A Trial of Poly-ICLC in the Management of Recurrent Pediatric Low Grade Gliomas

Background/Rationale The incidence of primary pediatric brain tumors in the United States is
about 1500 per year. Brain tumors are the most common solid tumor diagnosed in childhood and
thus account for significant childhood mortality in the United States. Low-grade astrocytomas
and gliomas are the most common type of brain tumor of childhood (36% of childhood brain
tumors). These tumors encompass a heterogeneous assortment of histological subtypes
including: fibrillary, protoplasmic, gemistocytic, and mixed variants. Pilocytic
astrocytomas, pleomorphic xanthoastrocytomas and subependymal giant cell astrocytomas are
also included. Furthermore, in young children there are some unique rare entities that behave
like low-grade tumors, including infantile desmoplastic gangliogliomas, and desmoplastic
astrocytomas. Although children with low-grade astrocytomas often survive many years after
conventional treatment with surgery and sometimes radiotherapy, some children will not fare
as well. These tumors constitute a heterogeneous group because of differing locations within
the brain and varying biological behavior of different subtypes. For those where total
excision is possible, such as cerebellar astrocytomas, prognosis is excellent with over 90%
ten-year survival rates with surgical excision alone. In contrast, survival rates in children
with cerebral or diencephalic tumors are 40-70% at five years with irradiation, but decline
to 11-50% at 10 years (Mundigers, 1990). Some tumors however may be unresectable/partially
resectable, and radiation can have undesirable side effects in young children. While the most
significant intellectual deficits occur in young children less than 5 years treated with
cranial irradiation, the deficits recognized even in young adults warrant extending the age
to 10 years for avoiding radiation. Chemotherapy regimens are used for high-risk patients
(progressive tumor, residual tumor) as a means to avoid or delay radiation in young patients,
but side effects of chemotherapy are frequently reported.

Newer forms of effective treatment that will have lesser side effects are much needed in
childhood brain tumors especially low-grade gliomas. We propose to study the efficacy and
toxicity of poly-ICLC, a biological response modifier in children with low-grade gliomas.

PROTEOMICS

Current diagnostic and therapeutic monitoring of brain tumor patients are significantly
hindered due to limited understanding of brain tumor biology and response to therapy. The
majority of CNS tumors cannot be identified or followed by expression of serum or CSF
markers. However, if available, such markers would be highly desirable and could be used to:

- Detect minimal residual disease

- Predict response to specific targeted therapies

- Predict or anticipate tumor progression

- Distinguish tumor recurrence from post surgical changes or post-radiation changes on
neuro-imaging

- Augment current histopathologic classification systems

- Improve current clinical and pathological treatment stratification schemata

- Assess efficacy of and tumor response to specific biologic targeted therapies that may
not impact tumor size as a primary tumor endpoint (e.g., small molecule inhibitors or
anti-angiogenic strategies) While such markers would be useful to prognosticate, monitor
and treat all CNS tumors, its use in glial tumors including recurrent low grade
astrocytomas is critical since these tumors are often biopsied at presentation, but not
at recurrence. Often these tumors are not amenable complete resection or biopsy due to
the eloquence of brain tissue they infiltrate (e.g., optic pathway, brainstem or
hypothalamic gliomas), or the blood vessels that they encase.

CNS biologic material in CSF Glial tumors tend to disseminate locally along white matter
tracts rather than through sub-arachnoid seeding. Dissemination of low grade gliomas along
the sub-arachnoid space has been reported in children with low grade gliomas. Even focal
tumors are frequently adjacent to CSF pathways (e.g., intrapeduncular fossa, third and fourth
ventricles) resulting in direct contact between tumor tissue and spinal fluid. Yet
examination of CSF cytology for these tumors is not standard. Given limitations of
identifying tumor cells in the CSF, methodologies that could improve our understanding of CNS
tumors of all types are needed. This would provide a significant improvement in currently
available knowledge about the biology of these tumors, and could elucidate potential
therapeutic avenues.

Proteomics, a relatively new area of research whereby total protein complement of a tissue
compartment is analyzed, has successfully been used to identify novel biomarkers in solid
tumors (Zheng, 2003);( Khwaja, 2007). Because proteins are effectors of all cellular
functions, their measurement should represent the most direct means of cellular
characterization and hence tumor biology. Because cells and their environment exist in an
integrated state, it has been possible to interrogate the proteins of extra-cellular
compartments to assess the presence and impact of tumor cells. This has been done primarily
using serum or plasma to establish a method of screening for the presence of low stage
tumors. An analogous extra-cellular compartment for use in brain tumors would be
cerebrospinal fluid (CSF). It circulates throughout the CNS and exchanges proteins with the
extra-cellular fluid of the brain and spinal cord.

