Nihms 1745031

HHS Public Access
Author manuscript
Cancer Res. Author manuscript; available in PMC 2022 December 06.
Author Manuscript
Published in final edited form as:

Cancer Res. 2021 December 01; 81(23): 5783–5799. doi:10.1158/0008-5472.CAN-21-1191.
Recent advances in pediatric cancer research

Troy A. McEachron1,*, Lee J Helman2,3
1Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892
2Osteosarcoma Institute, Dallas, TX 75201
3Cancer and Blood Disease Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027
Author Manuscript
Abstract
Over the past few years, the field of pediatric cancer has experienced a shift in momentum and this
has led to new and exciting findings that have relevance beyond pediatric malignancies. Here we
present the current status of key aspects of pediatric cancer research. We have focused on genetic
and epigenetic drivers of disease, cellular origins of different pediatric cancers, disease models, the
tumor microenvironment, and cellular immunotherapies.
Keywords
Pediatric Cancer; Genomics; Epigenetics; Microenvironment; Immunotherapy
Author Manuscript
INTRODUCTION:
The treatment of pediatric cancers has been a success story, with current overall survival
of ~80% in the U.S. It is estimated that there are currently >500,000 survivors of pediatric
cancer in the U.S. Nonetheless, this success has occurred at a significant price, as the
prevalence of severe chronic health disorders among long-term survivors of pediatric cancer
is 3-fold higher than in matched controls. In addition, there are still close to 13,000 pediatric
cancer deaths per year in the U.S, and there are several tumors for which long-term survival
remains poor. Recently, the adult cancer research community has seen major advances
in detailing the molecular and cellular mechanisms of disease that have resulted in the
generation of novel targeted therapies and immunotherapies, some of which are applicable
to pediatric tumors. Here, we have reviewed several major topics that we believe have had
a major impact already on fundamental understanding of the biology of pediatric cancers
Author Manuscript
and will continue to impact the field going forward. To maintain the current relevance of
this review, we have restricted most of our literature search to the last 5–6 years and have
included a few older references for context. The following sections focus on new insights
into the genetic and epigenetic underpinnings of these cancers, therapeutic breakthroughs,
developmental and cellular origins of these tumors, model systems that have provided
new perspectives, microenvironmental influences on tumor biology, and immunotherapy
*
Corresponding Author: Troy A. McEachron, Pediatric Oncology Branch, National Cancer Institute, 10 Center Drive, Building 10,
Room 1W-3872, Bethesda, MD 20892, Phone: 1-240-858-3638, Troy.McEachron@NIH.gov.
Conflict of Interest: The authors have no financial interests to declare as it pertains to this manuscript
McEachron and Helman Page 2
approaches to treatment. While a comprehensive review of the current status of pediatric

Author Manuscript
cancer research is of course beyond the scope of this manuscript, we believe that each of the
areas discussed will continue to generate new ideas and hypotheses in the near future that
hopefully will lead to new and improved therapies that ultimately improve both short-term
and long-term outcomes of childhood cancer.
GENOMIC AND EPIGENOMIC PROFILING STUDIES

Pediatric Pan-Cancer Profiling Studies:
Large scale genomic profiling of various hematologic and non-hematologic tumors, so
called “pan cancer studies”, have been instrumental in revealing the overall landscape of
adult malignancies allowing for the identification of genetic drivers of disease, discovery of
mutational signatures and complex structural aberrations, and investigation of drug targets
(1,2). Recently, several pediatric pan-cancer genomic studies have been completed and have
Author Manuscript
reported that pediatric tumors contain fewer coding mutations than adult tumors, that TP53
remains the most frequently recurring mutation, and that mutations in epigenetic-associated
genes were common events (3–6). Profiling close to 1,700 pediatric leukemias and solid
tumors revealed 142 somatic driver gene mutations and found that copy number alterations
and structural variants were the most prevalent types of alterations to impact these driver
genes (3). Furthermore, this study discovered two new mutation signatures in addition to
the existing signatures within the COSMIC (Catalogue Of Somatic Mutations In Cancer)
database.
Analysis of DNA sequencing data obtained from 961 tumors across 24 different pediatric,
adolescent, and young adult patient histologies identified 34 genomic loci recurrently
impacted by copy number alterations (4). This study also demonstrated that of the 77
Author Manuscript
significantly mutated genes identified across the entire sample cohort, the majority of these
mutations were exclusive to specific tumor types (4). Furthermore, the authors highlighted
the differences between the genomic landscapes of adults and pediatrics showing that only
30% of the significantly mutated genes identified in pediatric tumors were present in adults.
Of the 24 tumor types profiled in this study, osteosarcomas and adrenocortical carcinomas
were the most genomically complex, hallmarked by an increased rate of hyperdiploidy
and chromothripsis, a catastrophic shattering of chromosomes with subsequent error-prone
repair (4,7). Whether the observed genomic complexity in adrenocortical carcinoma and
osteosarcoma represents the culmination of persistent chromosomal instability or the
product of a single early cataclysmic event is yet to be experimentally determined.
Collectively, these pan-cancer studies further highlight the unique biology driving several
pediatric cancers (Figure 1). Moreover, the data from these studies validate the seminal
Author Manuscript
publication by Bert Vogelstein and colleagues illustrating the age-associated differences in

the mutational landscapes across multiple cancer types (8).
DNA Methylation Profiling of Brain Tumors

DNA methylation refers to the addition of methyl groups to chromosomal DNA to regulate
gene transcription. Recently, numerous studies have emerged demonstrating the utility
of DNA methylation analysis to subcategorize central nervous system (CNS) tumors.

Genome-wide DNA methylation profiling of 3,093 meningiomas revealed that clear cell
Author Manuscript
meningiomas, a histology predominantly observed in children and young adults, clustered

independently of all other subtypes (9). DNA methylation arrays subdivided Group 3 and
Group 4 medulloblastomas into 8 different molecular subgroups (10). DNA methylation
analysis of pilocytic astrocytoma specimens showed that tumors arising in the infratentorial,
midline, and cortical regions of the brain had distinct methylation profiles (11). A separate
study comparing diffuse astrocytoma and pilocytic astrocytoma specimens showed that the
pilocytic specimens were hypomethylated and had a different methylation profile than the
diffuse astrocytoma specimens (12). Integrated epigenetic profiling of CNS atypical teratoid/
rhabdoid tumors, identified three disease subgroups with distinct DNA methylation profiles
and epigenetic landscapes (13,14). Together, these data illustrate the power and applicability
of DNA methylation to subcategorize CNS tumors which has clinical implications pertaining
to the refinement of ambiguous diagnoses (15,16).
Author Manuscript
Mutational Activation of RAS-MAPK Pathway

Upstream and downstream components of the RAS and MAPK pathways are recurrently
mutated in various pediatric CNS and solid tumors (4,5,17). Analysis of the Foundation
Medicine sequencing data generated from 1,215 different pediatric tumors showed MAPK
mutations were common in hypermutant tumors driven by replication repair deficiency
and that the RAS/MAPK pathway was indeed active in these tumors (5). By performing
functional experiments using pediatric hypermutant glioma and pediatric hypermutant
colorectal cancer patient-derived xenograft (PDX) models, the authors showed that these
RAS-MAPK mutant replication repair deficient tumors were susceptible to MEK inhibition,
highlighting a potential context for synthetic lethality (5).
Whole genome sequencing of matched primary-relapsed neuroblastoma specimens led to the

Author Manuscript
discovery that 78% of relapsed specimens contained mutations in genes involved in RAS-
MAPK signaling (18). Similarly, mutations in PHOX2B, CIC, and DMD activated RAS-
MAPK signaling in neuroblastoma cell lines and tumor specimens (19). Importantly, both
studies demonstrated that mutations in genes involved in RAS-MAPK signaling conferred
increased sensitivity to pharmacologic MEK inhibition both in vitro and in vivo (5,19).
In the largest study of its kind, mutational analysis of DNA from 641 rhabdomyosarcoma
patients indicated that mutations in the RAS pathway occurred at a frequency of 56% in
fusion negative tumors that lack the pathogenomic FOXO1 fusion oncogenes (20). This
study also showed that isoform-specific RAS mutations associated with age where HRAS
mutations were enriched in infants, KRAS in toddlers, and NRAS in adolescents (20). This
incredibly intriguing finding implies that the different RAS isoforms may impart specific
Author Manuscript
age-restricted oncogenic programs.
Somatic mutations to the RAS-MAPK pathway have also been reported in aggressive
pediatric hematological malignancies. Survivors of pediatric cancer are at increased risk
for subsequent malignancies due to the late effects of their highly cytotoxic chemotherapy
treatments (21,22). Genomic analysis revealed a high frequency of KRAS and NRAS
mutations in pediatric cancer survivors who had developed therapy related myeloid
neoplasms (23). A combination of exome and whole genome sequencing discovered that

44% of relapsed acute lymphoblastic leukemia (ALL) specimens had somatic mutations
Author Manuscript
in NRAS, KRAS, or PTPN11 (24). Using isogeneic leukemia cell lines expressing
either wild-type or the G12D KRAS mutant, this study showed that oncogenic KRAS
conferred resistance to methotrexate and an unexpected sensitivity to vincristine. Although
juvenile myelomonocytic leukemia has long been associated with oncogenic RAS signaling,
integrated genomic and DNA methylation analysis revealed that mutations in NRAS, KRAS,
and PTPN11 were differentially enriched in independent molecular subgroups (25). While
each of the subgroups were associated with different clinical and molecular features,
the extent to which the respective somatic RAS pathway mutations contributed to these
subgroup-specific features was not reported.
Germline Variants Associated with Predisposition and Increased Disease Risk:

The importance of understanding the deleterious effects of germline mutations in pediatric
Author Manuscript
cancer patients can be traced back to Knudson’s original 1971 publication describing the
two-hit hypothesis of tumorigenesis in patients with retinoblastoma (26). Five decades
later, the prevalence and impact of germline variants in patients with pediatric tumors is
still an active area of research due, in large part, to the advances in genome sequencing
technology. A comprehensive summary of the various germline variants associated with
pediatric cancers and cancer predisposition syndromes is beyond the scope of this
manuscript but have been thoroughly reviewed (27–31). Analysis of germline DNA
sequencing data from 1,120 pediatric cancer patients diagnosed with leukemia, CNS
tumors, neuroblastoma, retinoblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma,
adrenocortical carcinoma, and melanoma revealed that the overall rate of pathogenic or
likely pathogenic germline variants was a striking 8.5% (32). Two additional studies
estimate that the frequency of causative germline variants in pediatric cancer patients to
Author Manuscript
be slightly lower at 6% and 6.9%, respectively (4,33). When hematologic malignancies were
excluded, the percentage of pediatric patients diagnosed with solid tumors and CNS tumors
that carried germline variants in known cancer predisposition genes increased to 12% (34).
Strikingly, germline variants were frequently observed in the following DNA repair genes:
TP53, BRCA2, PMS2, CHECK2, MSH2, and MSH6 (32,35).
The frequency of germline variants in TP53 varies by tumor type. Germline variants in
TP53 increased the risk of developing sonic hedgehog (SHH) medulloblastomas and were
associated with an increased prevalence of chromothripsis in these patients (36). This is
consistent with the finding that germline variants in TP53 were associated with a higher
number of somatic chromosomal structural variations (4). In adrenocortical carcinoma,
germline TP53 variants were observed in approximately 50% of patients and rather than
clustering in hotspots, these variants were distributed throughout the gene (37). Between 4–
Author Manuscript
5.3% of patients with osteosarcoma harbored pathogenic or likely pathogenic germline TP53
variants, with almost half of these mutations occurring de novo (38,39). These osteosarcoma
patients tended to be younger, have disease within the axial skeleton, were more likely
to present with metastatic disease upon diagnosis, and had an inferior prognosis (38).
Germline TP53 variants were observed at a frequency of 2% in pediatric B-cell ALL (40).
According to this study, pathogenic TP53 variants were enriched in older children and those
diagnosed with hypodiploid ALL, a more aggressive form of this disease. Moreover, these

patients experienced inferior outcomes and were at a higher risk of developing a secondary
Author Manuscript
malignancy (40).
As stated above, there are several germline variants in genes other than TP53 that
have also been documented in pediatric cancers. Patients with osteosarcoma were found
to have pathogenic or likely pathogenic germline variants in CDKN2A, MEN1, VHL,
POT1, APC, MSH2, and ATRX (38). Amazingly, this study concluded that 28% of
patients with osteosarcoma had a pathogenic or likely pathogenic germline variant in a
cancer predisposition gene (38). Furthermore, the GLDC/IL33 locus has been identified
as an osteosarcoma susceptibility locus and two single nucleotide polymorphisms (SNPs),
rs3765555 and rs55933544, were associated with significantly decreased survival and
decreased IL33 expression (41,42). Data from the Children’s Oncology Group identified
germline variants in 15 genes at a frequency of 7.3% in children with rhabdomyosarcoma
(43). Similar results were obtained in a separate rhabdomyosarcoma study showing that
Author Manuscript
approximately 7% of patients had a germline variant in a known cancer predisposition

gene (44). Interestingly, in both studies these germline variants were enriched in
patients with FOXO1 fusion-negative disease. Separate genome wide association studies
(GWAS) have identified new SNPs associated with Ewing sarcoma risk at the 6p25.1,
20p11.22, and 20p11.23 loci (45,46). In patients with Ewing sarcoma, pathogenic or
likely pathogenic germline variants occurred at a frequency of 13% and were enriched
in several genes associated with DNA damage repair (47). GWAS of 707 neuroblastoma
patients revealed that the rs6720708 SNP at the BARD1 locus, a known high-risk
neuroblastoma susceptibility locus, was significantly associated with the development of
adrenal neuroblastoma versus developing thoracic disease (48).
A study consisting of 280 California patients with glioblastoma, anaplastic astrocytoma, and
Author Manuscript
astrocytoma not otherwise specified reported that pathogenic germline variants were present
in approximately 11% of the patients and that these variants were enriched in patients
with glioblastoma (49). In medulloblastoma, germline variants were differentially enriched
according to disease subgroups. The SHH subgroup of medulloblastoma had the highest
rate of germline alterations affecting various genes including TP53, SUFU, PTCH1, PABL2,
BRCA2, GPR161, and ELP1 (36,50,51). Germline variants in APC were enriched in the
WNT subgroup whereas PALB2 and BRCA2 germline variants were observed in SHH,
Group 3, and Group 4 medulloblastomas (36).
ETV6 germline variants were associated with increased risk of developing childhood ALL
and patients with these predisposing germline variants were more likely to be older at
the time of diagnosis and have hyperdiploid ALL, a leukemia subtype with a favorable
Author Manuscript
prognosis (52). The rs3824662 SNP in GATA3 was recently discovered to confer increased
risk of relapse in patients with ALL (53). New ALL risk loci were also identified at 5q31.1,
6p21.31, 9q21.31, and 17q21.32 and associated with specific ALL subtypes (54). Two SNPs
rs886285 (at 5q31.1) and rs210143 (at 6p21.31) were associated with high-hyperdiploidy
ALL, rs10853104 (at 17q21.32) with the ETV6-RUNX1 subtype, and rs76925697 (at
9q21.31) with B-cell ALL (54). Patients with Down syndrome have an extraordinarily
high risk for developing various leukemias during childhood, including acute myeloid
leukemia (AML), ALL, and megakaryoblastic leukemia (55). ALL patients with Down

syndrome exhibited an increased frequency of a risk allele in CDKN2A, a previously

Author Manuscript
established susceptibility locus (56). The IKZF1 SNP rs58923657, which maps to an active
B-cell enhancer, was also enriched in patients with Down syndrome and the risk allele
decreased the enhancer activity (56). Moreover, silencing IKZF1 preferentially increased
the proliferation of lymphoblastoid cells from individuals with Down syndrome versus cells
from those without Down syndrome (56). Together, these data suggest that presence of
trisomy 21 modifies the phenotypic effect of the rs58923657 risk allele.
Telomeres are complexes of proteins bound to repetitive DNA sequences positioned at the
end of each chromosome that shorten with each cell division, thus acting as a cellular
clock recording each replicative event. Telomere length is disproportionately associated with
cancer incidence where shorter telomeres seem to be protective while longer telomeres
confer an increased risk (57–59). For instance, longer telomere length was associated
with a higher risk of developing ependymoma with adolescent and adult onset (60).
Author Manuscript
In osteosarcoma, a weighted polygenic risk score revealed that longer telomere length
was a risk factor for developing disease in Hispanic, Asian/Pacific Islander, and African
American children (61). The association between telomere length and osteosarcoma risk
was recapitulated in a separate study where the data were not stratified by ethnicity (62).
Additionally, this GWAS study also found that SNPs in known telomere maintenance genes
increased the risk of developing neuroblastoma and ALL (62).
Given that pediatric tumors are rare, germline variants that moderately increase risk in
adult disease may indeed have a larger affect size in certain pediatric tumors. That said,
not all germline variants in known cancer predisposition genes are causative of disease
but may impact disease severity and/or other phenotypic aspects of the tumor. Similarly,
germline variants in genes not previously associated with adult diseases may have biological
Author Manuscript
and/or clinical relevance in children and vice versa (63). However, the accessibility to
large scale genomic databases combined with growing institutional genomic profiling
efforts affords researchers a collaborative opportunity to perform clinically annotated
genotyping studies to further investigate the impact of germline variants in pediatric
cancers. For example, the MSK-IMPACT study (NCT01775072) is performing prospective
clinical germline sequencing on pediatric solid tumor patients and using the resultant
data to identify actionable targeted therapeutics and prioritize patients and families that
may benefit from further screening/surveillance (64). These types of clinical studies can
also inform the surveillance protocols for these patients. In 2017, the Pediatric Cancer
Working Group of the American Association for Cancer Research convened a Childhood
Cancer Predisposition Workshop where a set of screening and surveillance recommendations
were established. This meeting included the discussions on the use and interpretation of
Author Manuscript
whole-body MRI, the need for registries to enable natural history studies, the consultancy
role of syndrome-specific centralized centers of excellence, and modifications to existing
surveillance protocols (65–71).
In summary, the widespread adoption of advanced genomic technologies in both the clinic
and the research laboratory have led to the discovery of numerous germline variants.
Experimental investigations aimed at determining the functional impact of these and other
reported variants in racially and ethnically diverse cohorts will further our understanding

of the biological and clinical significance of germline genetics in pediatric malignancies.

