Clinical trial NCT02372409
Conditions: Glioma Pilocytic Astrocytoma Anaplastic Astrocytoma Glioblastoma Mixed Oligoastrocytoma Mixed Glioma Oligodendroglioma Optic Glioma Astrocytoma
Sponsor: Washington University School of Medicine
Start date: 2015-08-14; Completion date: 2023-03-23
Enrollment: 6 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
By employing a combination of advanced MRI techniques and correlative serum biomarkers of blood brain barrier (BBB) disruption, the investigators plan to develop a powerful, first of its kind clinical algorithm in pediatrics whereby the investigators can measure and identify the window of maximal BBB disruption post MLA to 1) allow for an alternative to surgery in incompletely resected tumors, 2) allow for optimal chemotherapeutic dosing to achieve the greatest benefits and the least systemic side effects and 3) distinguish subsequent tumor progression from long-term MLA treatment effects. Preliminary data in adult imaging studies have shown that the BBB disruption lasts for several weeks following treatment before returning to a low baseline. This pilot therapeutic study will provide preliminary validation in pediatric patients.

Detailed description:

Arms: arm 1: * MLA is a minimally invasive laser surgery currently FDA approved for cytoreductive treatment of brain tumors, both primary and metastatic. MLA employs a small incision in the scalp and skull, through which a thin laser probe is inserted and guided by MR imaging to the core of a tumor mass where it delivers hyperthermic ablation from the core to the rim.
* Participants will undergo DCE and DSC-MRI imaging at the following time points:

  * no more than 3 weeks prior to MLA (OPTIONAL)
  * within approximately 4 days after MLA
  * 2-4 weeks after MLA
  * Every 12 weeks (+/- 7 days) for the first year or until disease progression arm 2: * MLA is a minimally invasive laser surgery currently FDA approved for cytoreductive treatment of brain tumors, both primary and metastatic. MLA employs a small incision in the scalp and skull, through which a thin laser probe is inserted and guided by MR imaging to the core of a tumor mass where it delivers hyperthermic ablation from the core to the rim.
* Within 7 days of MLA (range 2-14 days) doxorubicin will be given intravenously on an outpatient basis weekly for 6 weeks at a dose of 25 mg/m\^2 over 5-30 minutes
* Following the completion of doxorubin, etoposide 50 mg/m\^2/day will be given orally for 21 days of each 28-day cycle (treatment can continue up to 24 cycles)
* Participants will undergo DCE and DSC-MRI imaging at the following time points:

  * no more than 3 weeks prior to MLA (OPTIONAL)
  * within approximately 4 days after MLA
  * 2-4 weeks after MLA
  * every 8 weeks (+/- 7 days) until 2 years have elapsed or disease progression, whichever comes first
Interventions: intervention 1: MRI-guided laser ablation intervention 2: Doxorubicin intervention 3: Etoposide intervention 4: Dynamic contrast-enhanced (DCE) MRI intervention 5: Dynamic susceptibility contrast (DSC) MRI
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None intervention 4: None intervention 5: None

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Version: 08/11/20 
 
Page 1 of 51 
 
 
 
 
 
 
 
 
A Pilot Study of Using MRI-Guided Laser Heat Ablation to Induce Disruption of the 
Peritumoral Blood Brain Barrier to Enhance Delivery and Efficacy of Treatment of 
Pediatric Brain Tumors 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Campus Box 8056 
St. Louis, MO  63110 
 
Protocol #: 201502062 
Version Date: 08/11/20 
 
Principal Investigator: 
Margaret Shatara, MD 
 
 
 
 
660 S. Euclid, Campus Box 8116 
 
 
 
 
St. Louis, MO  63110 
 
 
 
 
Phone:  (314) 454-6018 
 
 
 
 
Fax:  (314) 454-2737 
 
 
 