CSF is continuously created and reabsorbed, providing a real time steady state proteome.
Unlike serum, which contains a highly complex protein mixture ranging from very low abundance
proteins in the 10-30 pg/mL range to very abundant proteins in the 35-55 mg/mL range, CSF
contains a less complex protein mixture (Omenn, 2005).

Therefore, the CSF is more likely to contain higher relative concentrations of tumor-specific
proteins (higher signal to noise ratio) than serum. Taken together this makes CSF and
attractive alternative to serum for detection of brain tumor related biomarkers. Unlike
leukemia and many solid tumors outside the CNS, where serial biopsies are readily performed,
tumors of the CNS are not easily accessible other than at the time of initial or repeat
resection or biopsy. While studies on these samples provide important findings regarding
tumor biology, serial analyses during treatment are not reasonable. By contrast, the CSF of
tumor patients can be more readily sampled in most pediatric patients. With the development
of proteomic technology, investigation of tumor related signals at the time of diagnosis
through treatment, and then in remission and/or at the time of recurrence or progression is
possible.

While CSF for seeding tumors is readily available and routinely obtained for cytology, the
systematic evaluation of the proteins within these samples could be of considerable
scientific importance. In addition to identifying potential makers of disease or response to
therapy, the glycosylation and phosphorylation status of many proteins can also be evaluated.
Studies in tumor tissue show that such information reveals activity of different enzymes that
correlate with treatment response (Mellinghoff, 2005); (Helgi, 2005) or progression of
leptomeningeal metastases (Brandsma, 2006).

Proteomics CSF proteomics has been applied to many neurological disorders including
Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, acute brain injury
and Creutzfeldt-Jakob disease (Rohlff, 2001).

Reports of its use in neuro-oncology are limited, but demonstrate the potential of this
technology to effectively identify tumor biomarkers. One study used two dimensional
polyacrylamide (2-D) gel electrophoresis to measure the relative quantities of two
pre-selected markers, N-Myc and l-CaD, in the CSF of brain tumor patients (Zheng, 2003).
Another used ELISA of CSF to identify Osteopontin as predictive of AT/RT and correlated with
response to therapy (Kao, 2005). CSF proteomics using 2-D gel electrophoresis in combination
with mass spectroscopy and cleavable isotope Coded Affinity Tag (cICAT) was used to evaluate
60 samples of CSF and tumor cyst fluid taken from adults with brain tumors and non-neoplastic
controls. These techniques were used to find a panel of proteins differentially expressed in
lower vs. higher-grade gliomas. Findings were confirmed using Western Blot analysis probing
for eight selected proteins based on implied role in gliomagenesis and availability of
antibodies. This report, which has been accepted for publication pending revisions,
identified 21 potential CSF biomarkers for astrocytoma.

As mentioned above, there is evidence that gliomas disseminate through the subarachnoid
space. Currently there are several consortia actively studying protein expression in the
spinal fluid of children with malignant glial and embryonal tumors (Pediatric Brain Tumor
Consortium, Pediatric Oncology Experimental Therapeutics Consortium). Proteins interrogated
in these protocols include those involved with angiogenesis and neovascularity (EGF, VEGF and
bFGF), those involved in tumor growth and migration (Secreted protein with acidic and
cysteine rich domains (SPARC), attractin). There is no consortium actively collecting spinal
fluid sample in children with lower grade tumors. A secondary goal of this study is to
examine these proteins in the CSF of children with low grade gliomas who have tumor
progression. Comparison of CSF protein expression of in high grade and low grade tumors is
likely to help identify biological markers specific for tumor progression, or for tumor
pathology.

Inclusion Criteria:

- Age:Patients must be between 0 - 21 years of age when registered on this protocol.

- Diagnosis:Patients must have pathologically confirmed low grade glioma with histologic
subtypes interpreted as WHO grade I and II including:

- juvenile pilocytic astrocytoma (JPA)

- pleomorphic JPA

- diffuse astrocytoma (fibrillary, gemistocytic, giant cell, or pleomorphic
xanthoastrocytoma)

- low grade oligoastrocytoma

- low grade oligodendroglioma

- low grade glioma NOS Tumors of all regions of the CNS, with appropriate histology are
eligible for study. However patients with tumors intrinsic to the optic nerve and
involvement of the optic nerve cannot be biopsied/resected are eligible without
histological confirmation.

Patients with neurofibromatosis type 1(NF1) are also eligible.