Author Manuscript
Nonetheless, the current data clearly indicate that subsets of pediatric cancer patients are
genetically predisposed to developing disease and supports the observed inverse relationship
between germline variants and age (37,38,72) (Figure 2).
Liquid Biopsies
Disease monitoring is an important aspect to the management of both pediatric and adult
disease. However, unlike the adult setting where repeated biopsies are more readily obtained,
performing serial biopsies in pediatric patients has traditionally been discouraged unless
deemed medically necessary. This “one and done” approach constrains the ability to identify
and/or develop reliable biomarkers for the longitudinal study of central nervous system
(CNS) and solid tumors in children. Liquid biopsies are minimally invasive tests that
utilize accessible body fluids (i.e.: blood, urine, saliva, and/or cerebral spinal fluid) to
Author Manuscript
detect cellular and/or molecular biomarkers of a given disease such as cell free DNA
(cfDNA), circulating tumor cells, microRNAs, proteins, and metabolites (73–75). Emerging
data suggests that liquid biopsies may circumvent these sampling limitations and allow for
longitudinal assessments of disease burden. Detection and quantification of cfDNA has been
documented in various solid and CNS tumors of childhood, with the earlier studies largely
relying on polymerase chain reaction-based approaches (76). These methods were reliable
in detecting cfDNA from diseases in which known recurrent driver mutations existed, for
example histone mutations in high grade glioma (HGG) (77,78).
More recently, next generation sequencing (NGS) based methods have been employed
and have the potential to identify mutations in tumors a priori. NGS profiling studies of
cfDNA from patients with neuroblastoma, Ewing sarcoma, Wilms tumor, osteosarcoma,
alveolar rhabdomyosarcoma, medulloblastoma, and high-grade glioma have consistently
Author Manuscript
reported positive associations between cfDNA abundance and disease burden (78–88). The
anatomical site from which cfDNA is obtained can impact the cfDNA yield for subsequent
downstream analyses. Studies have shown improved detection of cfDNA isolated from
cerebral spinal fluid versus that of plasma in patients with medulloblastoma and brain stem
glioma (84,88). For Wilms tumor, detecting mutations in cfDNA isolated from urine was not
only achievable, but more mutations were detected in the urine versus plasma from these
patients (83). Optimization of this urine-based Wilms tumor cfDNA profiling approach is
particularly appealing as it presents zero risk to the patient, allows for serial collections
in large quantities, and can be done at home. Moreover, this study provides rationale to
investigate the applicability of cfDNA profiling in other childhood kidney tumors such as
renal cell carcinoma, malignant rhabdoid tumors, and clear cell sarcoma of the kidney.
Author Manuscript
cfDNA has also been used to gain important biological insight. Longitudinal profiling
of cfDNA from patients with neuroblastoma demonstrated the ability to identify clonal
populations with distinct mutational patterns, model clonal evolution throughout the
treatment regimen, and identify relapse specific clones (79). Analysis of gene-specific
5-hydroxymethylcytosine modified cfDNA from neuroblastoma patients identified unique
profiles associated with disease burden (81). Further analysis revealed differentially enriched
biological processes and pathways in patients with and without active disease. Methylation

analysis of cfDNA isolated from the cerebral spinal fluid of patients with medulloblastoma
Author Manuscript
was used to derive an epigenetic signature based on differentially methylated CpG sites that
could specifically and sensitively identify medulloblastoma subtypes (85).
Several companies have gained, or are seeking, US Food and Drug Administration (FDA)
approval for their respective adult oncology liquid biopsy assays including Guardant,
FoundationOne, Grail, and Thrive (89–95). Currently, liquid biopsies have not yet been
approved for use with pediatric patients but trials investigating the clinical utility of liquid
biopsies to monitor disease burden and treatment response in various pediatric cancers are
underway. It is likely that additional studies on the utility of liquid biopsies will increase
in the coming years and ultimately integrate into standard of care for patients with certain
tumor types.
Filling in the Knowledge Gaps

Author Manuscript
Despite the recent advances in exploring the genomic landscapes of various pediatric
cancers, knowledge gaps remain. One such knowledge gap is that there are only a few
studies that detail the genomic and transcriptomic profiles of specimens from patients with
recurrent, refractory, and/or relapsed CNS and solid tumors as the majority of published
genomic studies were performed using treatment naive specimens. The emerging data
from the few studies performed on recurrent, refractory, and/or relapsed specimens from
pediatric patients with CNS and solid tumors suggest a comparatively higher mutation
burden compared to treatment naïve patient specimens (4,18,96–98). Additional descriptive
and functional studies are needed to uncover the mechanisms associated with disease
progression in each of these tumors and tumor subtypes.
Another knowledge gap is that most of the genomic profiling data has been generated
Author Manuscript
using short-read sequencing methods. While the identification of single nucleotide variants
from these data is well established, many short-read sequencing analytical tools struggle
to resolve complex genomic aberrations, necessitating the implementation of sophisticated
algorithms, each with their own biases and limitations (99,100). Long-read sequencing
platforms routinely produce continuous sequencing read lengths of >10kb and have
been demonstrated to resolve complex genomic rearrangements, including chromothripsis
(101,102). As part of the Gabriella Miller Kids First (GMKF) Pediatric Research Program,
long-read sequencing technologies will be used to uncover the genomic complexities
associated with certain pediatric cancers and birth defects. It is anticipated that long-read
sequencing technologies may supplement the existing data to better resolve the complex
structural variants inherent to tumors such as osteosarcoma and adrenocortical carcinoma.
Author Manuscript
BREAKTHROUGHS IN TARGETED THERAPIES

Unlike adults, few efficacious targeted therapies exist for childhood cancers. Recently,
larotrectinib and selumetinib received FDA and European Medicines Agency (EMA)
approval, a significant therapeutic advance for the treatment pediatric cancer patients bearing
driver mutations targeted by these kinase inhibitors. Larotrectinib targets TRK proteins and
is indicated for cancers with oncogenic TRK-fusions such as those found in 2.22% of
pediatric patients (103,104). The overall response rate for patients with TRK-fusion positive

tumors treated with Larotrectinib was 79% with 16% having complete responses (105). In
Author Manuscript
addition to its potent and durable antineoplastic effect, larotrectinib improved the quality of
life for patients, the gold standard for any therapeutic agent (106).
Selumetinib is a MEK1/2 inhibitor indicated for patients with neurofibromatosis type 1 with
inoperable plexiform neurofibromas (107–109). Of the patients treated with selumetinib,
>70% exhibited a confirmed durable partial response of >20% reduction in tumor size
(107,108). Impressively, all the patients who had received selumetinib had experienced
some degree of tumor shrinkage. Selumetinib is also currently under clinical investigation
for pediatric patients with BRAF mutant pilocytic astrocytoma and NF1-associated low-
grade gliomas (110). The approval of selumetinib marks the first ever approved targeted
therapy for patients with neurofibromatosis. Moreover, these two targeted agents validate the
necessity of a pediatric drug development pipeline.
Author Manuscript
EPIGENETIC DRIVERS OF PEDIATRIC CANCER

Histone H3K27:
Since the first reports of somatic histone H3 mutations at lysine 27 (H3K27M) and
glycine 34 (H3G34) in pediatric high-grade gliomas (HGG) in 2012, the interest in these
“onco-histones” has sparked numerous investigations aimed at understanding their role in
tumorigenesis (111,112). Functional studies show that the H3K27M mutation increased
the self-renewal capacity of neurospheres in vitro whereas CRISPR-mediated correction
of the H3K27M mutation in glioma cell lines decreased cell proliferation (113,114).
In genetically engineered murine HGG models, the presence of the H3K27M mutation
decreased tumor latency (113,115). Multiple studies have shown that the H3K27M mutation
regulated genes involved in neurogenesis and differentiation (113,114,116–118). Bivalent
Author Manuscript
promoters are chromatin domains that are marked by both repressive and activating histone
modifications. Mutant H3K27M histones colocalized with the H3K27Ac mark at bivalent
promoters and enhancers, diminished the deposition and spreading of H3K27me3 marks,
and allowed for transcription of polycomb repressed genes (113,114,117,118). Interestingly,
the H3K27M mutation did not sequester polycomb repressor complex 2 (PRC2) (117,118).
Correction of the mutation restored the balance between H3K27me3 and H3K27Ac marked
loci, reestablished polycomb mediated repression of developmental genes, and promoted
glial lineage commitment (114,116). Therapeutically, H3K27M mutant cell lines were
sensitive to pharmacologic inhibition of EZH2, the PRC2 histone methyltransferase, and
did so in a p16INK4a-dependent manner and such interventions prolonged the survival of
tumor bearing mice (115). Bromodomain and extra-terminal (BET) proteins are “chromatin
readers” that bind to acetylated lysines to recruit chromatin remodelers that subsequently
Author Manuscript
regulate transcription. The BET inhibitor JQ1 forced the differentiation of H3K27M mutant
glioma cells in vitro and prolonged survival in vivo (118). Interestingly, an aggressive
subset of pediatric posterior fossa ependymomas have been identified as having low
levels of H3K27me3 and increased levels of H3K27Ac (119). These tumors exhibited
DNA methylation profiles like that of H3K27M mutant gliomas as well as overlapping
H3K27me3 ChIP-seq peaks. This signifies that histone mutations are not the only means

by which to achieve the molecular and phenotypic characteristics attributed to the H3K27M
Author Manuscript
mutation.
Enhancer Dysregulation:
Enhancers are regulatory chromatin domains that recruit transcriptional machinery to
promote or repress gene expression from a specific locus. The accessibility and activity
of enhancers are developmentally regulated and associated with lineage commitment but
can be dysregulated to aberrantly drive oncogenic transcriptional profiles. In pediatric HGG,
the presence of the H3G34 mutation arrested the cells in a developmental state where the
chromatin conformation juxtaposed the GSX2 enhancer and PDGFRA locus and allowed
for PDGFRA to hijack the enhancer of this developmentally regulated transcription factor to
drive pathologic PGDFRA expression (120). Enhancer hijacking has also been observed in
MYCN-amplified neuroblastoma. An integrative genomic approach identified two classes of
Author Manuscript
MYCN amplicons (121). Class I amplicons were simple amplification events that contained
both MYCN and its local enhancer whereas class II amplicons contained MYCN without
the local enhancer. Further investigation revealed that these Class II amplicons were double
minute chromosomes and circumvented the lack of the local MYCN enhancer by hijacking
distal enhancers through complex chromosomal structural rearrangements (121).
Differential enhancer activity was observed in matched primary-metastatic osteosarcoma

tumors and isogenic cell line pairs, revealing and that these metastatic-specific enhancers
promoted transcriptional programs necessary for metastatic seeding (122). Further analysis
indicated that an enhancer regulating the expression of coagulation factor 3 was positively
selected for in the metastatic cell lines and that perturbation of this enhancer reduced
pulmonary metastasis in vivo (122). The EWS-FLI1 chimeric oncoprotein is the product
of the EWSR1-FLI1 translocation that defines 85% of Ewing sarcoma tumors. GGAA
Author Manuscript
repeats are the known EWS-FLI1 binding motif in Ewing sarcoma (123). Recent data
demonstrated that these GGAA repeats are enriched at super-enhancers, were bound by
EWS-FLI1, and identified MEIS1, a developmentally regulated homeobox protein, as
a super-enhancer driven oncogenic transcription factor (124). Additionally, MEIS1 and
EWS-FLI1 co-localized at a subset of super-enhancers to further drive transcriptional
dysregulation.
Recently both mesenchymal stem-like and adrenergic-like cells were identified in primary
and established neuroblastoma cell lines, and each subpopulation of cells had unique super-
enhancer landscapes that were responsible for maintaining their respective lineage identities
(125). Additional functional analysis showed that ectopic expression of the mesenchymal
stem-like super-enhancer associated transcription factor PRRX1 partially reprogramed
Author Manuscript
the adrenergic-like neuroblastoma cells towards a more mesenchymal stem-like state as

evidenced by global shifts in the super-enhancer landscape and transcriptional profiles (125).
The PAX3/7-FOXO1 translocations, the hallmark chromosomal aberrations in alveolar

rhabdomyosarcoma, brings PAX3/7 gene transcription under the control of the FOXO1
super-enhancer (126). Mutations in the RAS-MAPK pathway drive a subset of fusion
negative rhabdomyosarcomas tumors (127). In this context, RAS-mutant cells were
developmentally arrested due to oncogenic MAPK signaling which imparted a RAS-

dependent super-enhancer landscape (128). Inhibition of MAPK signaling by trametinib

Author Manuscript
altered the chromatin accessibility and restored the myogenic super-enhancer landscape to
drive differentiation of fusion negative rhabdomyosarcoma cells (128). SNAI2 was shown
to compete with MYOD1, a master myogenic transcription factor, and inactivate myogenic
enhancers to halt differentiation in fusion negative rhabdomyosarcoma (129). A separate
study demonstrated that TWIST2 repressed myogenesis by both upregulating SNAI2
expression and competing with MYOD1 binding at target genes associated with muscle
differentiation (130). The loss of H3K27Ac and deposition of H3K27me3 at TWIST2-bound
myogenic loci indicated that TWIST is capable of recruiting PRC2 to inhibit differentiation
in fusion negative rhabdomyosarcoma cells.
Pioneer Factors:
Pioneer factors are the first transcription factors to bind specific heterochromatic loci
Author Manuscript
and alter the local chromatin accessibility by recruiting additional chromatin remodelers.
OTX2 is a developmentally regulated transcription factor that was found to occupy several
active enhancers in Group 3 medulloblastoma cells (131). Additionally, NEUROD1, an
established neuronal pioneering factor, was shown to interact with OTX2 at enhancers
(131,132). Ectopic expression of OTX2 induced a Group 3 medulloblastoma chromatin
accessibility and enhancer signature in mesenchymal stem cells, suggesting that OTX2
functions as a pioneer factor (131). The EWS-FLI1 oncogenic fusion gene endowed Ewing
sarcoma cells with a unique enhancer profile (133). Mechanistically, EWS-FLI1 bound to
GGAA repeats to remodel chromatin and generate de novo enhancers and displaced ETS
transcription factors to promote transcription of target genes (134). The PAX3-FOXO1
fusion was found to function as a pioneering factor in alveolar rhabdomyosarcoma and
created de novo super-enhancers that drove oncogenic and myogenic transcription programs
Author Manuscript
in a BRD4-dependent manner (135). BRD4 is a member of the BET family of “chromatin

readers” and CHD4 is a chromatin remodeler. CHD4 interacted with BRD4 and colocalized
with PAX3-FOXO1 at a subset of super-enhancers in alveolar rhabdomyosarcoma (136).
These data also demonstrated that PAX3-FOXO1 binding was, at least in part, dependent on
the presence of CHD4 chromatin remodeling (136).
Redirecting/Hijacking of Chromatin Remodelers:

BAF is the mammalian equivalent of the SWI/SNF chromatin remodeling complex, of
which SS18 is a member. The SS18-SSX chimeric oncoprotein, the gene product of the
translocation that defines synovial sarcoma, competed with SS18 to hijack the BAF complex
(137). The SS18-SSX fusion antagonized PRC2-mediated gene repression by directing the
BAF complex away from enhancers and towards bivalent genes to promote aberrant gene
Author Manuscript
transcription (138,139). Additionally, the histone demethylase KDM2B, a member of the

non-canonical PRC1.1 complex, has been shown to recruit BAF complexes containing
the SS18-SSX fusion to repressed developmentally regulated genes, resulting in their
transcription (140).
At active enhancers, the interaction between EWS-FLI1 and RING1B of the PRC1 complex
was necessary for target gene expression (141). These data also showed that RING1B
maintained its repressive function at non-EWS-FLI1 bound loci. Together, the data suggest

that RING1B targets EWS-FLI1 to repressed enhancers to recruit additional chromatin

Author Manuscript
modifiers and transcription factors to activate these enhancers and subsequent transcriptional
programs (141). The prion domain present in wild type EWS is retained in the EWS-FLI1
fusion and this domain was deemed critical for recruiting the BAF complex to activate
enhancers (142).
As previously mentioned, most pediatric tumors have a relatively low mutational burden.
It is becoming clear that in contradistinction, many pediatric tumors have significantly
reprogramed epigenomes, leading to widespread transcriptional dysregulation. Better
mechanistic understanding of the means by which this epigenetic reprograming occurs may
lead to new therapeutic “targeted” approaches in a variety of pediatric tumors.
DEVELOPMENTAL AND CELLULAR ORIGINS OF PEDIATRIC TUMORS

Author Manuscript
Despite decades of research and various hypotheses, the question regarding the cell of origin
of childhood tumors has plagued the field of pediatric cancer research. Recent advances in
single cell RNA sequencing (scRNA-seq) technologies combined with powerful informatic
analyses have allowed investigators to more deeply probe this question. Since many
pediatric tumors are “embryonal” in appearance, an integrated approach utilized across
various studies from multiple groups has involved comparing the single cell transcriptional
profiles of normal developing tissues with that of tumors derived from the same tissue.
Comparative single cell analysis of transcriptomes from normal human fetal adrenal glands
and dissociated neuroblastoma tumors suggested that, of the cells profiled, neuroblastoma
cells most closely resembled developmentally arrested sympathoblasts (143). Strikingly, this
comparison was consistent across the different neuroblastoma risk groups. A separate study
used scRNA-seq data from embryonic and post-natal murine adrenal glands to demonstrate
Author Manuscript
that neuroblastoma cells significantly correlated with neuroblasts, a cell type whose
signature partially overlapped with that of sympathoblasts (144). This study also highlighted
a subset of neuroblastoma cells that closely resembled Schwann cell precursors which
were hallmarked by decreased MYCN and ALK expression (144). Another study mapped
neuroblastoma cells to cells isolated from early human embryos and fetal adrenal glands
and showed that neuroblastoma cells most closely resembled undifferentiated chromaffin
cells (145). In these studies, chromaffin cells clustered adjacently to sympathoblasts, thus
approximating the potential cellular origins of neuroblastoma (143–145). The creation of a
single cell transcriptome atlas derived from healthy fetal, pediatric, adolescent, and adult
kidneys and ureters identified ureteric bud cells and primitive vesicle cells of the developing
nephron as the cells that most closely correlated with Wilms tumor cells (146).
Similar comparative scRNA-seq approaches have been used to identify the cellular origin
Author Manuscript
in pediatric brain tumors. Comparing scRNA-seq data from human medulloblastoma cells
to cells from the developing mouse brain revealed that WNT subgroup medulloblastoma
cells were heterogenous and did not explicitly correlate with any cell type in the murine
cerebellum but, in fact, resembled lower rhombic lip mossy-fiber neurons of the pons
(147,148). Group 4 medulloblastoma cells mapped to unipolar brush cells and glutamatergic
cerebellar nuclei (147,148). The SHH subgroup were enriched in granule neuron progenitors
that varied in their differentiation status in accordance with age, highlighting the differences

between adult and pediatric disease (147). scRNA-seq on both the developing brain and
Author Manuscript
tumor cells from a mouse model of SHH medulloblastoma revealed significant correlations
between cerebellar granule neuron progenitor cells and the murine tumor cells, thereby
independently validating the conclusions of similar studies (147–149). Projection of bulk
RNA-seq data onto a single cell atlas derived from scRNA-seq data of the developing human
brain stem, murine pons, and murine forebrain identified neural progenitor cells as the
potential cell of origin for H3K27M pontine gliomas (148). Similarly, the H3G34 mutation
arrested HGG tumor cells in a developmental state most consistent with interneuron
progenitor cells (120). Analysis of embryonal tumors with multilayered rosettes suggested
that these tumors were likely derived from fetal radial glial cells (148). The cellular origins
of atypical teratoid/rhabdoid tumors (ATRT) were more ambiguous and, while the precise
cell of origin was not identified, the data suggested that these tumors likely arose from early
progenitor cells of non-neuroectodermal origin (148).
Author Manuscript
EXPERIMENTAL MODEL SYSTEMS

Comprehensively Profiled Patient Derived Xenografts
In comparison to adult diseases, the rate of progress in the field of pediatric cancer research
has been somewhat constrained due to the relative rarity of these diseases, which ultimately
equates to a paucity of reliable models for experimentation. PDX models help to circumvent
this issue as the implanted tumor cells reflect the natural history of the disease. However,
assessing the fidelity of these models is of paramount importance in interpreting the
resultant data, thus necessitating comprehensive characterization of these models. Recently,
different groups have established large banks of clinically annotated and genomically
profiled pediatric PDX models (98,150–154). Collectively, these PDX models comprised
hematologic malignancies, solid tumors, CNS tumors, and rare histologies obtained from
Author Manuscript
patients with primary, relapsed, and/or metastatic disease and have been made available
to the research community (Table 1). A combination of genomic, transcriptomic, and
epigenomic profiling concluded that these PDX models closely resembled the original tumor
tissues from which they were derived.
Genomic data derived from molecularly characterized osteosarcoma PDX models were used
to identify druggable targets and demonstrated the efficacy of genome-informed therapeutic
approaches (150). Genomically characterized PDX models of high-risk rhabdomyosarcoma
were used to conduct Phase II and Phase III pre-clinical trials and identified that the addition
of a WEE1 inhibitor improved the therapeutic response to irinotecan and vincristine (151).
A separate Pediatric Preclinical Testing Consortium study evaluating the efficacy of WEE1
inhibition in combination with irinotecan in neuroblastoma, osteosarcoma, and Wilms tumor
Author Manuscript
xenografts yielded similar results (155). Importantly, these two independent studies provided
compelling pre-clinical data resulting in the establishment of a Phase I/II clinical trial to
investigate the combination of WEE1 inhibition with irinotecan in pediatric patients with
relapse or refractory solid tumors (NCT02095132).
While these thoroughly characterized models are useful in facilitating the study of cell
autonomous mechanisms of disease and preclinical identification/evaluation of therapeutic
targets, they are not ideal for mechanistic immuno-oncology investigations, which require an

intact immune system not present in PDX models. Expanding the use of these models into
Author Manuscript
humanized mice can partially address this deficiency, albeit certain species incompatibility
issues and other limitations remain (156). Furthermore, it is important to note that, although
PDX models exhibit high fidelity with respect to the source material, sampling bias still
exists, due to the variable extent of intratumoral heterogeneity present in CNS and solid
tumors. Therefore, depending on the abundance and distribution of clonal and/or sub-clonal
populations present in the tumor location from which the specimen was obtained, the PDX
generated from this material may or may not be representative of the bulk tumor, but rather a
regional sub-population. Nonetheless, PDX models are incredibly valuable in advancing the
field of pediatric cancer research.
Patient Derived Organoids

Several factors impact the engraftment of tumor tissues and thus the rate of successful PDX
Author Manuscript
generation varies markedly. The patient derived organoid is a newer model system that
fills the gap between PDX models and patient derived cancer cell lines (157). Recently a
pediatric renal tumor organoid biobank comprised of 54 organoids derived from tumors
including Wilms tumor, metanephric adenoma, malignant rhabdoid tumors, renal cell
carcinoma, congenital mesoblastic nephroma, and a hyperplastic intralobular nephrogenic
rest was established (158). Corresponding normal tissue organoids were also developed.
A hepatoblastoma tumor-normal organoid pair was also recently reported (159). In both
studies, genomic and transcriptomic profiling revealed that the organoid models resembled
the tumors from which they were derived and demonstrated the applicability of these tumor
derived organoids for in vitro drug screening (158,159).
Somatic Genome Engineering of Oncogenic Translocations

Author Manuscript
Numerous pediatric solid tumors are defined by hallmark translocations that give rise
to fusion oncoproteins that drive disease, most notably Ewing sarcoma (EWSR1-FLI1),
desmoplastic small round cell tumors (EWSR1-WT1), and rhabdomyosarcoma (PAX3/7-
FOXO1). Somatic genome engineering via CRISPR-Cas9 has been used to successfully
generate these translocations. Functional EWSR1-FLI1 translocations have been engineered
into HEK293 cells (160,161) as well as mesenchymal stem cells and induced pluripotent
stem cells (162). Functional EWSR1-WT1 translocations have also been engineered using
CRISPR-Cas9 (161,163). The PAX3-FOXO1 translocation was engineered into mouse
myoblasts using a Cre-Lox strategy to invert the FOXO1 locus followed by CRISPR-Cas9 to
create intronic double strand breaks in PAX3 and FOXO1 (164). Here, the rate of successful
translocation formation varied between forelimb and hindlimb myoblasts and was attributed
to the differences in the 3D genome organization and physical proximity of the PAX3 and
Author Manuscript
FOXO1 loci.
All experimental models have inherent flaws, and their utility is dependent upon the specific
questions being interrogated. However, a thorough comprehension of the benefits and
limitations of each model will allow for strategic utilization of the most appropriate model(s)
to generate useful data that will drive the field forward. It is likely that integrated studies that
utilize scRNA-seq of tumor tissues and/or organoids to map the cellular origins of disease
will be critical in the development of new models where disease-specific mutations are

engineered into the presumed permissive cells of origin. If successful, this approach could
Author Manuscript
provide new insights into diseases such as Ewing sarcoma, where despite immense effort
put forth by international groups of researchers, murine models have yet to be successfully
engineered (165).
IMMUNE MICROENVIRONMENT PROFILES OF PEDIATRIC TUMORS

The tumor microenvironment is comprised of neoplastic cells, immune cells, fibroblasts,
endothelial cells, pericytes, various extracellular matrix components, growth factors, soluble
stimulatory and inhibitory molecules, nutrient gradients, and variable oxygen tension that
work in concert to sustain tumor growth (166). Over the last decade, the number of
tumor microenvironment studies in adult cancers has increased dramatically. While still
less studied, recent reports documenting the landscape of the tumor microenvironment in
different pediatric cancers have increased. The use of computational tools to impute cell
Author Manuscript
types from RNA sequencing data has significantly contributed to this increase by allowing
for data mining and retrospective inquiry of established datasets (167–169). Functional
studies are also beginning to emerge, albeit at a reduced frequency which is likely
attributable to a scarcity of robust immunocompetent tumor models to study. In reviewing
the available literature, one more consistent finding is that the molecular and cellular
microenvironmental profiles of pediatric CNS and solid tumors is incredibly heterogeneous
and are thus deserving of their own discussion.
Osteosarcoma:
scRNA-seq analysis of osteosarcoma specimens revealed marked cellular heterogeneity
between primary, metastatic, and recurrent disease states (170). This study also reported
a decreased abundance of CD4+ and CD8+ lymphocytes in the recurrent and metastatic
Author Manuscript
specimens with CD8+ cells expressing markers of T-cell exhaustion (170). This data is
consistent with reports that metastatic osteosarcoma specimens contained fewer CD8+
lymphocytes than non-metastatic specimens, expressed low cytotoxicity scores, and
exhibited lymphocyte exclusion (171–173). Additionally, an integrated multi-omic analysis
showed that osteosarcomas exhibited an ineffective immune response, and low neoantigen
expression (174). This study also showed that copy number alterations inversely correlated
with immune cell abundance. One of the most striking findings from this study was the data
showing the correlation between patient age and tumor inflammation with tumor infiltrating
lymphocytes being more abundant in adult specimens versus pediatric specimens (174). This
data further highlights the differences between pediatric and adult disease.
Myeloid cells are an important feature of the osteosarcoma microenvironment. Analysis of

Author Manuscript
osteosarcoma specimens demonstrated that myeloid lineage cells were the most abundant
immune cells present in these tissues (170). Clustering analysis of this data identified 10
distinct subgroups of myeloid cells, highlighting the heterogeneity of myeloid lineage cells
in osteosarcoma. Experimental data showed that M2 tumor associated macrophages (TAMs)
promoted osteosarcoma metastasis and that treatment with all-trans retinoic acid reduced
TAM-dependent metastasis in vivo (175). Myeloid derived suppressor cells (MDSCs) are
highly suppressive immature myeloid cells with demonstrated ability to limit the efficacy

of chimeric antigen receptor (CAR) T-cells in osteosarcoma models in vivo (176,177). All-
Author Manuscript
trans retinoic acid was sufficient to eliminate monocytic MDSCs and relieve the suppressive
phenotype of granulocytic MDSCs (177). These studies highlight the biological significance,
phenotypic plasticity, and functional heterogeneity of myeloid lineage cells within the
osteosarcoma microenvironment.
Ewing sarcoma:
Ewing sarcoma specimens exhibited the lowest lymphocyte abundance when compared
to other pediatric solid tumors and the presence of CD8+ T-cells did not confer
a survival benefit to patients (178,179). Examination of pregnancy-associated plasma
protein-A (PAPPA) expression across various pediatric solid tumors illustrated that
Ewing sarcoma had the highest expression levels (180). Functional analysis demonstrated
that inhibition of PAPPA expression upregulated the expression of numerous genes
Author Manuscript
associated with an active immune response, suggesting that PAPPA is immunosuppressive.

Immunohistochemistry revealed that suppressive HLA-G+ lymphocytes outnumbered HLA-
G- lymphocytes in Ewing sarcoma biopsy specimens and that HLA-G was upregulated on
xenografted tumors cells in response to CAR T-cell treatment (181). Moreover, CCL21,
an immunostimulatory cytokine, was shown downregulated in metastatic patient specimens,
was inversely correlated with the CD4+/CD8+ T-cell ratio and was associated with a better
prognosis (182). Together, these data highlight the immunosuppressive nature intrinsic
to Ewing sarcoma. Exactly how the EWS-FLI1 fusion oncoprotein contributes to the
immunosuppressive phenotype of Ewing sarcoma is not yet known.
Neuroblastoma:
Gene expression and immunohistochemical analysis of neuroblastoma specimens revealed
Author Manuscript
that T-cells positively correlated with intratumoral dendritic cells (DC) and natural killer
(NK) cells, both of which served as positive prognosticators of overall survival (183).
When compared to other pediatric solid tumors, the gene expression level of CD200 was
significantly higher in neuroblastoma specimens than in any of the other tumor types (184).
Moreover, CD200-high neuroblastoma specimens contained fewer CD4+ and CD8+ T-cells
and expressed lower levels of IFNγ and TNFα, associating CD200 with a dampened T-cell
response. MYCN-amplified neuroblastoma tumors were shown to be less immunogenic than
non-amplified tumors as evidenced by lower immune scores and decreased MHC class
I gene expression (185,186). Interestingly, a separate study using RNA sequencing data
from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments)
and GMKF cohorts demonstrated that the activation of downstream MYCN transcriptional
programs, rather than amplification of MYCN itself was inversely correlated with T-cell
Author Manuscript
abundance and tumor inflammation (187). This study also showed that increased T-cell
infiltration and a higher neoantigen load were independently associated with improved
overall survival in these patients (187). For high-risk MYCN non-amplified neuroblastoma
specimens with a high MYCN gene signature, T-cell clonal expansion was associated with
improved outcomes while a subgroup of these specimens demonstrated T-cell exhaustion
(185). Increased abundance of CSF1R+ myeloid cells was associated with inferior relapse-
free survival for patients with neuroblastoma and therapeutically targeting these cells both
decreased tumor burden and sensitized animals to PD-1 inhibition (188).