 
E-mail:  rubin_j@wustl.edu 
 
Sub-Investigators 
 
 
Modality 
Jian Campian, MD, PhD 
 
Medical Oncology 
Eric C. Leuthardt, MD 
 
Neurosurgery 
David Limbrick, MD, PhD 
 
Pediatric Neurosurgery 
Joshua S. Shimony, MD, PhD 
Neuro-Radiology 
Gavin Dunn, MD, PhD 
 
Neurosurgery 
Allison King, MD, MPH 
 
Pediatric Hematology/Oncology 
Rachel Langley 
 
 
Pharmacy 
Esther Lu, PhD 
 
 
Biostatistics 
 
Study Drug(s): 
 
Doxorubicin  
Etoposide 
 
Clinical Trials.gov #: 
NCT02372409 
 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to 
obtain informed consent, may not be disclosed to another party unless law or regulations require such 
disclosure.  Persons to whom the information is disclosed must be informed that the information is 
confidential and may not be further disclosed by them 
 
 


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A Pilot Study of Using MRI-Guided Laser Heat Ablation in to Induce Disruption of the 
Peritumoral Blood Brain Barrier to Enhance Delivery and Efficacy of Treatment of 
Pediatric Brain Tumors 
 
Protocol Revision History 
 
Initial Approval Version 
 
 
 
 
 
 
02/03/15 
Amendment #1 Version 
 
 
 
 
 
 
06/05/15 
Amendment #2 Version 
 
 
 
 
 
 
08/09/17 
Amendment #3 Version 
 
 
 
 
 
 
04/19/18 
Amendment #4 Version 
 
 
 
 
 
 
08/11/20 
 
 
 


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A Pilot Study of Using MRI-Guided Laser Heat Ablation in to Induce Disruption of the 
Peritumoral Blood Brain Barrier to Enhance Delivery and Efficacy of Treatment of 
Pediatric Brain Tumors 
 
SCHEMA 
 
Arm A 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Arm B 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment Plan 
MLA followed by standard of care therapy 
Eligible Patients 
Patients with presumed glioma on MRI who are candidates for MLA 
therapy 
Treatment Plan 
MLA followed by induction therapy with 
doxorubicin and maintenance etoposide 
Eligible Patients 
Patients with relapsed malignant primary CNS tumor, grade III or 
IV, any histologic subtype who are candidates for MLA therapy 


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Glossary of Abbreviations 
 
AE 
Adverse event 
ALT (SGPT) 
Alanine transaminase (serum glutamate pyruvic transaminase) 
ANC 
Absolute neutrophil count 
AST (SGOT) 
Aspartate transaminase (serum glutamic oxaloacetic transaminase) 
BBB 
Blood brain barrier 
B-HCG 
Beta human chorionic gonadotropin  
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CNS 
Central nervous system 
CR 
Complete remission 
CRF 
Case report form 
CT 
Computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DNA 
deoxyribonucleic acid 
DSM 
Data and Safety Monitoring  
DSMC 
Data Safety Monitoring Committee 
ECG (or EKG) 
Electrocardiogram 
EDTA 
ethylenediaminetetraacetic acid 
EE 
Efficacy-Evaluable 
EFS 
Event free survival 
FDA 
Food and Drug Administration 
G-CSF 
Granulocyte colony stimulating factor, filgrastim (Neupogen) 
HIV 
Human Immunodeficiency Virus 
HRPO 
Human Research Protection Office (IRB) 
IRB 
Institutional Review Board 
IV 
Intravenous (i.v.) 
MRI 
Magnetic resonance imaging 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
OHRP 
Office of Human Research Protections  
ORR 
Overall response rate 
OS 
Overall survival 
PB 
Peripheral blood 
PBMC 
Peripheral blood mononuclear cell 
PD 
Progressive disease 
PFS 
Progression free survival 
6PFS 
Progression Free Survival at 6 months 
PI 
Principal investigator 


