes. Nat Med. 1995;1(3):249-55.
59. Samy M, Gattolliat CH, Pendino F, et al. Loss of the malignant phenotype of
human neuroblastoma cells by a catalytically inactive dominant-negative Htert
mutant. Mol Cancer Ther. 2012;11(11):2384-93.
60. Russo C, Cohn SL, Petruzzi MJ, et al. Long-term neurologic outcome in
children with opsoclonus-myoclonus associated with neuroblastoma: a report
from the Pediatric Oncology Group. Med Pediatr Oncol. 1997;28(4):284-8.
61. Antunes NL, Khakoo Y, Matthay KK, et al. Antineuronal antibodies in patients
with neuroblastoma and paraneoplastic opsoclonus-myoclonus. J Pediatr
Hematol Oncol. 2000;22(4):315-20.
62. Rudnick E, Khakoo Y, Antunes NL, et al. Opsoclonusmyoclonus-ataxia
syndrome in neuroblastoma: clinical outcome and antineuronal antibodies-a

81
report from the Children’s Cancer Group study. Med Pediatr Oncol.
2001;36(6):612-22.
63. Cooper R, Khakoo Y, Matthay KK, et al. Opsoclonusmyoclonus-ataxia
syndrome in neuroblastoma: histopathologic features-a report from the
Children’s Cancer Group. Med Pediatr Oncol. 2001;36(6):623-9.
64. Astuti D, Agathanggelou A, Honorio S, et al. RASSF1A promoter region CpG
island hypermethylation in phaeochromocytomas and neuroblastoma tumours.
Oncogene. 2001;20(51):7573-7.
65. Takita J, Ishii M, Tsutsumi S, et al. Gene expression profiling and identification
of novel prognostic marker genes in neuroblastoma. Genes Chromosomes
Cancer. 2004;40(2):120-32.
66. Barbieri E, De Preter K, Capasso M, et al. Histone chaperone CHAF1A inhibits
differentiation and promotes aggressive neuroblastoma. Cancer Res.
2014;74(3):765-74.
67. Olsson M, Beck S, Kogner P, et al. Genome-wide methylation profiling
identifies novel methylated genes in neuroblastoma tumors. Epigenetics.
2016;11(1):74-84.
68. Alaminos M, Davalos V, Cheung NK, et al. Clustering of gene
hypermethylation associated with clinical risk groups in neuroblastoma. J Natl
Cancer Inst. 2004;96(16):1208-19.
69. Henrich KO, Bender S, Saadati M, et al. Integrative genomescale analysis
identifies epigenetic mechanisms of transcriptional deregulation in unfavorable
neuroblastomas. Cancer Res. 2016;76(18):5523-37.
70. Hellstrom IE, Hellstrom KE, Pierce GE, et al. Demonstration of cell-bound and
humoral immunity against neuroblastoma cells. Proc Natl Acad Sci U S A.
1968;60(4):1231-8.
71. Lauder I, Aherne W. The significance of lymphocytic infiltration in
neuroblastoma. Br J Cancer. 1972;26(4):321-30.
72. D’Angio GJ, Evans AE, Koop CE. Special pattern of widespread neuroblastoma
with a favourable prognosis. Lancet. 1971;1(7708):1046-9.
73. Hero B, Simon T, Spitz R, et al. Localized infant neuroblastomas often show
spontaneous regression: results of the prospective trials NB95-S and NB97. J
Clin Oncol. 2008;26(9):1504-10.

neuroblastoma

Recommended

Recommended

More Related Content

What's hot

What's hot (15)

Similar to neuroblastoma

Similar to neuroblastoma (20)

Recently uploaded

Recently uploaded (20)

neuroblastoma