Wilms Tumor
Author Manuscript
In Wilms tumor specimens, an inverse relationship between M1 macrophage abundance

and tumor stage was observed, whereas the opposite was true for M2 macrophages
(189). Functional studies show that loss of the tumor suppressor WT1 upregulated COX2
expression in normal kidneys and that Wilms tumors derived from the WT1-IGF2 murine
model demonstrated robust COX2 expression, elucidating one possible mechanism by
which Wilms tumor cells promote a suppressive microenvironment (190). This study also
highlighted the immunosuppressive microenvironment of Wilms tumors as indicated by
increased abundance of regulatory T-cells, the expression of suppressive cytokines, and a
diminished Th1 response when compared to normal kidneys (190).
Rhabdomyosarcoma
While the abundance of infiltrating immune cells did not dramatically differ between
Author Manuscript
embryonal and alveolar rhabdomyosarcoma, their distribution throughout the tumor tissue
did indeed vary. The vast majority of both CD3+ and CD163+ cells localized within 15um
of tumor blood vessels in alveolar specimens whereas the perivascular localization in
embryonal specimens was more diffuse (191). Additionally, tertiary lymphoid structures
were more frequently associated with the alveolar histology. Consistent across the
histologies was that both embryonal and alveolar rhabdomyosarcoma contained higher
numbers of TAMs than T-cells (191,192). Fibrocytes have been shown to promote an
immunosuppressive microenvironment and undermine the efficacy of immune checkpoint
inhibition in a syngeneic embryonal rhabdomyosarcoma model (193).
Medulloblastoma
Analysis of immune infiltrates across multiple CNS tumors revealed a consistent inverse
Author Manuscript
correlation between tumor grade and immune cell content (194). In comparison to
the other major brain tumor histologies, medulloblastomas were characterized as being
immunologically “cold” (195). However, within medulloblastomas, the relative abundance
of immune cells varied by subgroup with SHH and WNT tumors having higher proportion
of CD8+ T-cells than Group 3 and Group 4 tumors (194). In low-risk Group 3
medulloblastoma, a decreased T-reg abundance was associated with inferior progression
free survival (194). A murine model of SHH and Group 3 medulloblastoma also
revealed subtype-specific differences where SHH medulloblastoma tumors contained higher
numbers of infiltrating lymphocytes and myeloid-lineage cells than Group 3 tumors (196).
Interestingly, a murine model of SHH medulloblastoma demonstrated that tumoricidal
TAMs were recruited to the tumors in a CCL2/CCR2-dependent manner, suggesting a
beneficial role of macrophages in the SHH medulloblastoma microenvironment (197). A
Author Manuscript
separate study using a p53-mutant medulloblastoma model demonstrated that mutant p53
inhibited the presentation of MHC class-I by negatively regulating the expression of TAP1
and ERAP1, molecules needed for successful antigen loading (198). Importantly, activation
of TNFR2 and LTβR signaling was sufficient to restore MHC class-I expression in the tumor
cells in vitro.

Glioma
Author Manuscript
The microenvironmental heterogeneity of pediatric gliomas varied in accordance with

grade, subgroup, and histone mutation status (194,199,200). Multiplexed immunofluorescent
imaging showed that low grade gliomas (LGG) had a higher density of CD3+ cells than
HGG and that, amongst the LGGs, pilocytic astrocytomas had the lowest T-cell density
(200). This study also reported increased vascularity in recurrent versus primary pilocytic
astrocytomas. Immunohistochemical evaluation of infiltrating immune cells demonstrated
that there were no statistically significant differences in the abundance of natural killer
cells or CD163+ myeloid cells between LGG and HGG (201). In a separate study, diffuse
intrinsic pontine gliomas were shown to be “immunologically cold” tumors with little
immune infiltrate (202,203). Proteomic and phospho-proteomic analysis revealed marked
heterogeneity in the immune microenvironment of both LGG and HGG and showed that
different subgroups of gliomas were classified as “hot” or “cold” irrespective of histology
Author Manuscript
and/or diagnosis (195).
The pediatric tumor microenvironment field, while gaining interest, is still emerging and
additional studies are needed to comprehend the dynamic interaction between tumor cells
and the various components of their microenvironment. Studies directed at answering
important questions pertaining to the microenvironmental mechanisms that promote
immunosuppression and drive resistance to current immunotherapies are of critical clinical
importance and will contribute to shaping the therapeutic landscape.
IMMUNOTHERAPEUTIC APPROACHES FOR PEDIATRIC TUMORS

The 2011 approval of ipilimumab by both FDA and EMA marked the start of the
immunotherapy revolution (204,205). This rapidly evolving therapeutic landscape has
Author Manuscript
dramatically changed the treatment paradigm for adult and select pediatric malignancies
(Table 2). With the various late effects associated with certain prolonged cytotoxic
chemotherapy and radiation therapy regimens, it is the hope that immunotherapy may
increase the quality of life for pediatric and adolescent patients in addition to providing
durable responses, but this will require long-term follow-up.
Immune Checkpoint Inhibition in CNS and Solid Tumors

Data shows that robust expression of PD-L1 is not a universally common finding in most
pediatric tumors, although a subset of 11q-deleted neuroblastomas exhibited increased
expression of PD-L1 (202,206–208). Furthermore, somatic copy number gains at loci
encompassing genes that encode for PD-L1/PD-L2 has been shown in a subset of
osteosarcoma specimens (209). Two patients with hypermutant glioblastoma driven by
Author Manuscript
biallelic mismatch repair deficiency syndrome were successfully treated with single agent
nivolumab and exhibited durable clinical and radiologic responses (210). In a separate
study, a patient with a hypermutant glioma and constitutional mismatch repair deficiency
was treated with pembrolizumab yet succumbed to their disease (64). These two studies
support the findings that mismatch repair deficient tumors exhibit variable responses to
checkpoint inhibition (211). Nonetheless, immune checkpoint inhibition targeting PD-1 or
CTLA4 has shown limited therapeutic efficacy for most pediatric patients with CNS and

solid tumors with some notable exception (206,212–214). Given the clinical inefficacy of
Author Manuscript
currently available immune checkpoint inhibition, adoptive cell therapies have played a
much larger role to date in the immunotherapy of pediatric tumors.
CD19 CAR T-Cell Therapy in Leukemia

One of the most remarkable advances in cancer research has been the development, clinical
implementation, and efficacy of chimeric antigen receptor (CAR) T-cell therapy in high-
risk pediatric B-cell ALL. Furthermore, the fact that the first FDA approved the use of
CAR T-cell therapy, tisagenlecleucel, was for a pediatric and adolescent indication before
being approved for adults was a ground-breaking milestone for field of pediatric oncology.
Infusion of these autologous CD19-directed CAR T-cells into pediatric and young adult
patients with relapsed/refractory B-cell ALL achieved a remission rate of >80% with no
evidence of residual disease at 3 months post-infusion (215). Additionally, these patients
Author Manuscript
demonstrated 1-year event free survival (EFS) and overall survival (OS) rates of 50% and
76%, respectively. Nonetheless, this therapy is not without side effects as a high incidence of
cytokine release syndrome was observed with 40% of these patients experiencing neurologic
events (215). CD19 CAR T-cell therapy has also demonstrated efficacy in pediatric patients
with CNS B-cell ALL (216,217). In these patients, neurotoxicity was associated with an
increased CNS disease burden prior to treatment (217). The acute effects of cytokine release
syndrome can be managed but late effects may exist in children and longitudinal monitoring
is needed in these patients (218).
Leukemic B-cells can escape killing by CAR T-cells by employing a variety of

mechanisms, including downregulating the expression of CD19 (219). To address this,
studies investigating sequential infusion of CD19 and CD22 CAR T-cells and preclinical
efficacy of CD19/CD22 bivalent CAR T-cells are underway (220–222). Additionally, the
Author Manuscript
use of low affinity CD19 CAR T-cells demonstrated superior in vitro and in vivo T-cell
responses when compared to high affinity CAR T-cells (223). In patients, these lower
affinity CAR T-cells were associated with increased persistence and expansion of the
adoptively transferred cells, lower toxicity, a remission rate of 85%, a 1-year EFS rate
of 64%, and a 1-year OS rate of 46% (223). With a median follow-up of 4.8 years, a
recent study found that allogeneic hematopoietic stem cell transplant after CD19 CAR T-cell
infusion significantly reduced the relapse rate in patients with recurrent B-ALL (224).
GD2-targeted CAR T-Cell Therapy

GD2 is a carbohydrate-containing sphingolipid that is expressed by many cell types
throughout the body and highly expressed by neuroblastoma cells (225). GD2-targeted CAR
T-cells engineered to co-express IL15 reduced T-cell exhaustion in vitro and enhanced the
Author Manuscript
therapeutic efficacy in vivo (226). Interestingly, GD2-targeted CAR T-cells were effective
at killing neuroblastoma xenografts but were ineffective in osteosarcoma and Ewing
sarcoma xenografts, both of which expressed GD2 (177). Moreover, the expansion and
immunosuppressive effects of MDSCs in the sarcoma microenvironment was responsible for
the lack of therapeutic effect of the CAR T-cells (177).

Tumor cells can evade GD2 CAR T-cells. Upon encountering GD2-targeted CAR T-cells,
Author Manuscript
osteosarcoma cells upregulated PD-L1 to induce exhaustion and apoptosis of the CAR
T-cells (227). Systemic administration of GD2 CAR T-cells in orthotopic DIPG xenografts
resulted in a durable reduction in tumor burden and improved survival, however a small
subset of xenografted tumor cells lost antigen expression (228). Retinoblastoma cells that
were initially responsive to GD2 CAR T-cell therapy escaped killing by downregulating
GD2 expression and increasing the expression of PD-L1 and PD-1 on the surface of tumor
cells and CAR T-cells, respectively (229,230). Sequential exposure of retinoblastoma cells to
CAR T-cells against different targets (GD2 and CD171) enhanced tumor cell killing in vivo
(230). In neuroblastoma xenograft models, engineering GD2 CAR T-cells to express IL15
improved the therapeutic efficacy and reduced the expression of PD-1 on the CAR T-cells
(231).
In phase 1 studies of patients with relapsed/refractory neuroblastoma, lymphodepletion

Author Manuscript
prior to infusion of GD2 CAR T-cells was deemed safe (232,233). In one study, despite
demonstrated evidence of transient tumor regression in 25% of patients, the investigators
did not observe an objective clinical response (233). The second study showed a significant
CAR T-cell expansion in the patients who received prior lymphodepleting therapy versus
those show did not and that inhibition of PD1 did not improve the performance of the CAR
T-cells (232). This study also observed an expansion of CD11b+CD163+ myeloid cells after
GD2 CAR T-cell infusion. While the results are promising, comprehensive investigations of
the tumor-specific microenvironments needs to be performed in order to better understand
the complexities of immune escape and optimize immunotherapeutic interventions.
NY-ESO-1 Engineered T Cell Receptors

NY-ESO-1 is a cancer testes antigen expressed in the majority of synovial sarcoma tumors
Author Manuscript
and a subset of malignant peripheral nerve sheath tumors (234,235). Synovial sarcoma
patients treated with autologous T-cells expressing a NY-ESO-1 engineered T cell receptor
(NY-ESO-1 c259T-cells) resulted in an overall response rate of 50% (236). After infusion,
NY-ESO-1 c259T-cells generated a memory T-cell response, did not exhibit signs of T-cell
exhaustion, were clonally expanded, and persisted in vivo. A second study revealed that
increased expression of NY-ESO-1 was likely associated with more robust and durable
therapeutic responses (237). These studies provide a degree of optimism for a difficult to
manage adolescent/young adult tumor.
Despite some successes, immunotherapy for pediatric cancer patients is still in the early
phase of development. To realize the full potential of immunotherapy to provide efficacious
and durable responses for pediatric patients, especially those with CNS and solid tumors,
Author Manuscript
a better understanding of the interaction between the tumor, the microenvironment and
the immune cells will be required to move this field forward. To address this, the
Cancer Moonshot has created the Pediatric Immunotherapy Discovery and Development
Network and the Cancer Immunotherapy Trials Network. The goal of these networks is
to collaboratively advance the field of pediatric cancer immunotherapy by characterizing
new targets, generating suitable experimental models for preclinical evaluation, and better
understand pediatric tumor immunology.

CONCLUSIONS AND OUTLOOK

Author Manuscript
The last 5 years has witnessed significant advances in our understanding of the genetic and
epigenetic underpinning of pediatric cancers as well as the treatments of some pediatric
tumors. At the genetic level, it is clear that many genetic changes identified are distinct
from adult cancers and will require unique interventions (FIGURE 2). Furthermore, it is
also clear that pediatric tumors appear to have a high degree of epigenetic changes that lead
to widespread alterations in gene expression secondary to these changes. This in turn may
ultimately lead to novel therapeutic approaches in the future. Germline genetic variations
associated with predisposition to cancer appears to occur at a higher frequency in pediatric
patients with cancer compared to adult cancer patients, and these findings may ultimately
allow both earlier intervention in identified high-risk children and hopefully prevention in
the future. Finally, adoptive immunotherapy, particularly CAR-T cells have had a major
impact on the treatment of childhood ALL, but immune checkpoint inhibitors have to
Author Manuscript
date been of minimal use in pediatric tumors. Ongoing work identifying the impact of the
tumor microenvironment on trafficking of immune cells necessary for ICI activity may help
improve the effect of these agents in the future.
ACKNOWLEDGEMENTS
We are indebted to the patients and their families who have participated in the various studies, without which,
progress would not have been made. We are both grateful and thankful to our scientific and clinical colleagues
around the world that have dedicated their careers to investigating pediatric malignancies. Given the numerous
recent developments and achievements in pediatric cancer research, we regret that we were not able to encompass
all these studies into this review. Nonetheless, your collective accomplishments and efforts are noted and
appreciated. T.A.M. is supported by the Intramural Research Program of the National Institutes of Health,
National Cancer Institute, Center for Cancer Research. The views and opinions contained within this article do
not necessarily reflect those of the National Institutes of Health or the US Department of Health and Human
Services. The mention of trade names and/or commercialized products does not indicate endorsement by the US
Author Manuscript
government.
REFERENCES
1. Cieslik M, Chinnaiyan AM. Global genomics project unravels cancer’s complexity at unprecedented
scale. Nature 2020;578:39–40 [PubMed: 32025004]
2. Consortium ITP-CAoWG. Pan-cancer analysis of whole genomes. Nature 2020;578:82–93
[PubMed: 32025007]
3. Ma X, Liu Y, Alexandrov LB, Edmonson MN, Gawad C, Zhou X, et al. Pan-cancer genome and
transcriptome analyses of 1,699 paediatric leukaemias and solid tumours. Nature 2018;555:371–6
[PubMed: 29489755]
4. Grobner SN, Worst BC, Weischenfeldt J, Buchhalter I, Kleinheinz K, Rudneva VA, et al. The
landscape of genomic alterations across childhood cancers. Nature 2018;555:321–7 [PubMed:
29489754]
Author Manuscript
5. Campbell BB, Galati MA, Stone SC, Riemenschneider AN, Edwards M, Sudhaman S, et al.
Mutations in the RAS/MAPK pathway drive replication repair deficient hypermutated tumors and
confer sensitivity to MEK inhibition. Cancer Discov 2021
6. Wong M, Mayoh C, Lau LMS, Khuong-Quang DA, Pinese M, Kumar A, et al. Whole genome,
transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric
cancer. Nat Med 2020;26:1742–53 [PubMed: 33020650]
7. Hatch EM, Hetzer MW. Chromothripsis. Curr Biol 2015;25:R397–9 [PubMed: 25989073]
8. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler KW. Cancer genome
landscapes. Science 2013;339:1546–58 [PubMed: 23539594]

9. Sievers P, Sill M, Blume C, Tauziede-Espariat A, Schrimpf D, Stichel D, et al. Clear cell

meningiomas are defined by a highly distinct DNA methylation profile and mutations in
Author Manuscript
SMARCE1. Acta Neuropathol 2021;141:281–90 [PubMed: 33319313]