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PR 
Partial response (Partial remission) 
QASMC 
Quality Assurance and Safety Monitoring Committee 
RANO 
Response Assessment in Neuro-Oncology 
RBC 
Red blood cell (count) 
RR 
Response rate 
SAE 
Serious adverse event 
SCC 
Siteman Cancer Center 
SD 
Stable disease 
UPN 
Unique patient number 
WBC 
White blood cell (count) 
 
 
 
 


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Table of Contents 
SCHEMA ........................................................................................................................................ 3 
1.0 
BACKGROUND AND RATIONALE ................................................................................ 8 
1.1 
Pediatric Brain Tumors .................................................................................................... 8 
1.2 
Penetrating the Blood Brain Barrier (BBB) ..................................................................... 8 
1.3 
MRI-guided Laser Ablation (MLA) ............................................................................... 10 
1.4 
MLA followed by Standard of Care in Newly Diagnosed Glioma Patients (Arm A) ... 11 
1.5 
MLA followed by Doxorubicin and Etoposide in Relapsed Malignant Tumor Patients 
(Arm B) ..................................................................................................................................... 12 
1.6 
DCE and DSC-MRI ....................................................................................................... 13 
1.7 
Biomarkers of BBB Disruption ...................................................................................... 15 
1.8 
MLA and Immune Activation ........................................................................................ 15 
1.9 
Study Rationale .............................................................................................................. 16 
2.0 
OBJECTIVES .................................................................................................................... 17 
2.1 
Primary Objectives ......................................................................................................... 17 
2.2 
Secondary Objectives ..................................................................................................... 17 
2.3 
Exploratory Objective .................................................................................................... 17 
3.0 
PATIENT SELECTION .................................................................................................... 18 
3.1 
Inclusion Criteria – Arm A ............................................................................................. 18 
3.2 
Inclusion Criteria – Arm B ............................................................................................. 18 
3.3 
Exclusion Criteria – Arm A ........................................................................................... 19 
3.4 
Exclusion Criteria – Arm B ............................................................................................ 19 
3.5 
Inclusion of Women and Minorities ............................................................................... 20 
4.0 
REGISTRATION PROCEDURES ................................................................................... 20 
4.1 
Confirmation of Patient Eligibility ................................................................................. 20 
4.2 
Patient Registration in the Siteman Cancer Center Database ........................................ 21 
4.3 
Assignment of UPN ....................................................................................................... 21 
5.0 
TREATMENT PLAN ........................................................................................................ 21 
5.1 
Study Summary .............................................................................................................. 21 
5.2 
MRI-Guided Laser Ablation .......................................................................................... 22 
5.3 
Premedication Administration ........................................................................................ 22 
5.4 
Agent Administration ..................................................................................................... 23 
5.5 
Evaluability Criteria ....................................................................................................... 23 
5.6 
General Concomitant Medication and Supportive Care Guidelines .............................. 23 
5.7 
Women of Childbearing Potential .................................................................................. 23 
5.8 
Duration of Therapy ....................................................................................................... 24 
5.9 
Duration of Follow-up .................................................................................................... 24 
6.0 
DOSE DELAYS/DOSE MODIFICATIONS (ARM B) .................................................... 25 
7.0 
REGULATORY AND REPORTING REQUIREMENTS ............................................... 26 
7.1 
Adverse Events (AEs) .................................................................................................... 26 
7.2 
Unanticipated Problems ................................................................................................. 27 
7.3 
Noncompliance............................................................................................................... 27 
7.4 
Serious Noncompliance .................................................................................................. 27 
7.5 
Protocol Exceptions ........................................................................................................ 27 
7.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality Assurance 
and Safety Monitoring Committee (QASMC) at Washington University ................................ 27 