Patients must have demonstrated either tumor progression or recurrence by radiographic
criteria and/or clinical criteria as defined below:

1. Patients with progressive non-resectable disease regardless of location in the brain
or spine are eligible for this study. Patients with evidence of leptomeningeal
dissemination are eligible for this study. Patients do not require biopsy/histologic
confirmation at the time of progression or relapse.

2. Radiographic progression is defined as >40% increase in the product of the three
perpendicular diameters of initial tumor relative to the initial baseline measurement
- length (L)x width (W) x transverse (T) (current scan) > 1.4 x L x W x T (initial
scan), or the development of any new sites of disease independent of the response of
the initial tumor. See section 7.1.2 for methodology for tumor measurement.

3. Post radiation changes are often seen on post-treatment imaging studies, so that
classification of a patient as having progressive disease may require several serial
MRI's if the child has received radiation within the preceding 12 months.

4. Tumor volume includes the entire tumor volume seen on gadolinium enhanced T1 MR
imaging plus non-enhancing abnormality seen on T2 or FLAIR.

5. All tumor cysts will be included in the tumor volume

6. Clinical progression without radiographic progression includes children with optic
pathway gliomas who demonstrate sustained decrease in visual fields and/or acuity in
three serial vision examinations. Each of the vision examinations must be performed >2
weeks apart.

7. Children with previously negative cerebrospinal fluid (CSF) cytology who show evidence
of tumor cells in fluid obtained by lumbar puncture can be designated as having
progressive disease in the absence of radiographic evidence of progression.

Measurable disease: Patients must have measurable disease documented by radiographic
criteria prior to enrollment.

Performance Level and Life Expectancy:Patients must have a performance status of > 50%
(Appendix I). Use Karnofsky for patients > 16 years of age and Lansky for patients ≤ 16
years of age. Patients must have a life expectancy of ≥ 8 weeks.

Prior Therapy:Patients must have fully recovered from the acute toxic effects of all prior
chemotherapy, immunotherapy, or radiotherapy prior to entering this study and meet time
restrictions from end of prior therapy as stated below:

1. Myelosuppressive chemotherapy patients must have received the last dose of
myelosuppressive therapy at least 3 weeks prior to study registration or at least 6
weeks if nitrosourea.

2. Investigational / Biological agent: Patient must have received the last dose of other
investigational or biological agent >7 days prior to study registration

3. XRT: Patients must be ≥ 8 weeks since the completion of radiation therapy.

4. Study specific limitations on prior therapy: There is no limit on the number of prior
treatment regimens or received doses of radiation therapy.

5. Growth factor(s): Must not have received any hematopoetic growth factors within 7 days
of study entry or 21 days for neulasta.

6. Prior Surgery: Must be ≥ 2 weeks from prior surgery.

7. Steroids: Must be on a stable steroid dose for 7 days prior to study entry.

Organ Function Requirements:All patients must have adequate organ function defined as:

Hematologic Function:

1. Hemoglobin: > 8.0 gm/dl (may transfuse PRBCs)

2. ANC: > 750/mm3 Must be at least 7days after last dose of growth factor

3. Platelet Count: > 50,000 (transfusion independent; ≥ 7 days from last transfusion)

Renal Function:

Serum creatinine ≤ 2 x normal for age (see below) or Creatinine clearance/GFR > 60
cc/min/1.73 m2 [Urine Creatinine (mg/dL)][Volume collected (ml)]/[Serum Creatinine
9mg/dL)][Hours x 60]

Age (years) Maximum Serum Creatinine (mg/dL)

≤ 5 0.8 > 5 & ≤ 10 1.0 > 10 & ≤ 15 1.2 > 15 1.5

Liver Function:

1. Total bilirubin < 1.5 x ULN for age, AND

2. SGPT (ALT) < 2.5x ULN

3. SGOT (AST) < 2.5x ULN

Pulmonary Function:

No evidence of dyspnea at rest, no exercise intolerance, and a pulse oximetry ≥ 94% if
there is clinical indication for determination.

Coagulation Function:

Normal PT and PTT at enrollment per institutional range

Reproductive Function:Due to potential teratogenic effects of (poly-ICLC), negative serum
beta-HCG in females, and use of effective contraception in males and females of
childbearing potential, IS REQUIRED.

Exclusion Criteria:

- Pregnant or lactating females. Women of childbearing age will agree to use
contraception during the protocol.

- Patients receiving other experimental immunotherapy.

- Patients may not have fever (38.50C) within 7 days of enrollment.

- No concurrent XRT or chemotherapy is allowed.

- Patients who, in the opinion of the investigator, may not be able to comply with the
safety monitoring requirements of the study.