10. Northcott PA, Buchhalter I, Morrissy AS, Hovestadt V, Weischenfeldt J, Ehrenberger T, et al.
The whole-genome landscape of medulloblastoma subtypes. Nature 2017;547:311–7 [PubMed:
28726821]
11. Sexton-Oates A, Dodgshun A, Hovestadt V, Jones DTW, Ashley DM, Sullivan M, et al.
Methylation profiling of paediatric pilocytic astrocytoma reveals variants specifically associated
with tumour location and predictive of recurrence. Mol Oncol 2018;12:1219–32 [PubMed:
28388012]
12. Jeyapalan JN, Doctor GT, Jones TA, Alberman SN, Tep A, Haria CM, et al. DNA methylation
analysis of paediatric low-grade astrocytomas identifies a tumour-specific hypomethylation
signature in pilocytic astrocytomas. Acta Neuropathol Commun 2016;4:54 [PubMed: 27229157]
13. Torchia J, Golbourn B, Feng S, Ho KC, Sin-Chan P, Vasiljevic A, et al. Integrated (epi)-Genomic
Analyses Identify Subgroup-Specific Therapeutic Targets in CNS Rhabdoid Tumors. Cancer Cell
2016;30:891–908 [PubMed: 27960086]
Author Manuscript
14. Johann PD, Erkek S, Zapatka M, Kerl K, Buchhalter I, Hovestadt V, et al. Atypical Teratoid/
Rhabdoid Tumors Are Comprised of Three Epigenetic Subgroups with Distinct Enhancer
Landscapes. Cancer Cell 2016;29:379–93 [PubMed: 26923874]
15. Capper D, Jones DTW, Sill M, Hovestadt V, Schrimpf D, Sturm D, et al. DNA methylation-based
classification of central nervous system tumours. Nature 2018;555:469–74 [PubMed: 29539639]
16. Danielsson A, Nemes S, Tisell M, Lannering B, Nordborg C, Sabel M, et al. MethPed: a DNA
methylation classifier tool for the identification of pediatric brain tumor subtypes. Clin Epigenetics
2015;7:62 [PubMed: 26157508]
17. Ryall S, Zapotocky M, Fukuoka K, Nobre L, Guerreiro Stucklin A, Bennett J, et al.
Integrated Molecular and Clinical Analysis of 1,000 Pediatric Low-Grade Gliomas. Cancer Cell
2020;37:569–83.e5 [PubMed: 32289278]
18. Eleveld TF, Oldridge DA, Bernard V, Koster J, Colmet Daage L, Diskin SJ, et al. Relapsed
neuroblastomas show frequent RAS-MAPK pathway mutations. Nat Genet 2015;47:864–71
[PubMed: 26121087]
19. Eleveld TF, Schild L, Koster J, Zwijnenburg DA, Alles LK, Ebus ME, et al. RAS-MAPK Pathway-
Author Manuscript
Driven Tumor Progression Is Associated with Loss of CIC and Other Genomic Aberrations in
Neuroblastoma. Cancer Res 2018;78:6297–307 [PubMed: 30115695]
20. Shern JF, Selfe J, Izquierdo E, Patidar R, Chou HC, Song YK, et al. Genomic Classification and
Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium. J Clin
Oncol 2021:JCO2003060
21. Moke DJ, Song Z, Liu L, Hamilton AS, Deapen D, Freyer DR. A Population-Based Analysis
of 30-Year Mortality among Five-Year Survivors of Adolescent and Young Adult Cancer: The
Roles of Primary Cancer, Subsequent Malignancy, and Other Health Conditions. Cancers (Basel)
2021;13 [PubMed: 35008178]
22. Zahnreich S, Schmidberger H. Childhood Cancer: Occurrence, Treatment and Risk of Second
Primary Malignancies. Cancers (Basel) 2021;13 [PubMed: 35008178]
23. Schwartz JR, Ma J, Kamens J, Westover T, Walsh MP, Brady SW, et al. The acquisition of
molecular drivers in pediatric therapy-related myeloid neoplasms. Nat Commun 2021;12:985
[PubMed: 33579957]
Author Manuscript
24. Oshima K, Khiabanian H, da Silva-Almeida AC, Tzoneva G, Abate F, Ambesi-Impiombato A, et

al. Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute
lymphoblastic leukemia. Proc Natl Acad Sci U S A 2016;113:11306–11 [PubMed: 27655895]
25. Lipka DB, Witte T, Toth R, Yang J, Wiesenfarth M, Nöllke P, et al. RAS-pathway mutation patterns
define epigenetic subclasses in juvenile myelomonocytic leukemia. Nat Commun 2017;8:2126
[PubMed: 29259247]
26. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A
1971;68:820–3 [PubMed: 5279523]

27. Miller DB, Piccolo SR. Compound Heterozygous Variants in Pediatric Cancers: A Systematic
Review. Front Genet 2020;11:493 [PubMed: 32508881]
Author Manuscript
28. Capasso M, Montella A, Tirelli M, Maiorino T, Cantalupo S, Iolascon A. Genetic Predisposition to

Solid Pediatric Cancers. Front Oncol 2020;10:590033
29. Kratz CP, Jongmans MC, Cavé H, Wimmer K, Behjati S, Guerrini-Rousseau L, et al.
Predisposition to cancer in children and adolescents. Lancet Child Adolesc Health 2021;5:142–54
[PubMed: 33484663]
30. Plon SE, Lupo PJ. Genetic Predisposition to Childhood Cancer in the Genomic Era. Annu Rev
Genomics Hum Genet 2019;20:241–63 [PubMed: 31082280]
31. Mitchell SG, Pencheva B, Porter CC. Germline Genetics and Childhood Cancer: Emerging Cancer
Predisposition Syndromes and Psychosocial Impacts. Curr Oncol Rep 2019;21:85 [PubMed:
31414239]
32. Zhang J, Walsh MF, Wu G, Edmonson MN, Gruber TA, Easton J, et al. Germline Mutations in
Predisposition Genes in Pediatric Cancer. N Engl J Med 2015;373:2336–46 [PubMed: 26580448]
33. Wagener R, Taeubner J, Walter C, Yasin L, Alzoubi D, Bartenhagen C, et al. Comprehensive
germline-genomic and clinical profiling in 160 unselected children and adolescents with cancer.
Author Manuscript
Eur J Hum Genet 2021;29:1301–11 [PubMed: 33840814]

34. Akhavanfard S, Padmanabhan R, Yehia L, Cheng F, Eng C. Comprehensive germline genomic
profiles of children, adolescents and young adults with solid tumors. Nat Commun 2020;11:2206
[PubMed: 32371905]
35. Gröbner SN, Worst BC, Weischenfeldt J, Buchhalter I, Kleinheinz K, Rudneva VA, et al. The
landscape of genomic alterations across childhood cancers. Nature 2018;555:321–7 [PubMed:
29489754]
36. Waszak SM, Northcott PA, Buchhalter I, Robinson GW, Sutter C, Groebner S, et al. Spectrum
and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study
and prospective validation in a clinical trial cohort. Lancet Oncol 2018;19:785–98 [PubMed:
29753700]
37. Wasserman JD, Novokmet A, Eichler-Jonsson C, Ribeiro RC, Rodriguez-Galindo C, Zambetti
GP, et al. Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical
carcinoma: a children’s oncology group study. J Clin Oncol 2015;33:602–9 [PubMed: 25584008]
Author Manuscript
38. Mirabello L, Zhu B, Koster R, Karlins E, Dean M, Yeager M, et al. Frequency of Pathogenic
Germline Variants in Cancer-Susceptibility Genes in Patients With Osteosarcoma. JAMA Oncol
2020;6:724–34 [PubMed: 32191290]
39. Diessner BJ, Pankratz N, Hooten AJ, Mirabello L, Sarver AL, Mills LJ, et al. Nearly Half of TP53
Germline Variants Predicted To Be Pathogenic in Patients With Osteosarcoma Are De Novo: A
Report From the Children’s Oncology Group. JCO Precis Oncol 2020;4
40. Qian M, Cao X, Devidas M, Yang W, Cheng C, Dai Y, et al. TP53 Germline Variations Influence
the Predisposition and Prognosis of B-Cell Acute Lymphoblastic Leukemia in Children. J Clin
Oncol 2018;36:591–9 [PubMed: 29300620]
41. Koster R, Panagiotou OA, Wheeler WA, Karlins E, Gastier-Foster JM, Caminada de Toledo SR,
et al. Genome-wide association study identifies the GLDC/IL33 locus associated with survival of
osteosarcoma patients. Int J Cancer 2018;142:1594–601 [PubMed: 29210060]
42. Lin Q, Han J, Sun Q, Wen L, Wang S. Functional variant of IL33 is associated with survival of
osteosarcoma patients. J Bone Oncol 2020;20:100270
43. Li H, Sisoudiya SD, Martin-Giacalone BA, Khayat MM, Dugan-Perez S, Marquez-Do DA, et
Author Manuscript
al. Germline Cancer-Predisposition Variants in Pediatric Rhabdomyosarcoma: A Report from the

Children’s Oncology Group. J Natl Cancer Inst 2020
44. Kim J, Light N, Subasri V, Young EL, Wegman-Ostrosky T, Barkauskas DA, et al. Pathogenic
Germline Variants in Cancer Susceptibility Genes in Children and Young Adults With
Rhabdomyosarcoma. JCO Precis Oncol 2021;5
45. Machiela MJ, Grunewald TGP, Surdez D, Reynaud S, Mirabeau O, Karlins E, et al. Genome-wide
association study identifies multiple new loci associated with Ewing sarcoma susceptibility. Nat
Commun 2018;9:3184 [PubMed: 30093639]

46. Lin SH, Sampson JN, Grunewald TGP, Surdez D, Reynaud S, Mirabeau O, et al. Low-frequency
variation near common germline susceptibility loci are associated with risk of Ewing sarcoma.
Author Manuscript
PLoS One 2020;15:e0237792

47. Brohl AS, Patidar R, Turner CE, Wen X, Song YK, Wei JS, et al. Frequent inactivating germline
mutations in DNA repair genes in patients with Ewing sarcoma. Genet Med 2017;19:955–8
[PubMed: 28125078]
48. Oldridge DA, Truong B, Russ D, DuBois SG, Vaksman Z, Mosse YP, et al. Differences in
Genomic Profiles and Outcomes Between Thoracic and Adrenal Neuroblastoma. J Natl Cancer
Inst 2019;111:1192–201 [PubMed: 30793172]
49. Muskens IS, de Smith AJ, Zhang C, Hansen HM, Morimoto L, Metayer C, et al. Germline cancer
predisposition variants and pediatric glioma: a population-based study in California. Neuro Oncol
2020;22:864–74 [PubMed: 31970404]
50. Begemann M, Waszak SM, Robinson GW, Jager N, Sharma T, Knopp C, et al. Germline GPR161
Mutations Predispose to Pediatric Medulloblastoma. J Clin Oncol 2020;38:43–50 [PubMed:
31609649]
51. Waszak SM, Robinson GW, Gudenas BL, Smith KS, Forget A, Kojic M, et al. Germline Elongator
Author Manuscript
mutations in Sonic Hedgehog medulloblastoma. Nature 2020;580:396–401 [PubMed: 32296180]

52. Moriyama T, Metzger ML, Wu G, Nishii R, Qian M, Devidas M, et al. Germline genetic variation
in ETV6 and risk of childhood acute lymphoblastic leukaemia: a systematic genetic study. Lancet
Oncol 2015;16:1659–66 [PubMed: 26522332]
53. Zhang H, Liu AP, Devidas M, Lee SH, Cao X, Pei D, et al. Association of GATA3 Polymorphisms
With Minimal Residual Disease and Relapse Risk in Childhood Acute Lymphoblastic Leukemia. J
Natl Cancer Inst 2021;113:408–17 [PubMed: 32894760]
54. Vijayakrishnan J, Qian M, Studd JB, Yang W, Kinnersley B, Law PJ, et al. Identification of four
novel associations for B-cell acute lymphoblastic leukaemia risk. Nat Commun 2019;10:5348
[PubMed: 31767839]
55. Marlow EC, Ducore J, Kwan ML, Cheng SY, Bowles EJA, Greenlee RT, et al. Leukemia Risk in a
Cohort of 3.9 Million Children with and without Down Syndrome. J Pediatr 2021
56. Brown AL, de Smith AJ, Gant VU, Yang W, Scheurer ME, Walsh KM, et al. Inherited genetic
susceptibility to acute lymphoblastic leukemia in Down syndrome. Blood 2019;134:1227–37
[PubMed: 31350265]
Author Manuscript
57. McNally EJ, Luncsford PJ, Armanios M. Long telomeres and cancer risk: the price of cellular
immortality. J Clin Invest 2019;129:3474–81 [PubMed: 31380804]
58. Ackermann S, Fischer M. Telomere Maintenance in Pediatric Cancer. Int J Mol Sci 2019;20
[PubMed: 31861461]
59. Haycock PC, Burgess S, Nounu A, Zheng J, Okoli GN, Bowden J, et al. Association Between
Telomere Length and Risk of Cancer and Non-Neoplastic Diseases: A Mendelian Randomization
Study. JAMA Oncol 2017;3:636–51 [PubMed: 28241208]
60. Zhang C, Ostrom QT, Semmes EC, Ramaswamy V, Hansen HM, Morimoto L, et al. Genetic
predisposition to longer telomere length and risk of childhood, adolescent and adult-onset
ependymoma. Acta Neuropathol Commun 2020;8:173 [PubMed: 33115534]
61. Zhang C, Hansen HM, Semmes EC, Gonzalez-Maya J, Morimoto L, Wei Q, et al. Common genetic
variation and risk of osteosarcoma in a multi-ethnic pediatric and adolescent population. Bone
2020;130:115070
62. Walsh KM, Whitehead TP, de Smith AJ, Smirnov IV, Park M, Endicott AA, et al. Common
Author Manuscript
genetic variants associated with telomere length confer risk for neuroblastoma and other childhood
cancers. Carcinogenesis 2016;37:576–82 [PubMed: 27207662]
63. Huang KL, Mashl RJ, Wu Y, Ritter DI, Wang J, Oh C, et al. Pathogenic Germline Variants in
10,389 Adult Cancers. Cell 2018;173:355–70.e14 [PubMed: 29625052]
64. Fiala EM, Jayakumaran G, Mauguen A, Kennedy JA, Bouvier N, Kemel Y, et al. Prospective
pan-cancer germline testing using MSK-IMPACT informs clinical translation in 751 patients with
pediatric solid tumors. Nat Cancer 2021;2:357–65 [PubMed: 34308366]

65. Brodeur GM, Nichols KE, Plon SE, Schiffman JD, Malkin D. Pediatric Cancer Predisposition and
Surveillance: An Overview, and a Tribute to Alfred G. Knudson Jr. Clin Cancer Res 2017;23:e1–
Author Manuscript
e5 [PubMed: 28572261]
66. Porter CC, Druley TE, Erez A, Kuiper RP, Onel K, Schiffman JD, et al. Recommendations for
Surveillance for Children with Leukemia-Predisposing Conditions. Clin Cancer Res 2017;23:e14–
e22 [PubMed: 28572263]
67. Walsh MF, Chang VY, Kohlmann WK, Scott HS, Cunniff C, Bourdeaut F, et al. Recommendations
for Childhood Cancer Screening and Surveillance in DNA Repair Disorders. Clin Cancer Res
2017;23:e23–e31 [PubMed: 28572264]
68. Greer MC, Voss SD, States LJ. Pediatric Cancer Predisposition Imaging: Focus on Whole-Body
MRI. Clin Cancer Res 2017;23:e6–e13 [PubMed: 28572262]
69. Druker H, Zelley K, McGee RB, Scollon SR, Kohlmann WK, Schneider KA, et al. Genetic
Counselor Recommendations for Cancer Predisposition Evaluation and Surveillance in the
Pediatric Oncology Patient. Clin Cancer Res 2017;23:e91–e7 [PubMed: 28674117]
70. Kratz CP, Achatz MI, Brugières L, Frebourg T, Garber JE, Greer MC, et al. Cancer Screening
Recommendations for Individuals with Li-Fraumeni Syndrome. Clin Cancer Res 2017;23:e38–e45
Author Manuscript
[PubMed: 28572266]
71. Tabori U, Hansford JR, Achatz MI, Kratz CP, Plon SE, Frebourg T, et al. Clinical Management and
Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood.
Clin Cancer Res 2017;23:e32–e7 [PubMed: 28572265]
72. Qing T, Mohsen H, Marczyk M, Ye Y, O’Meara T, Zhao H, et al. Germline variant burden
in cancer genes correlates with age at diagnosis and somatic mutation burden. Nat Commun
2020;11:2438 [PubMed: 32415133]
73. Alix-Panabières C, Pantel K. Liquid Biopsy: From Discovery to Clinical Application. Cancer
Discov 2021;11:858–73 [PubMed: 33811121]
74. Kahana-Edwin S, Cain LE, Karpelowsky J. Roadmap to Liquid Biopsy Biobanking from Pediatric
Cancers-Challenges and Opportunities. Biopreserv Biobank 2021;19:124–9 [PubMed: 33493007]
75. Trigg RM, Shaw JA, Turner SD. Opportunities and challenges of circulating biomarkers in
neuroblastoma. Open Biol 2019;9:190056
76. Andersson D, Fagman H, Dalin MG, Stahlberg A. Circulating cell-free tumor DNA analysis in
Author Manuscript
pediatric cancers. Mol Aspects Med 2020;72:100819