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7.7 
Timeframe for Reporting Required Events .................................................................... 28 
8.0 
PHARMACEUTICAL INFORMATION .......................................................................... 28 
8.1 
Doxorubicin (Adriamycin) ............................................................................................. 28 
8.2 
Etoposide (VP-16) .......................................................................................................... 30 
9.0 
CORRELATIVE STUDIES .............................................................................................. 32 
9.1 
DCE and DSC-MRI ....................................................................................................... 32 
9.2 
Blood for Correlative Studies ......................................................................................... 32 
9.3 
Blood for Serum Biomarkers of BBB Disruption .......................................................... 33 
9.4 
Blood for Phenotyping of Peripheral Blood Immune Cells ........................................... 33 
9.5 
OPTIONAL Biopsy for Tumor-Specific Functional Activity of Peripheral Blood 
Immune Cells ............................................................................................................................ 33 
9.6 
Quality of Life Assessments .......................................................................................... 34 
10.0 STUDY CALENDAR ....................................................................................................... 35 
10.1 Arm A Study Calendar ................................................................................................... 35 
10.2 Arm B Study Calendar ................................................................................................... 36 
11.0 DATA SUBMISSION SCHEDULE ................................................................................. 37 
12.0 MEASUREMENT OF EFFECT........................................................................................ 37 
12.1 Antitumor Effect – Solid Tumors ................................................................................... 37 
12.2 Disease Parameters ......................................................................................................... 40 
12.3 Methods for Evaluation of Measurable Disease ............................................................ 41 
13.0 DATA AND SAFETY MONITORING ............................................................................ 43 
14.0 STATISTICAL CONSIDERATIONS............................................................................... 44 
15.0 REFERENCES .................................................................................................................. 46 
APPENDIX 1: Karnofsky and Lansky Performance Status Scale ............................................... 50 
APPENDIX 2: PATIENT’S MEDICATION DIARY ................................................................. 51 
 
 


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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
Pediatric Brain Tumors 
 
Pediatric brain tumors comprise a heterogeneous group of tumors, including gliomas (high 
and low grade), medulloblastoma, ependymoma, primitive neuroectodermal tumor 
(PNET), atypical teratoid/rhabdoid tumor (ATRT), choroid plexus carcinoma, as well as 
other rarer tumors.  Gliomas are the most common tumors in pediatrics, especially grade I 
(pilocytic astrocytoma) and grade II. High grade gliomas include anaplastic astrocytoma, 
glioblastoma (GBM), and mixed oligoastrocytomas. As with adults, high grade gliomas in 
pediatrics confer a poor prognosis. The current standard chemoradiotherapy for newly 
diagnosed high grade glioma produces only a modest survival benefit in pediatric patients, 
with less than 20% alive at five years [1]. All recurrent pediatric malignant tumors have a 
very poor prognosis with no standard and few effective therapeutic options available. Most 
children with relapsed disease will succumb within 3 years of recurrence with any 
malignant tumors. One of the challenges in treating brain tumors is the limitation of many 
agents to penetrate the blood brain barrier (BBB).  Several methods to overcome BBB 
disruption have been attempted and currently undergoing studies, including intra-arterial 
chemotherapy administration, direct intratumoral/intracavitary placement of chemotherapy 
agents, and convection-enhanced delivery of agents, although a method with clear survival 
benefit has yet to be demonstrated [2]. 
 
1.2 
Penetrating the Blood Brain Barrier (BBB) 
 
Despite the fact that several cytotoxic and targeted agents have shown significant anti-
growth activity in cultured cells, when tested in clinical trials there has been with minimal 
success, particularly in high grade glioma patients [3, 4]. In one study of pediatric high 
grade glioma using primary tumor cell cultures, it was shown that several classic 
chemotherapy agents, including anthracyclines, have a high cytotoxicity in vitro [4]. The 
high failure rate is in part due to the redundancy in key growth pathways and in drug 
resistance of malignant cells. Another explanation is the poor CNS penetration many of 
these drugs have due to the blood brain barrier (BBB). As a result, high doses of drugs 
were used in studies to achieve therapeutic drug concentrations in the CNS, which led to 
significant systemic toxicities and limited their clinical usefulness [5]. Preclinical models 
have shown that the delivery across the blood brain barrier depends on the size and 
molecular weight of the compound, even after blood brain disruption by mannitol [6].  
Thus, an outstanding challenge in neuro-oncology has been to generate drugs that have 
excellent CNS penetration or methods that can compromise the BBB to enhance drug 
delivery.  
 
