Pediatric Solid Tumor Metabolism [A Prospective Study Exploring Metabolism of Solid Tumors in Pediatrics]

The principal objective of this study is the metabolic characterization of pediatric solid
tumors, with a particular focus on neuroblastoma (NBL) and fusion positive sarcoma (FPS),
which will allow the detection of tumor specific metabolic alterations that can be exploited
with the aim of developing novel therapeutic strategies and biomarkers. The rationale behind
this study and the reasons for its clinical significance are described below:

Neuroblastoma:

Neuroblastoma, a malignancy of the sympathetic nervous system, is the most common
extra-cranial solid tumor in children, accounting for 7% of childhood cancers and 15% of
childhood cancer related deaths. Neuroblastoma has a wide range of clinical outcomes ranging
from spontaneous maturation with regression, to death from widespread metastatic disease. The
outcomes and prognoses for children with neuroblastoma depend on their specific risk group
classification. Risk stratification is dependent on subject age, tumor histology and tumor
genetic characteristics and leads to vastly different therapies and outcomes. Currently the
therapy for low risk disease includes surgery and possibly chemotherapy with a 5 year EFS
>85%. However, despite intensive cytotoxic chemotherapy, double autologous stem cell
transplantation, and targeted radiopharmaceutical delivery of methyl-iodo-benzyl-guanidine
(MIBG), children with high risk disease have a 5 year EFS <50%.

The relevance of neuroblastoma genotype to clinical outcome is well established, as evidenced
by the poor prognosis in children with MYCN amplification. An important mechanism by which
oncogenes promote tumorigenesis, including increased proliferation and decreased
differentiation, is by regulating cellular metabolism. While non-malignant cells typically
generate cellular energy through use of oxidative phosphorylation, malignant cells emphasize
aerobic glycolysis (Warburg Effect) as a source of cellular energy since this process also
provide substrates for macromolecule synthesis and redox pathways. More specifically, in the
presence of oxygen, differentiated, non-malignant, cells metabolize glucose through oxidative
phosphorylation, creating an increased amount of energy, specifically 36 molecules of ATP.
However, in malignant cells, the majority of glucose is converted to lactate despite the
presence of oxygen, resulting in less energy and ATP production (2 molecules). More recent
research suggests that in fact both processes are increased in malignant cells. This finding
has been identified in cell-based neuroblastoma systems with MYCN amplification. MYNC
amplified tumors have alterations in mitochondrial metabolism that cause cells to be
dependent on glutamine for survival. If glutamine is depleted, these cells undergo apoptosis
leading to cell death. Studies are currently underway looking at the use of Fenretinide, a
synthetic retinoid, and its ability to cause a glutamine deplete environment leading to
apoptosis of these cells. Altered metabolism, in cell-based neuroblastoma systems, is also
evidenced by reduced activity of the metabolic enzyme succinate dehydrogenase (SDH) in
correlation to loss of heterozygosity (LOH) of 1p36 and increased expression of multiple
genes involved in glycolysis, glutamine, fatty acid and mitochondrial metabolism in
correlation to MYCN expression. However, MYCN amplification is only present in about 20% of
neuroblastoma tumors and there are multiple other genetic mutations whose metabolic
consequences at yet to be determined. When NBL occurs in a hereditary fashion, there is
classically a mutation in the ALK oncogene or more rarely a mutation in PHOX2B. When
neuroblastoma occurs sporadically, it is associated with amplification of MYCN (50% of
high-risk patients), LOH of 1p36 (35% of primary NBL), LOH of 11q (35-40% of newly diagnosed
patients), and gains at 17q. Therefore, the investigators feel it is imperative to continue
to explore the metabolism of neuroblastoma to discover further alterations in their
metabolism and possible links to these genetic alterations.

Fusion Positive Sarcoma:

Fusion positive sarcoma, including Ewing sarcoma (EWS), Ewing-like sarcoma, alveolar
rhabdomyosarcoma (aRMS) and many childhood non-rhabdomyosarcoma soft tissue sarcoma (NRSTS),
occurs in approximately 1,100 children in the United States annually. Eighty percent of those
with metastatic or recurrent FPS will die of disease, and only 55-75% of those with more
favorable risk can expect long-term survival. Juxtaposition of the EWS gene to ETS-family
FLI-1 gene occurs in 85% of patients with EWS, while alternate ETS family fusion-partners
including ERG, ETV1, E1AF and FEV occur less commonly. More recently a new class of fusion
positive Ewing-like sarcoma has been described, with translocations such as CIC:DUX4.
Alveolar rhabdomyosarcoma is now characterized as either PAX-FOXO1 fusion positive or fusion
negative. The group of malignancies categorized as NRSTS in children, includes many
histological subsets driven by oncogenic fusion proteins, such as the SS18-SSX in synovial
sarcoma. Genomic studies have revealed critical understanding of FPS disease biology but to
date have not laid the foundation for new therapies. In contrast, metabolism studies have the
potential to provide new insights. EWS cell lines demonstrate aerobic glycolysis as evidenced
by measures of glucose uptake, lactate dehydrogenase activity, ATP, and mitochondrial
membrane potential. Alveolar RMS cell lines also display aerobic glycolysis as demonstrated
by indirect measures of oxygen consumption and extracellular acidification and by metabolic
flux analysis. Cell line verification of aerobic glycolysis in FPS and emerging data
demonstrating that metabolic alterations are different in culture and in-vivo renders in-vivo
study an essential complement to metabolic studies in cultured cells. Molecular studies in
EWS and aRMS have focused on nucleic acid sequencing and identified only rare examples that
point toward new therapeutics. In contrast, metabolic studies can identify potential
vulnerabilities. A profound example of such a vulnerability relates to 2-hydroxyglutarate
(2HG), a putative "onco-metabolite". In 2009, Dang et al. demonstrated the strong correlation
between increased 2HG in glioma with mutations in isocitrate dehydrogenase-1 (IDH1). More
than 20 studies have confirmed this finding, and investigators in the UTSW Advanced Imaging
Research Center developed this finding into a clinical test in which 2HG can be imaged as a
non-invasive marker for IDH1/2 mutation and as a response marker for IDH1/2 inhibitors in
glioma. There are very few other examples in cancer, similar to IDH-1 in glioma, in which
genetic mutations occur in a metabolic enzyme resulting in an onco-metabolite that influences
cell activity. However, an early response biomarker, as exemplified by IDH1/2, is essential
for modification of therapy in FPS, and may be similarly useful in NBL.

Cellular metabolism studies provide insight, in a complementary way to genomics, into
processes acting downstream from oncogenes and oncogenic fusion proteins, and such insight
may point toward previously unrecognized therapeutic targets or onco-metabolites that are
traceable as robust biomarkers for response. The investigator's new approach to use an
in-vivo comprehensive analysis of metabolic reprograming in FPS/NBL has never been performed
in childhood FPS/NBL and will complement genomics studies for these cancers. For this study
the investigators plan to obtain tumor samples at time of surgical biopsy/resection and study
their metabolic signatures.

They will specifically evaluate the metabolomic profiles and perform metabolic flux analyses
of these tumors. Metabolomic profiles provide snapshots of up to 50 metabolites from pathways
including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and both
amino acid and nucleotide metabolism, all of which are known to be under oncogenic control in
cancer as noted above. Metabolic flux analyses provide dynamic evaluations of glucose entry
into major energetic and biosynthetic pathways. Pathway analysis depends on the ability to
detect metabolic alterations in these cells. To do this, they will infuse the patient with
13C-glucose pre-operatively. They will then trace the utilization of 13C-glucose of the tumor
cells by evaluating the samples with mass spectrometry after obtained at time of biopsy.
Biopsies will be performed by either surgeons in the operating room or interventional
radiologists in the interventional radiology suites.

13C, a stable, naturally occurring isotope of carbon is uniquely suited for this examination
since it does not undergo radioactive decay and has been given to humans safely in prior
studies. Some of these analyses may be performed without the concomitant perioperative
delivery of 13C-glucose as well. The investigators are ideally positioned to accomplish this
here at UT Southwestern, which is one of a very few centers worldwide to have developed
intra-operative delivery of stable isotope tracers to characterize metabolic flux in human
tumors in children. Furthermore, whenever feasible, patients will undergo preoperative
imaging studies, such as PET scan, MIBG, or MRI studies. The results of these imaging studies
if obtained will be correlated with the metabolic phenotype to generate a comprehensive
non-invasive view of the tumor with the goal of identifying infiltrative, metabolically
active solid tumor cells. In addition, a comprehensive molecular profile of the tumor will be
reviewed to enable a genotype-metabolic phenotype comparative analysis.

Inclusion Criteria:

- Suspected malignancy

- Age ≤ 26 years and being cared for at Children's Medical Center

- Ability to undergo standard of care diagnostic procedure, including biopsy or
resection of the tumor, in the OR or IR at CMC

Exclusion Criteria:

- Poorly controlled diabetes

- Any other medical condition that prevents administration of glucose