77. Stallard S, Savelieff MG, Wierzbicki K, Mullan B, Miklja Z, Bruzek A, et al. CSF H3F3A K27M
circulating tumor DNA copy number quantifies tumor growth and in vitro treatment response.
Acta Neuropathol Commun 2018;6:80 [PubMed: 30111355]
78. Huang TY, Piunti A, Lulla RR, Qi J, Horbinski CM, Tomita T, et al. Detection of Histone H3
mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma.
Acta Neuropathol Commun 2017;5:28 [PubMed: 28416018]
79. Chicard M, Colmet-Daage L, Clement N, Danzon A, Bohec M, Bernard V, et al. Whole-Exome
Sequencing of Cell-Free DNA Reveals Temporo-spatial Heterogeneity and Identifies Treatment-
Resistant Clones in Neuroblastoma. Clin Cancer Res 2018;24:939–49 [PubMed: 29191970]
80. Trigg RM, Turner SD, Shaw JA, Jahangiri L. Diagnostic accuracy of circulating-free DNA for
the determination of MYCN amplification status in advanced-stage neuroblastoma: a systematic
review and meta-analysis. Br J Cancer 2020;122:1077–84 [PubMed: 32015512]
81. Applebaum MA, Barr EK, Karpus J, West-Szymanski DC, Oliva M, Sokol EA, et al. 5-
Hydroxymethylcytosine Profiles in Circulating Cell-Free DNA Associate with Disease Burden
Author Manuscript
in Children with Neuroblastoma. Clin Cancer Res 2020;26:1309–17 [PubMed: 31852832]

82. Shulman DS, Klega K, Imamovic-Tuco A, Clapp A, Nag A, Thorner AR, et al. Detection of
circulating tumour DNA is associated with inferior outcomes in Ewing sarcoma and osteosarcoma:
a report from the Children’s Oncology Group. Br J Cancer 2018;119:615–21 [PubMed: 30131550]
83. Miguez ACK, Barros BDF, de Souza JES, da Costa CML, Cunha IW, Barbosa P, et al. Assessment
of somatic mutations in urine and plasma of Wilms tumor patients. Cancer Med 2020;9:5948–59
[PubMed: 32592321]

84. Escudero L, Llort A, Arias A, Diaz-Navarro A, Martinez-Ricarte F, Rubio-Perez C, et al.

Circulating tumour DNA from the cerebrospinal fluid allows the characterisation and monitoring
Author Manuscript
of medulloblastoma. Nat Commun 2020;11:5376 [PubMed: 33110059]

85. Li J, Zhao S, Lee M, Yin Y, Li J, Zhou Y, et al. Reliable tumor detection by whole-genome
methylation sequencing of cell-free DNA in cerebrospinal fluid of pediatric medulloblastoma. Sci
Adv 2020;6
86. Klega K, Imamovic-Tuco A, Ha G, Clapp AN, Meyer S, Ward A, et al. Detection of Somatic
Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in
Children With Solid Tumors. JCO Precis Oncol 2018;2018
87. Cimmino F, Lasorsa VA, Vetrella S, Iolascon A, Capasso M. A Targeted Gene Panel for Circulating
Tumor DNA Sequencing in Neuroblastoma. Front Oncol 2020;10:596191
88. Pan C, Diplas BH, Chen X, Wu Y, Xiao X, Jiang L, et al. Molecular profiling of tumors
of the brainstem by sequencing of CSF-derived circulating tumor DNA. Acta Neuropathol
2019;137:297–306 [PubMed: 30460397]
89. Woodhouse R, Li M, Hughes J, Delfosse D, Skoletsky J, Ma P, et al. Clinical and analytical
validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive
Author Manuscript
genomic profiling assay for cancers of solid tumor origin. PLoS One 2020;15:e0237802
90. Li G, Pavlick D, Chung JH, Bauer T, Tan BA, Peguero J, et al. Genomic profiling of cell-free
circulating tumor DNA in patients with colorectal cancer and its fidelity to the genomics of the
tumor biopsy. J Gastrointest Oncol 2019;10:831–40 [PubMed: 31602320]
91. Leighl NB, Page RD, Raymond VM, Daniel DB, Divers SG, Reckamp KL, et al. Clinical Utility of
Comprehensive Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with Newly
Diagnosed Metastatic Non-small Cell Lung Cancer. Clin Cancer Res 2019;25:4691–700 [PubMed:
30988079]
92. Strickler JH, Loree JM, Ahronian LG, Parikh AR, Niedzwiecki D, Pereira AAL, et al. Genomic
Landscape of Cell-Free DNA in Patients with Colorectal Cancer. Cancer Discov 2018;8:164–73
[PubMed: 29196463]
93. Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV, Consortium C. Sensitive and specific
multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol
2020;31:745–59 [PubMed: 33506766]
94. Cohen JD, Li L, Wang Y, Thoburn C, Afsari B, Danilova L, et al. Detection and localization of
Author Manuscript
surgically resectable cancers with a multi-analyte blood test. Science 2018;359:926–30 [PubMed:
29348365]
95. Lennon AM, Buchanan AH, Kinde I, Warren A, Honushefsky A, Cohain AT, et al. Feasibility
of blood testing combined with PET-CT to screen for cancer and guide intervention. Science
2020;369
96. Chang W, Brohl AS, Patidar R, Sindiri S, Shern JF, Wei JS, et al. MultiDimensional ClinOmics
for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory
Cancer: A Report from the Center for Cancer Research. Clin Cancer Res 2016;22:3810–20
[PubMed: 26994145]
97. Zhang Y, Chen F, Donehower LA, Scheurer ME, Creighton CJ. A pediatric brain tumor atlas
of genes deregulated by somatic genomic rearrangement. Nat Commun 2021;12:937 [PubMed:
33568653]
98. Rokita JL, Rathi KS, Cardenas MF, Upton KA, Jayaseelan J, Cross KL, et al. Genomic Profiling
of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design.
Author Manuscript
Cell Rep 2019;29:1675–89 e9 [PubMed: 31693904]

99. Cameron DL, Di Stefano L, Papenfuss AT. Comprehensive evaluation and characterisation of short
read general-purpose structural variant calling software. Nat Commun 2019;10:3240 [PubMed:
31324872]
100. van Belzen IAEM, Schönhuth A, Kemmeren P, Hehir-Kwa JY. Structural variant detection in
cancer genomes: computational challenges and perspectives for precision oncology. NPJ Precis
Oncol 2021;5:15 [PubMed: 33654267]

101. Lei M, Liang D, Yang Y, Mitsuhashi S, Katoh K, Miyake N, et al. Long-read DNA sequencing
fully characterized chromothripsis in a patient with Langer-Giedion syndrome and Cornelia de
Author Manuscript
Lange syndrome-4. J Hum Genet 2020;65:667–74 [PubMed: 32296131]

102. Mitsuhashi S, Ohori S, Katoh K, Frith MC, Matsumoto N. A pipeline for complete
characterization of complex germline rearrangements from long DNA reads. Genome Med
2020;12:67 [PubMed: 32731881]
103. Federman N, McDermott R. Larotrectinib, a highly selective tropomyosin receptor kinase (TRK)
inhibitor for the treatment of TRK fusion cancer. Expert Rev Clin Pharmacol 2019;12:931–9
[PubMed: 31469968]
104. Zhao X, Kotch C, Fox E, Surrey LF, Wertheim GB, Baloch ZW, et al. NTRK Fusions Identified
in Pediatric Tumors: The Frequency, Fusion Partners, and Clinical Outcome. JCO Precis Oncol
2021;1 [PubMed: 34994591]
105. Laetsch TW, DuBois SG, Mascarenhas L, Turpin B, Federman N, Albert CM, et al. Larotrectinib
for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre,
open-label, phase 1/2 study. Lancet Oncol 2018;19:705–14 [PubMed: 29606586]
106. Kummar S, Berlin J, Mascarenhas L, van Tilburg CM, Geoerger B, Lassen UN, et al. Quality of
Author Manuscript
Life in Adult and Pediatric Patients with Tropomyosin Receptor Kinase Fusion Cancer Receiving
Larotrectinib. Curr Probl Cancer 2021:100734
107. Dombi E, Baldwin A, Marcus LJ, Fisher MJ, Weiss B, Kim A, et al. Activity of Selumetinib in
Neurofibromatosis Type 1-Related Plexiform Neurofibromas. N Engl J Med 2016;375:2550–60
[PubMed: 28029918]
108. Gross AM, Wolters PL, Dombi E, Baldwin A, Whitcomb P, Fisher MJ, et al. Selumetinib in
Children with Inoperable Plexiform Neurofibromas. N Engl J Med 2020;382:1430–42 [PubMed:
32187457]
109. Jackson S, Baker EH, Gross AM, Whitcomb P, Baldwin A, Derdak J, et al. The MEK
inhibitor selumetinib reduces spinal neurofibroma burden in patients with NF1 and plexiform
neurofibromas. Neurooncol Adv 2020;2:vdaa095
110. Fangusaro J, Onar-Thomas A, Young Poussaint T, Wu S, Ligon AH, Lindeman N, et al.
Selumetinib in paediatric patients with BRAF-aberrant or neurofibromatosis type 1-associated
recurrent, refractory, or progressive low-grade glioma: a multicentre, phase 2 trial. Lancet Oncol
2019;20:1011–22 [PubMed: 31151904]
Author Manuscript
111. Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K, et al. Driver mutations in
histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 2012;482:226–
31 [PubMed: 22286061]
112. Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, et al. Somatic histone H3
alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat
Genet 2012;44:251–3 [PubMed: 22286216]
113. Larson JD, Kasper LH, Paugh BS, Jin H, Wu G, Kwon CH, et al. Histone H3.3 K27M
Accelerates Spontaneous Brainstem Glioma and Drives Restricted Changes in Bivalent Gene
Expression. Cancer Cell 2019;35:140–55 e7 [PubMed: 30595505]
114. Chen KY, Bush K, Klein RH, Cervantes V, Lewis N, Naqvi A, et al. Reciprocal H3.3 gene
editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling.
Commun Biol 2020;3:363 [PubMed: 32647372]
115. Mohammad F, Weissmann S, Leblanc B, Pandey DP, Hojfeldt JW, Comet I, et al. EZH2 is a
potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med 2017;23:483–92
Author Manuscript
[PubMed: 28263309]
116. Silveira AB, Kasper LH, Fan Y, Jin H, Wu G, Shaw TI, et al. H3.3 K27M depletion increases
differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo. Acta
Neuropathol 2019;137:637–55 [PubMed: 30770999]
117. Harutyunyan AS, Krug B, Chen H, Papillon-Cavanagh S, Zeinieh M, De Jay N, et al.
H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and
is essential for glioma tumorigenesis. Nat Commun 2019;10:1262 [PubMed: 30890717]

118. Piunti A, Hashizume R, Morgan MA, Bartom ET, Horbinski CM, Marshall SA, et al. Therapeutic
targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. Nat
Author Manuscript
Med 2017;23:493–500 [PubMed: 28263307]

119. Bayliss J, Mukherjee P, Lu C, Jain SU, Chung C, Martinez D, et al. Lowered H3K27me3
and DNA hypomethylation define poorly prognostic pediatric posterior fossa ependymomas. Sci
Transl Med 2016;8:366ra161
120. Chen CCL, Deshmukh S, Jessa S, Hadjadj D, Lisi V, Andrade AF, et al. Histone H3.3G34-
Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis. Cell 2020;183:1617–33
e22 [PubMed: 33259802]
121. Helmsauer K, Valieva ME, Ali S, Chamorro Gonzalez R, Schopflin R, Roefzaad C, et al.
Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in
neuroblastoma. Nat Commun 2020;11:5823 [PubMed: 33199677]
122. Morrow JJ, Bayles I, Funnell APW, Miller TE, Saiakhova A, Lizardo MM, et al. Positively
selected enhancer elements endow osteosarcoma cells with metastatic competence. Nat Med
2018;24:176–85 [PubMed: 29334376]
123. Guillon N, Tirode F, Boeva V, Zynovyev A, Barillot E, Delattre O. The oncogenic EWS-FLI1
Author Manuscript
protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation
function. PLoS One 2009;4:e4932 [PubMed: 19305498]
124. Lin L, Huang M, Shi X, Mayakonda A, Hu K, Jiang YY, et al. Super-enhancer-associated
MEIS1 promotes transcriptional dysregulation in Ewing sarcoma in co-operation with EWS-
FLI1. Nucleic Acids Res 2019;47:1255–67 [PubMed: 30496486]
125. van Groningen T, Koster J, Valentijn LJ, Zwijnenburg DA, Akogul N, Hasselt NE, et al.
Neuroblastoma is composed of two super-enhancer-associated differentiation states. Nat Genet
2017;49:1261–6 [PubMed: 28650485]
126. Gryder BE, Wachtel M, Chang K, El Demerdash O, Aboreden NG, Mohammed W, et al.
Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage. iScience 2020;23:101103
127. Shern JF, Chen L, Chmielecki J, Wei JS, Patidar R, Rosenberg M, et al. Comprehensive genomic
analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic
axis in fusion-positive and fusion-negative tumors. Cancer Discov 2014;4:216–31 [PubMed:
24436047]
128. Yohe ME, Gryder BE, Shern JF, Song YK, Chou HC, Sindiri S, et al. MEK inhibition induces
Author Manuscript
MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma. Sci Transl Med

2018;10
129. Pomella S, Sreenivas P, Gryder BE, Wang L, Milewski D, Cassandri M, et al. Interaction between
SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative
Rhabdomyosarcoma. Nat Commun 2021;12:192 [PubMed: 33420019]
130. Li S, Chen K, Zhang Y, Barnes SD, Jaichander P, Zheng Y, et al. Twist2 amplification in
rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA
binding. Genes Dev 2019;33:626–40 [PubMed: 30975722]
131. Boulay G, Awad ME, Riggi N, Archer TC, Iyer S, Boonseng WE, et al. OTX2 Activity at Distal
Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma. Cancer
Discov 2017;7:288–301 [PubMed: 28213356]
132. Matsuda T, Irie T, Katsurabayashi S, Hayashi Y, Nagai T, Hamazaki N, et al. Pioneer Factor
NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron
Conversion. Neuron 2019;101:472–85 e7 [PubMed: 30638745]
Author Manuscript
133. Tomazou EM, Sheffield NC, Schmidl C, Schuster M, Schonegger A, Datlinger P, et al.
Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer
reprogramming by the oncogenic fusion protein EWS-FLI1. Cell Rep 2015;10:1082–95
[PubMed: 25704812]
134. Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suva ML, et al. EWS-FLI1 utilizes
divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in
Ewing sarcoma. Cancer Cell 2014;26:668–81 [PubMed: 25453903]

135. Gryder BE, Yohe ME, Chou HC, Zhang X, Marques J, Wachtel M, et al. PAX3-FOXO1
Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability. Cancer
Author Manuscript
Discov 2017;7:884–99 [PubMed: 28446439]