The impact of the BBB on primary brain tumors has been most studied in GBM. GBM 
typically appear on MR imaging as rim-enhancing masses, suggesting that the BBB within 
the growth-intensive rim is impaired because contrast enhancement reflects increased BBB 
permeability [7, 8]. Consequently, delivery of cytotoxic agents to the tumor rim is higher 
than that to normal brain tissue. However, beyond the enhancing rim – the peritumoral 


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region – where most micrometastatic GBM cells reside, the BBB remains relatively intact 
as demonstrated by the lack of contrast enhancement. As a result, access of cytotoxic drugs 
to this region is predicted to be more limited. Similar findings have also been observed in 
brain metastases arising from extracranial tumors. In many cancers, responses of brain 
metastases to systemic chemotherapy tend to closely parallel those of extra-cranial tumors 
[9-14]. Yet brain metastasis still represents a significantly poorer prognostic indicator than 
extracranial metastasis. This is likely because of the inherent nature of brain metastatic foci 
compromising critical neurological functions. Another possible explanation for this 
apparent paradox is that although cytotoxic agents can readily access brain metastases 
(BBB disrupted), they fail to reach therapeutic levels in the peritumoral or distant foci of 
brain micrometastases (BBB intact). Evidence supporting this theory came from studies in 
which drug levels of several cytotoxic agents were sampled in tumors and the surrounding 
normal brain tissue at the time of surgery or autopsy. Drug concentrations were at the 
highest in the tumors, and then rapidly decreased up to 40 fold lower within 2 cm distance 
from the viable tumor edge [15-17]. Evidence also suggests that efflux transporters in the 
endothelial cells of the BBB, such as multi drug resistance gene (MDR1) and multi drug 
resistance-related protein gene (MRP1), prevent agents from crossing the BBB [4, 18], and 
expression of these proteins negatively correlates with achieved concentration of 
chemotherapy within tumor cells [18]. Overall, these results support two notions: 1) the 
BBB and its integrity negatively correlate with delivery and therapeutic effect of cytotoxic 
agents; and 2) if we can disrupt the BBB in the peritumoral region, we could improve 
cytotoxic chemotherapy delivery in this area. In GBM, most recurrent tumors arise within 
the peritumoral region – in one series, 90% of GBM relapses occurred within 2-3 cm 
margin of the primary site [19] - therefore elimination of micrometastatic GBM cells in 
this area will likely improve long-term disease control. 
 
To circumvent the BBB problem in local drug delivery, recent approaches have focused on 
bypassing it.  A common method is the use of Gliadel wafers, a polymer implant 
impregnated with the chemotherapeutic agent BCNU and placed intraoperatively in the 
resection cavity to evade the BBB.  This approach resulted in a statistically significant but 
modest survival advantage in both newly diagnosed and recurrent GBM [20-22].  The 
modest benefit of Gliadel could be due to the short duration of drug delivery – most BCNU 
is released over a period of 5 days[23]. However, the fact that direct delivery of a cytotoxic 
drug into the resection cavity for as little as 5 days could improve survival of GBM patients 
to a degree approaching that achieved by 8 months of systemic temozolomide 
chemotherapy is remarkable in itself, supporting the theory that the BBB is critical to 
cytotoxic chemotherapy effect.  Unfortunately, Gliadel is not widely utilized as it requires 
a major surgery and can impair wound healing.  Another approach of bypassing the BBB 
is the convection enhanced delivery system, in which a catheter is surgically inserted into 
the tumor to deliver chemotherapy [24]. This invasive procedure requires prolonged 
hospitalization, meticulous maintenance of the external catheter to prevent serious 
complications, and as a result remains investigational and is rarely used. 
 