136. Marques JG, Gryder BE, Pavlovic B, Chung Y, Ngo QA, Frommelt F, et al. NuRD subunit
CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general
tumor dependency. Elife 2020;9
137. Kadoch C, Crabtree GR. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX
oncogenic fusion in synovial sarcoma. Cell 2013;153:71–85 [PubMed: 23540691]
138. Boulay G, Cironi L, Garcia SP, Rengarajan S, Xing YH, Lee L, et al. The chromatin
landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms
and dependencies. Life Sci Alliance 2021;4
139. McBride MJ, Pulice JL, Beird HC, Ingram DR, D’Avino AR, Shern JF, et al. The SS18-SSX
Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial Sarcoma.
Cancer Cell 2018;33:1128–41 e7 [PubMed: 29861296]
140. Banito A, Li X, Laporte AN, Roe JS, Sanchez-Vega F, Huang CH, et al. The SS18-SSX
Oncoprotein Hijacks KDM2B-PRC1.1 to Drive Synovial Sarcoma. Cancer Cell 2018;33:527–41
Author Manuscript
e8 [PubMed: 29502955]
141. Sanchez-Molina S, Figuerola-Bou E, Blanco E, Sanchez-Jimenez M, Taboas P, Gomez S, et al.
RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for
Ewing sarcoma tumorigenesis. Sci Adv 2020;6
142. Boulay G, Sandoval GJ, Riggi N, Iyer S, Buisson R, Naigles B, et al. Cancer-Specific Retargeting
of BAF Complexes by a Prion-like Domain. Cell 2017;171:163–78 e19 [PubMed: 28844694]
143. Kildisiute G, Kholosy WM, Young MD, Roberts K, Elmentaite R, van Hooff SR, et al. Tumor to
normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell. Sci Adv 2021;7
144. Hanemaaijer ES, Margaritis T, Sanders K, Bos FL, Candelli T, Al-Saati H, et al. Single-cell
atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to
neuroblastoma phenotype. Proc Natl Acad Sci U S A 2021;118
145. Dong R, Yang R, Zhan Y, Lai HD, Ye CJ, Yao XY, et al. Single-Cell Characterization of
Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma. Cancer Cell
2020;38:716–33 e6 [PubMed: 32946775]
Author Manuscript
146. Young MD, Mitchell TJ, Vieira Braga FA, Tran MGB, Stewart BJ, Ferdinand JR, et al. Single-
cell transcriptomes from human kidneys reveal the cellular identity of renal tumors. Science
2018;361:594–9 [PubMed: 30093597]
147. Hovestadt V, Smith KS, Bihannic L, Filbin MG, Shaw ML, Baumgartner A, et al. Resolving
medulloblastoma cellular architecture by single-cell genomics. Nature 2019;572:74–9 [PubMed:
31341285]
148. Jessa S, Blanchet-Cohen A, Krug B, Vladoiu M, Coutelier M, Faury D, et al. Stalled
developmental programs at the root of pediatric brain tumors. Nat Genet 2019;51:1702–13
[PubMed: 31768071]
149. Ocasio J, Babcock B, Malawsky D, Weir SJ, Loo L, Simon JM, et al. scRNA-seq in
medulloblastoma shows cellular heterogeneity and lineage expansion support resistance to SHH
inhibitor therapy. Nat Commun 2019;10:5829 [PubMed: 31863004]
150. Sayles LC, Breese MR, Koehne AL, Leung SG, Lee AG, Liu HY, et al. Genome-Informed
Targeted Therapy for Osteosarcoma. Cancer Discov 2018
151. Stewart E, Federico SM, Chen X, Shelat AA, Bradley C, Gordon B, et al. Orthotopic patient-
Author Manuscript
derived xenografts of paediatric solid tumours. Nature 2017;549:96–100 [PubMed: 28854174]

152. Smith KS, Xu K, Mercer KS, Boop F, Klimo P, DeCupyere M, et al. Patient-derived orthotopic
xenografts of pediatric brain tumors: a St. Jude resource. Acta Neuropathol 2020;140:209–25
[PubMed: 32519082]
153. Brabetz S, Leary SES, Grobner SN, Nakamoto MW, Seker-Cin H, Girard EJ, et al. A biobank of
patient-derived pediatric brain tumor models. Nat Med 2018;24:1752–61 [PubMed: 30349086]
154. Richter-Pechanska P, Kunz JB, Bornhauser B, von Knebel Doeberitz C, Rausch T, Erarslan-Uysal
B, et al. PDX models recapitulate the genetic and epigenetic landscape of pediatric T-cell
leukemia. EMBO Mol Med 2018;10 [PubMed: 29191946]

155. Kolb EA, Houghton PJ, Kurmasheva RT, Mosse YP, Maris JM, Erickson SW, et al. Preclinical
evaluation of the combination of AZD1775 and irinotecan against selected pediatric solid tumors:
Author Manuscript
A Pediatric Preclinical Testing Consortium report. Pediatr Blood Cancer 2020;67:e28098

156. Stripecke R, Munz C, Schuringa JJ, Bissig KD, Soper B, Meeham T, et al. Innovations,
challenges, and minimal information for standardization of humanized mice. EMBO Mol Med
2020;12:e8662 [PubMed: 32578942]
157. Drost J, Clevers H. Organoids in cancer research. Nat Rev Cancer 2018;18:407–18 [PubMed:
29692415]
158. Calandrini C, Schutgens F, Oka R, Margaritis T, Candelli T, Mathijsen L, et al. An organoid
biobank for childhood kidney cancers that captures disease and tissue heterogeneity. Nat
Commun 2020;11:1310 [PubMed: 32161258]
159. Saltsman JA, Hammond WJ, Narayan NJC, Requena D, Gehart H, Lalazar G, et al. A Human
Organoid Model of Aggressive Hepatoblastoma for Disease Modeling and Drug Testing. Cancers
(Basel) 2020;12 [PubMed: 33375055]
160. Torres R, Martin MC, Garcia A, Cigudosa JC, Ramirez JC, Rodriguez-Perales S. Engineering
human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9
Author Manuscript
system. Nat Commun 2014;5:3964 [PubMed: 24888982]

161. Spraggon L, Martelotto LG, Hmeljak J, Hitchman TD, Wang J, Wang L, et al. Generation of
conditional oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing and
homology-directed repair. J Pathol 2017;242:102–12 [PubMed: 28188619]
162. Torres-Ruiz R, Martinez-Lage M, Martin MC, Garcia A, Bueno C, Castano J, et al. Efficient
Recreation of t(11;22) EWSR1-FLI1(+) in Human Stem Cells Using CRISPR/Cas9. Stem Cell
Reports 2017;8:1408–20 [PubMed: 28494941]
163. Vanoli F, Tomishima M, Feng W, Lamribet K, Babin L, Brunet E, et al. CRISPR-Cas9-guided
oncogenic chromosomal translocations with conditional fusion protein expression in human
mesenchymal cells. Proc Natl Acad Sci U S A 2017;114:3696–701 [PubMed: 28325870]
164. Lagutina IV, Valentine V, Picchione F, Harwood F, Valentine MB, Villarejo-Balcells B, et al.
Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in
mouse myoblasts using CRISPR-Cas9 nuclease. PLoS Genet 2015;11:e1004951
165. Minas TZ, Surdez D, Javaheri T, Tanaka M, Howarth M, Kang H-J, et al. Combined experience
of six independent laboratories attempting to create an Ewing sarcoma mouse model. Oncotarget
Author Manuscript
2016
166. Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment.
Science 2015;348:74–80 [PubMed: 25838376]
167. Aran D, Hu Z, Butte AJ. xCell: digitally portraying the tissue cellular heterogeneity landscape.
Genome Biol 2017;18:220 [PubMed: 29141660]
168. Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, et al. Robust enumeration of cell
subsets from tissue expression profiles. Nat Methods 2015;12:453–7 [PubMed: 25822800]
169. Newman AM, Steen CB, Liu CL, Gentles AJ, Chaudhuri AA, Scherer F, et al. Determining
cell type abundance and expression from bulk tissues with digital cytometry. Nat Biotechnol
2019;37:773–82 [PubMed: 31061481]
170. Zhou Y, Yang D, Yang Q, Lv X, Huang W, Zhou Z, et al. Single-cell RNA landscape of
intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma.
Nat Commun 2020;11:6322 [PubMed: 33303760]
171. Gomez-Brouchet A, Illac C, Gilhodes J, Bouvier C, Aubert S, Guinebretiere JM, et
Author Manuscript
al. CD163-positive tumor-associated macrophages and CD8-positive cytotoxic lymphocytes

are powerful diagnostic markers for the therapeutic stratification of osteosarcoma patients:
An immunohistochemical analysis of the biopsies fromthe French OS2006 phase 3 trial.
Oncoimmunology 2017;6:e1331193
172. Ligon JA, Choi W, Cojocaru G, Fu W, Hsiue EH, Oke TF, et al. Pathways of immune exclusion
in metastatic osteosarcoma are associated with inferior patient outcomes. J Immunother Cancer
2021;9

173. Sorenson L, Fu Y, Hood T, Warren S, McEachron TA. Targeted transcriptional profiling of the
tumor microenvironment reveals lymphocyte exclusion and vascular dysfunction in metastatic
Author Manuscript
osteosarcoma. Oncoimmunology 2019;8:e1629779

174. Wu CC, Beird HC, Andrew Livingston J, Advani S, Mitra A, Cao S, et al. Immuno-genomic
landscape of osteosarcoma. Nat Commun 2020;11:1008 [PubMed: 32081846]
175. Zhou Q, Xian M, Xiang S, Xiang D, Shao X, Wang J, et al. All-Trans Retinoic Acid Prevents
Osteosarcoma Metastasis by Inhibiting M2 Polarization of Tumor-Associated Macrophages.
Cancer Immunol Res 2017;5:547–59 [PubMed: 28515123]
176. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system.
Nat Rev Immunol 2009;9:162–74 [PubMed: 19197294]
177. Long AH, Highfill SL, Cui Y, Smith JP, Walker AJ, Ramakrishna S, et al. Reduction of MDSCs
with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas. Cancer Immunol
Res 2016;4:869–80 [PubMed: 27549124]
178. Machado I, Lopez-Guerrero JA, Scotlandi K, Picci P, Llombart-Bosch A. Immunohistochemical
analysis and prognostic significance of PD-L1, PD-1, and CD8+ tumor-infiltrating lymphocytes
in Ewing’s sarcoma family of tumors (ESFT). Virchows Arch 2018;472:815–24 [PubMed:
Author Manuscript
29445891]
179. Mochizuki K, Kawana S, Yamada S, Muramatsu M, Sano H, Kobayashi S, et al. Various
checkpoint molecules, and tumor-infiltrating lymphocytes in common pediatric solid tumors:
Possibilities for novel immunotherapy. Pediatr Hematol Oncol 2019;36:17–27 [PubMed:
30870043]
180. Heitzeneder S, Sotillo E, Shern JF, Sindiri S, Xu P, Jones R, et al. Pregnancy-Associated Plasma
Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion. J Natl
Cancer Inst 2019;111:970–82 [PubMed: 30698726]
181. Spurny C, Kailayangiri S, Altvater B, Jamitzky S, Hartmann W, Wardelmann E, et al. T cell
infiltration into Ewing sarcomas is associated with local expression of immune-inhibitory HLA-
G. Oncotarget 2018;9:6536–49 [PubMed: 29464090]
182. Sand LG, Berghuis D, Szuhai K, Hogendoorn PC. Expression of CCL21 in Ewing sarcoma
shows an inverse correlation with metastases and is a candidate target for immunotherapy. Cancer
Immunol Immunother 2016;65:995–1002 [PubMed: 27369431]
183. Melaiu O, Chierici M, Lucarini V, Jurman G, Conti LA, De Vito R, et al. Cellular and
Author Manuscript
gene signatures of tumor-infiltrating dendritic cells and natural-killer cells predict prognosis of
neuroblastoma. Nat Commun 2020;11:5992 [PubMed: 33239635]
184. Xin C, Zhu J, Gu S, Yin M, Ma J, Pan C, et al. CD200 is overexpressed in neuroblastoma
and regulates tumor immune microenvironment. Cancer Immunol Immunother 2020;69:2333–43
[PubMed: 32514618]
185. Wei JS, Kuznetsov IB, Zhang S, Song YK, Asgharzadeh S, Sindiri S, et al. Clinically Relevant
Cytotoxic Immune Cell Signatures and Clonal Expansion of T-Cell Receptors in High-Risk
MYCN-Not-Amplified Human Neuroblastoma. Clin Cancer Res 2018;24:5673–84 [PubMed:
29784674]
186. Layer JP, Kronmuller MT, Quast T, van den Boorn-Konijnenberg D, Effern M, Hinze
D, et al. Amplification of N-Myc is associated with a T-cell-poor microenvironment in
metastatic neuroblastoma restraining interferon pathway activity and chemokine expression.
Oncoimmunology 2017;6:e1320626
187. Bao R, Spranger S, Hernandez K, Zha Y, Pytel P, Luke JJ, et al. Immunogenomic determinants
Author Manuscript
of tumor microenvironment correlate with superior survival in high-risk neuroblastoma. J

Immunother Cancer 2021;9
188. Mao Y, Eissler N, Blanc KL, Johnsen JI, Kogner P, Kiessling R. Targeting Suppressive Myeloid
Cells Potentiates Checkpoint Inhibitors to Control Spontaneous Neuroblastoma. Clin Cancer Res
2016;22:3849–59 [PubMed: 26957560]
189. Tian K, Wang X, Wu Y, Wu X, Du G, Liu W, et al. Relationship of tumour-associated
macrophages with poor prognosis in Wilms’ tumour. J Pediatr Urol 2020;16:376 e1- e8

190. Maturu P, Jones D, Ruteshouser EC, Hu Q, Reynolds JM, Hicks J, et al. Role of
Cyclooxygenase-2 Pathway in Creating an Immunosuppressive Microenvironment and in
Author Manuscript
Initiation and Progression of Wilms’ Tumor. Neoplasia 2017;19:237–49 [PubMed: 28254151]

191. Chen L, Oke T, Siegel N, Cojocaru G, Tam AJ, Blosser RL, et al. The Immunosuppressive Niche
of Soft-Tissue Sarcomas is Sustained by Tumor-Associated Macrophages and Characterized
by Intratumoral Tertiary Lymphoid Structures. Clin Cancer Res 2020;26:4018–30 [PubMed:
32332015]
192. Kather JN, Horner C, Weis CA, Aung T, Vokuhl C, Weiss C, et al. CD163+ immune
cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with
embryonal rhabdomyosarcoma. Sci Rep 2019;9:9211 [PubMed: 31239476]
193. Highfill SL, Cui Y, Giles AJ, Smith JP, Zhang H, Morse E, et al. Disruption of CXCR2-mediated
MDSC tumor trafficking enhances anti-PD1 efficacy. Sci Transl Med 2014;6:237ra67
194. Grabovska Y, Mackay A, O’Hare P, Crosier S, Finetti M, Schwalbe EC, et al. Pediatric
pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of
antitumor immunity. Nat Commun 2020;11:4324 [PubMed: 32859926]
195. Petralia F, Tignor N, Reva B, Koptyra M, Chowdhury S, Rykunov D, et al. Integrated
Author Manuscript
Proteogenomic Characterization across Major Histological Types of Pediatric Brain Cancer. Cell
2020;183:1962–85 e31 [PubMed: 33242424]
196. Pham CD, Flores C, Yang C, Pinheiro EM, Yearley JH, Sayour EJ, et al. Differential Immune
Microenvironments and Response to Immune Checkpoint Blockade among Molecular Subtypes
of Murine Medulloblastoma. Clin Cancer Res 2016;22:582–95 [PubMed: 26405194]
197. Maximov V, Chen Z, Wei Y, Robinson MH, Herting CJ, Shanmugam NS, et al. Tumour-
associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma.
Nat Commun 2019;10:2410 [PubMed: 31160587]
198. Garancher A, Suzuki H, Haricharan S, Chau LQ, Masihi MB, Rusert JM, et al. Tumor necrosis
factor overcomes immune evasion in p53-mutant medulloblastoma. Nat Neurosci 2020;23:842–
53 [PubMed: 32424282]
199. Bockmayr M, Klauschen F, Maire CL, Rutkowski S, Westphal M, Lamszus K, et al. Immunologic
Profiling of Mutational and Transcriptional Subgroups in Pediatric and Adult High-Grade
Gliomas. Cancer Immunol Res 2019;7:1401–11 [PubMed: 31266783]
200. Robinson MH, Vasquez J, Kaushal A, MacDonald TJ, Velazquez Vega JE, Schniederjan M, et
Author Manuscript
al. Subtype and grade-dependent spatial heterogeneity of T-cell infiltration in pediatric glioma. J
Immunother Cancer 2020;8
201. Haberthur K, Brennan K, Hoglund V, Balcaitis S, Chinn H, Davis A, et al. NKG2D ligand
expression in pediatric brain tumors. Cancer Biol Ther 2016;17:1253–65 [PubMed: 27834580]
202. Lieberman NAP, DeGolier K, Kovar HM, Davis A, Hoglund V, Stevens J, et al. Characterization
of the immune microenvironment of diffuse intrinsic pontine glioma: implications for
development of immunotherapy. Neuro Oncol 2019;21:83–94 [PubMed: 30169876]
203. Lin GL, Nagaraja S, Filbin MG, Suva ML, Vogel H, Monje M. Non-inflammatory tumor
microenvironment of diffuse intrinsic pontine glioma. Acta Neuropathol Commun 2018;6:51
[PubMed: 29954445]
204. Hanaizi Z, van Zwieten-Boot B, Calvo G, Lopez AS, van Dartel M, Camarero J, et al. The
European Medicines Agency review of ipilimumab (Yervoy) for the treatment of advanced
(unresectable or metastatic) melanoma in adults who have received prior therapy: summary of
the scientific assessment of the Committee for Medicinal Products for Human Use. Eur J Cancer
Author Manuscript
2012;48:237–42 [PubMed: 22030452]

205. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved
survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711–23
[PubMed: 20525992]
206. Davis KL, Fox E, Merchant MS, Reid JM, Kudgus RA, Liu X, et al. Nivolumab in children and
young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): a multicentre,
open-label, single-arm, phase 1–2 trial. Lancet Oncol 2020;21:541–50 [PubMed: 32192573]

207. Majzner RG, Simon JS, Grosso JF, Martinez D, Pawel BR, Santi M, et al. Assessment of
programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer
Author Manuscript
tissues. Cancer 2017;123:3807–15 [PubMed: 28608950]

208. Coronado E, Yanez Y, Vidal E, Rubio L, Vera-Sempere F, Canada-Martinez AJ, et al. Intratumoral
immunosuppression profiles in 11q-deleted neuroblastomas provide new potential therapeutic
targets. Mol Oncol 2021;15:364–80 [PubMed: 33252831]
209. McEachron TA, Triche TJ, Sorenson L, Parham DM, Carpten JD. Profiling targetable immune
checkpoints in osteosarcoma. Oncoimmunology 2018;7:e1475873
210. Bouffet E, Larouche V, Campbell BB, Merico D, de Borja R, Aronson M, et al. Immune
Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline
Biallelic Mismatch Repair Deficiency. J Clin Oncol 2016;34:2206–11 [PubMed: 27001570]
211. Mandal R, Samstein RM, Lee KW, Havel JJ, Wang H, Krishna C, et al. Genetic diversity of
tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response. Science
2019;364:485–91 [PubMed: 31048490]
212. Le Cesne A, Marec-Berard P, Blay JY, Gaspar N, Bertucci F, Penel N, et al. Programmed
cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the
Author Manuscript
PEMBROSARC study. Eur J Cancer 2019;119:151–7 [PubMed: 31442817]

213. Geoerger B, Kang HJ, Yalon-Oren M, Marshall LV, Vezina C, Pappo A, et al. Pembrolizumab
in paediatric patients with advanced melanoma or a PD-L1-positive, advanced, relapsed, or
refractory solid tumour or lymphoma (KEYNOTE-051): interim analysis of an open-label,
single-arm, phase 1–2 trial. Lancet Oncol 2020;21:121–33 [PubMed: 31812554]
214. Merchant MS, Wright M, Baird K, Wexler LH, Rodriguez-Galindo C, Bernstein D, et al. Phase I
Clinical Trial of Ipilimumab in Pediatric Patients with Advanced Solid Tumors. Clin Cancer Res
2016;22:1364–70 [PubMed: 26534966]
215. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in
Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 2018;378:439–
48 [PubMed: 29385370]
216. Zhang L, Zuo Y, Lu A, Wu J, Jia Y, Wang Y, et al. Safety and Efficacy of Chimeric Antigen
Receptor T-Cell Therapy in Children With Central Nervous System Leukemia. Clin Lymphoma
Myeloma Leuk 2021;21:e410–e4 [PubMed: 33526401]
217. Tan Y, Pan J, Deng B, Ling Z, Song W, Xu J, et al. Toxicity and effectiveness of CD19 CAR T
Author Manuscript
therapy in children with high-burden central nervous system refractory B-ALL. Cancer Immunol
Immunother 2021
218. Shalabi H, Gust J, Taraseviciute A, Wolters PL, Leahy AB, Sandi C, et al. Beyond the storm -
subacute toxicities and late effects in children receiving CAR T cells. Nat Rev Clin Oncol 2021
219. Li X, Chen W. Mechanisms of failure of chimeric antigen receptor T-cell therapy. Curr Opin
Hematol 2019;26:427–33 [PubMed: 31577606]
220. Pan J, Zuo S, Deng B, Xu X, Li C, Zheng Q, et al. Sequential CD19–22 CAR T therapy induces
sustained remission in children with r/r B-ALL. Blood 2020;135:387–91 [PubMed: 31725148]
221. Hua J, Qian W, Wu X, Zhou L, Yu L, Chen S, et al. Sequential Infusion of Anti-CD22 and
Anti-CD19 Chimeric Antigen Receptor T Cells for a Pediatric Ph-Like B-ALL Patient That
Relapsed After CART-Cell and Haplo-HSCT Therapy: A Case Report and Review of Literature.
Onco Targets Ther 2020;13:2311–7 [PubMed: 32256082]
222. Qin H, Ramakrishna S, Nguyen S, Fountaine TJ, Ponduri A, Stetler-Stevenson M, et al.
Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and
Author Manuscript
CD22. Mol Ther Oncolytics 2018;11:127–37 [PubMed: 30581986]

223. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, et al. Enhanced
CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a
low-affinity CD19 CAR. Nat Med 2019;25:1408–14 [PubMed: 31477906]
224. Shah NN, Lee DW, Yates B, Yuan CM, Shalabi H, Martin S, et al. Long-Term Follow-Up
of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL. J Clin Oncol
2021:JCO2002262
225. Nazha B, Inal C, Owonikoko TK. Disialoganglioside GD2 Expression in Solid Tumors and Role
as a Target for Cancer Therapy. Front Oncol 2020;10:1000 [PubMed: 32733795]

226. Xu X, Huang W, Heczey A, Liu D, Guo L, Wood M, et al. NKT Cells Coexpressing
a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In Vivo Persistence
Author Manuscript
and Antitumor Activity against Neuroblastoma. Clin Cancer Res 2019;25:7126–38 [PubMed:
31484667]
227. Chulanetra M, Morchang A, Sayour E, Eldjerou L, Milner R, Lagmay J, et al. GD2 chimeric
antigen receptor modified T cells in synergy with sub-toxic level of doxorubicin targeting
osteosarcomas. Am J Cancer Res 2020;10:674–87 [PubMed: 32195035]
228. Mount CW, Majzner RG, Sundaresh S, Arnold EP, Kadapakkam M, Haile S, et al. Potent
antitumor efficacy of anti-GD2 CAR T cells in H3-K27M(+) diffuse midline gliomas. Nat Med
2018;24:572–9 [PubMed: 29662203]
229. Sujjitjoon J, Sayour E, Tsao ST, Uiprasertkul M, Sanpakit K, Buaboonnam J, et al. GD2-specific
chimeric antigen receptor-modified T cells targeting retinoblastoma - assessing tumor and T cell
interaction. Transl Oncol 2021;14:100971
230. Andersch L, Radke J, Klaus A, Schwiebert S, Winkler A, Schumann E, et al. CD171- and
GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing.
BMC Cancer 2019;19:895 [PubMed: 31500597]
Author Manuscript
231. Chen Y, Sun C, Landoni E, Metelitsa L, Dotti G, Savoldo B. Eradication of Neuroblastoma

by T Cells Redirected with an Optimized GD2-Specific Chimeric Antigen Receptor and
Interleukin-15. Clin Cancer Res 2019;25:2915–24 [PubMed: 30617136]
232. Heczey A, Louis CU, Savoldo B, Dakhova O, Durett A, Grilley B, et al. CAR T Cells
Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with
Neuroblastoma. Mol Ther 2017;25:2214–24 [PubMed: 28602436]
233. Straathof K, Flutter B, Wallace R, Jain N, Loka T, Depani S, et al. Antitumor activity
without on-target off-tumor toxicity of GD2-chimeric antigen receptor T cells in patients with
neuroblastoma. Sci Transl Med 2020;12
234. Shurell E, Vergara-Lluri ME, Li Y, Crompton JG, Singh A, Bernthal N, et al. Comprehensive
adipocytic and neurogenic tissue microarray analysis of NY-ESO-1 expression - a promising
immunotherapy target in malignant peripheral nerve sheath tumor and liposarcoma. Oncotarget
2016;7:72860–7 [PubMed: 27655679]
235. Iura K, Maekawa A, Kohashi K, Ishii T, Bekki H, Otsuka H, et al. Cancer-testis antigen
expression in synovial sarcoma: NY-ESO-1, PRAME, MAGEA4, and MAGEA1. Hum Pathol
Author Manuscript
2017;61:130–9 [PubMed: 27993576]

236. D’Angelo SP, Melchiori L, Merchant MS, Bernstein D, Glod J, Kaplan R, et al. Antitumor
Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 (c259)T
Cells in Synovial Sarcoma. Cancer Discov 2018;8:944–57 [PubMed: 29891538]
237. Ramachandran I, Lowther DE, Dryer-Minnerly R, Wang R, Fayngerts S, Nunez D, et al. Systemic
and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial
sarcoma. J Immunother Cancer 2019;7:276 [PubMed: 31651363]
Author Manuscript

Author Manuscript
Author Manuscript
Figure 1: Features of pediatric tumors.

(A) SV’s are more common than SNVs. (B) Somatic mutations in TP53 are frequently
recurrent and mutations in epigenetic genes are also common. (C) The mutation spectra
between adult and pediatric tumors share little overlap. SNV, single nucleotide variant; SV,
structural variant; Epi; epigenetic associated genes.
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Figure 2: Comparison of germline and somatic mutations in pediatric versus adult cancers.
Germline variants are more frequently observed in pediatric cancer patients than in adult
cancer patients whereas adult tumors have an increased somatic mutation burden than
pediatric tumors.
Author Manuscript
Author Manuscript

Table 1:
PDX Repositories
Author Manuscript
Childhood Cancer Repository www.cccells.org/xenografts
EuroPDX Consortium www.europdx.eu
PDX Development and Trial Centers Research Network www.pdxnetwork.org
PDXFinder www.pdxfinder.org
Pediatric Preclinical Testing Consortium www.ncipptc.org
www.stjude.org/research/why-st-jude/data-tools/childhood-solid-
Childhood Solid Tumor Network at St Jude Children’s Research Hospital tumor-network.html
Author Manuscript
Author Manuscript
Author Manuscript

Table 2:
FDA approved immunotherapies for pediatric oncology patients

Author Manuscript
Drug Trade Name Pediatric Indication Target Type of Immunotherapy

Ipilimumab Yervoy Colorectal Cancer, Melnaoma CTLA-4 Monoclonal Antibody
Nivolumab Opdivo Colorectal Cancer PD-1 Monoclonal Antibody
Hodgkin Lymphoma, Merkel Cell

Pembrolizumab Keytruda Carcinoma, Non-Hodgkin Lymphoma, Solid PD-1 Monoclonal Antibody
Tumors
Blinatumomab Blincyto Acute Lymphoblastic Leukemia C19, CD3 Bi-specific T-cell engager
Tisagenlecleucel Kymriah Acute Lymphoblastic Leukemia, CD19 Chimeric Antigen Receptor T-cell
Gemtuzumab Ozogamicin Mylotarg Acute Myeloid Leukemia CD33 Antibody-drug conjugate
Avelumab Bavencio Merkel Cell Carcinoma PD-L1 Monoclonal Antibody
Dinutuximab Unituxin Neuroblastoma GD2 Monoclonal Antibody
Naxitamab-gqgk Danyelza Neuroblastoma GD2 Monoclonal Antibody

Author Manuscript
Table contents obtained from National Cancer Institute: cancer.gov/about-cancer/treatment/drugs/childhood-cancer-fda-approved-drugs

Author Manuscript
Author Manuscript

Nihms 1745031

Uploaded by

Copyright:

Available Formats

Nihms 1745031

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nihms 1745031

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

Recent advances in pediatric cancer research

approaches to treatment. While a comprehensive review of the current status of pediatric

GENOMIC AND EPIGENOMIC PROFILING STUDIES

publication by Bert Vogelstein and colleagues illustrating the age-associated differences in

DNA Methylation Profiling of Brain Tumors

Cancer Res. Author manuscript; available in PMC 2022 December 06.

meningiomas, a histology predominantly observed in children and young adults, clustered

Mutational Activation of RAS-MAPK Pathway

Whole genome sequencing of matched primary-relapsed neuroblastoma specimens led to the

age-restricted oncogenic programs.

Cancer Res. Author manuscript; available in PMC 2022 December 06.

Germline Variants Associated with Predisposition and Increased Disease Risk:

Cancer Res. Author manuscript; available in PMC 2022 December 06.

approximately 7% of patients had a germline variant in a known cancer predisposition

Cancer Res. Author manuscript; available in PMC 2022 December 06.

syndrome exhibited an increased frequency of a risk allele in CDKN2A, a previously

Cancer Res. Author manuscript; available in PMC 2022 December 06.

of the biological and clinical significance of germline genetics in pediatric malignancies.

Cancer Res. Author manuscript; available in PMC 2022 December 06.

Filling in the Knowledge Gaps

BREAKTHROUGHS IN TARGETED THERAPIES

Cancer Res. Author manuscript; available in PMC 2022 December 06.

EPIGENETIC DRIVERS OF PEDIATRIC CANCER

Cancer Res. Author manuscript; available in PMC 2022 December 06.

Differential enhancer activity was observed in matched primary-metastatic osteosarcoma

the adrenergic-like neuroblastoma cells towards a more mesenchymal stem-like state as

The PAX3/7-FOXO1 translocations, the hallmark chromosomal aberrations in alveolar

Cancer Res. Author manuscript; available in PMC 2022 December 06.

dependent super-enhancer landscape (128). Inhibition of MAPK signaling by trametinib

in a BRD4-dependent manner (135). BRD4 is a member of the BET family of “chromatin

Redirecting/Hijacking of Chromatin Remodelers:

transcription (138,139). Additionally, the histone demethylase KDM2B, a member of the

Cancer Res. Author manuscript; available in PMC 2022 December 06.

that RING1B targets EWS-FLI1 to repressed enhancers to recruit additional chromatin

DEVELOPMENTAL AND CELLULAR ORIGINS OF PEDIATRIC TUMORS

Cancer Res. Author manuscript; available in PMC 2022 December 06.

EXPERIMENTAL MODEL SYSTEMS

Cancer Res. Author manuscript; available in PMC 2022 December 06.

Patient Derived Organoids

Somatic Genome Engineering of Oncogenic Translocations

Cancer Res. Author manuscript; available in PMC 2022 December 06.

IMMUNE MICROENVIRONMENT PROFILES OF PEDIATRIC TUMORS

Myeloid cells are an important feature of the osteosarcoma microenvironment. Analysis of

Cancer Res. Author manuscript; available in PMC 2022 December 06.

associated with an active immune response, suggesting that PAPPA is immunosuppressive.

Cancer Res. Author manuscript; available in PMC 2022 December 06.

In Wilms tumor specimens, an inverse relationship between M1 macrophage abundance

Cancer Res. Author manuscript; available in PMC 2022 December 06.

The microenvironmental heterogeneity of pediatric gliomas varied in accordance with

and/or diagnosis (195).

IMMUNOTHERAPEUTIC APPROACHES FOR PEDIATRIC TUMORS

Immune Checkpoint Inhibition in CNS and Solid Tumors

Cancer Res. Author manuscript; available in PMC 2022 December 06.

CD19 CAR T-Cell Therapy in Leukemia

Leukemic B-cells can escape killing by CAR T-cells by employing a variety of

GD2-targeted CAR T-Cell Therapy

Cancer Res. Author manuscript; available in PMC 2022 December 06.