 


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1.3 
MRI-guided Laser Ablation (MLA) 
 
MLA 
is 
a 
minimally 
invasive 
laser 
surgery 
currently FDA approved 
for cytoreductive treatment 
of 
brain 
tumors, 
both 
primary 
and 
metastatic 
[25].  Dr. Eric Leuthardt at 
Washington 
University 
performed one of the first 
MLA procedures in the 
country 
and 
is 
an 
international expert in laser 
ablation treatment of brain 
tumors. 
 
Currently 
Washington University has 
one of the largest clinical 
experiences using this new 
technology 
(Monteris, 
Winnipeg, CA).  MLA 
employs a small incision in 
the scalp and skull, through 
which a thin laser probe is 
inserted and guided by MR 
imaging to the core of a 
tumor 
mass 
where 
it 
delivers 
hyperthermic 
ablation from the core to the 
rim. 
The 
maximal 
temperature in the core can 
reach greater than 70oC 
resulting in coagulative necrosis (Fig. 1). The temperature decreases in the peritumoral 
region but remains high enough (>40oC) to induce changes in the BBB as evidenced by 
new peritumoral contrast enhancement extending several centimeters from the tumor edge, 
while the original tumor enhancement is lost due to the heat ablation (Figs. 1 & 2). These 
observations suggest that an interesting side effect of MLA is the disruption of the 
peritumoral BBB. These changes often persist for several weeks (not shown), providing a 
rare window of opportunity during which drug delivery can be enhanced to eliminate 
infiltrative tumor cells residing in this region where most recurrences occur. 
 
Whether MLA indeed causes BBB disruption remains unexplored and is the main focus of 
this study. Interestingly, the role of hyperthermia in inducing increased BBB permeability 
has been previously described in several animal models.  In a rodent model of human 
glioma, the global heating of a mouse’s head to 42oC for 30 minutes in a warm water bath 


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significantly increased the 
maximal brain concentration 
of a thermosensitive liposome 
encapsulated 
with 
the 
chemotherapeutic 
drug 
Adriamycin [26]. To effect 
locoregional hyperthermia in 
the brain, retrograde infusion 
of a hyperthermic saline 
solution at 43oC into the left 
external carotid artery in the 
Wistar rat model reversibly 
increased BBB permeability 
to Evans-blue albumin in the 
left cerebral hemisphere[27]. 
In the most analogous method 
to the MLA, Nd:YAG laser-
induced thermo-therapy to 
the left forebrain of Fischer 
rats resulted in locoregional disruption of the BBB as evidenced by increased locoregional 
passage of the Evans blue dye, serum proteins (e.g. fibrinogen and IgM), and the cytotoxic 
drug paclitaxel [28]. Based on these results, we hypothesize that hyperthermia-induced 
disruption of the peritumoral BBB by MLA (Fig. 2) represents a potentially powerful tool 
to enhance delivery of chemotherapy to this region to effectively target residual disease in 
addition to maximal cytoreduction of the tumor.  
 
1.3.1 Prior Clinical Experience with MLA 
 
We have had one of the largest clinical experiences using MLA in the country.  To 
date we have done more than 80 cases, of which approximately two-thirds are high-
grade glial neoplasms. In regards to adverse events, patients have experienced 
transient aphasia, transient hemiparesis, transient hyponatremia, and a documented 
case of lower extremity deep venous thrombosis. Patients with aphasia or 
hemiparesis improved over time with steroid therapy. Hyponatremia resolved 
spontaneously. One patient experienced fatal meningitis. Subsequent in-depth 
analysis revealed that this patient’s meningitis was due to an operating room 
infrastructure-related contamination and not due to equipment contamination 
related to performance of the MLA procedure. 
 
1.4 
MLA followed by Standard of Care in Newly Diagnosed Glioma Patients 
(Arm A) 
 
MLA offers an alternative to some patients with tumors that are unresectable. Complete 
surgical resection of grade I and II gliomas have a >90% overall survival, although 
complete resection is not always possible due to the location of the tumor. For tumors with 
less than a complete resection, chemotherapy or radiation therapy is offered for progressive 


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and/or symptomatic tumors. For high grade gliomas, patients who have a complete 
resection of the enhancing tumor at time of diagnosis [29, 30] or at relapse have a longer 
survival compared to those with residual disease [31]. In this arm of the study, patients 
with any grade glioma who are candidates for MLA will undergo this procedure followed 
by standard of care treatment.   
 
1.5 
MLA followed by Doxorubicin and Etoposide in Relapsed Malignant Tumor 
Patients (Arm B) 
 
The ideal cytotoxic drugs for the purpose of this proposal should have potent activities 
against malignant cell lines in vitro, and in vivo activity that is limited by blood brain barrier 
function. Doxorubicin has been shown to kill a large number of high grade glioma cell 
lines in vitro [18, 32]. However, it has poor CNS penetration and has not been used 
extensively in CNS tumors. Doxorubicin and other anthracyclines induce cytotoxicity 
through intercalating between DNA base pairs, thereby interfering with strand elongation 
by DNA and RNA polymerase. Doxorubicin also affects topoisomerase II, which creates 
temporary double-strand DNA breaks during DNA replication. Doxorubicin stabilizes the 
DNA-topoII complex leading to double-strand DNA breaks and cell death.  Doxorubicin 
has a wide volume of distribution with tissue levels proportional to the DNA content of the 
tissue. Doxorubicin is 75% bound to plasma proteins. Doxorubicin is mostly metabolized 
in the liver and eliminated mainly as glucuronide or hydroxylated conjugates in the bile 
and feces. The half-life of doxorubicin is 1 to 3 hours.  
 
Maintenance chemotherapy using low-dose etoposide (21-day cycles) will be given 
following the course of doxorubicin.  Low-dose etoposide has been found effective in 
treating relapsed brain tumor patients by extending PFS [33-36]. Etoposide is a 
topoisomerase II inhibitor and has been shown to inhibit tumor cell proliferation at high 
doses and to function as an anti-angiogenic agent when administered in low daily doses 
(metronomic chemotherapy) [37].  In vitro and pre-clinical studies demonstrate that low 
dose daily etoposide inhibits secretion of angiogenic factors, proliferation of endothelial 
cells, and endothelial tube formation [37]. This agent has been well tolerated in pediatric 
patients when given as low-dose, metronomic therapy. 
 
In this arm, we are testing the concept that increased delivery of cytotoxic chemotherapy 
to the peritumoral region after MLA will result in increased peritumoral disease control. 
Although this approach appears to be similar to the dose escalation method, one clear 
difference is that in the dose escalation method, increasing systemic doses of drugs are 
used to achieve adequate drug concentrations in the CNS at the expense of significant 
systemic and global CNS toxicities, especially at doses near or exceeding the maximal 
tolerated dose. Therefore, the lack of benefits at high systemic doses may in part be due to 
excessive toxicities. On the other hand, in the MLA-enhanced drug delivery system, lower 
doses of drugs will be given more frequently to limit systemic toxicities and to selectively 
concentrate drugs in the peritumoral region where therapeutic action is desired, thus also 
reducing CNS toxicities.  
 
In pediatric malignancies, doxorubicin is well tolerated and is given in combination with 


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other cytotoxic drugs. In high risk acute lymphoblastic leukemia, doxorubicin is given at 
25 mg/m2 IV weekly for 4 weeks in combination with vincristine. In Ewing’s sarcoma, 
doxorubicin is given at 75 mg/m2 IV in combination with vincristine and 
cyclophosphamide, alternating with ifosfamide and etoposide every 2 weeks. Similar 
dosing and schedules are given in other solid tumors, such as neuroblastoma and 
osteosarcoma. In ATRT, doxorubicin at 60 mg/m2 (over 2 days) 2-3 weeks apart in 
combination with multiple other cytoxic agents [38]. We expect doxorubicin 25 
mg/m2/dose weekly for 6 weeks to be well tolerated. This is to minimize systemic toxicity 
while still achieving adequate drug delivery in the peritumoral region if the BBB is 
disrupted and to give continuous treatment during the window of MLA-induced 
peritumoral BBB disruption (6 weeks). 
    
1.6 
DCE and DSC-MRI 
 
The measurement of perfusion in the brain using MRI is now commonplace and traces its 
origin to the seminal paper by Ostergaard et al.[39]. This technique is now referred to as 
dynamic susceptibility contrast (DSC) and relies on measuring the T2* signal changes that 
occur in the brain as a bolus of contrast material dynamically passes through the capillary 
circulation. DSC methods have since proved valuable in the diagnosis of stroke and brain 
tumors and are routinely used in our radiology practice. The mathematical model that 
describes DSC assumes that the blood brain barrier (BBB) is intact, but this assumption 
does not often hold in enhancing high-grade glial tumors, which are often supplied by leaky 
neo-vascularity.  The leakage of contrast into the extracellular space causes changes in the 
T1 signal [40], which can invalidate uncorrected DSC measurements in tumors. Several 
methods, such as DCE, have been proposed to correct for this leakage [40, 41], some of 
which rely on a 2-compartment pharmacokinetic model that can estimate the vascular 
transfer constant (Ktrans)[41]. Ktrans is a parameter that describes the ability of contrast 
to move from the intravascular compartment to the extracellular compartment (the 2 
compartments in the model) and thus provides a quantitative measure of the degree of BBB 
leakage. Law et al. used this model to measure Ktrans and cerebral blood volume (CBV) 
in 74 patients with glial tumors and demonstrated that the combination of both measures 
Figure 3. Perfusion measurements in patients with GBM. See text for details. 


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provided 
the 
best 
discriminator 
for high-grade 
gliomas, 
thus 
providing 
validation 
for 
this model[42]. 
 
We 
implemented a 
modified 
version of the 
Johnson 
model[41] and 
applied it to 
several patients 
with high-grade 
gliomas 
as 
illustrated 
in 
Figure 3. Figure 
3A is a T1-
weighted post contrast image of the tumor, seen as a ring enhancing mass on the left side 
of the brain (right side of the image). Figure 3B is the same slice in a T2*-weighted image 
that is used to measure the signal change with the passage of a bolus of contrast material. 
Several regions of interest (ROI) are drawn on this image where measurements were made. 
The red ROI was placed in an area of relatively large BBB leakage, the blue ROI in an area 
of less leakage, and the green ROI in normal white matter. Figure 3C demonstrates the 
normalized tissue signal curves during the bolus tracking period that correspond to the 
drawn ROIs. The large signal drop seen centered at time frame 15 is taken at the peak of 
the contrast bolus transition. The depth and recovery of the signal during and after the bolus 
passage provides information on the degree of BBB leakage and on the value of Ktrans. In 
the normal white matter (green) the signal nearly recovers fully to its original value, 
consistent with the residual contrast left in the blood stream after the passage of the primary 
bolus. In the enhancing portion of the tumor (red and blue) the signal has a larger drop and 
does not fully recover providing us with estimates of the Ktrans.  
 
As part of the Monteris study on the adult tumor population we have employed another 
method to measure the break down of the BBB called dynamic contrast-enhanced MRI 
(DCE-MRI). DCE-MRI is another common method used to evaluate for permeability of 
blood vessels in the brain[8]. Similar to DSC-MRI, DCE-MRI consists of injection of an 
MRI contrast agent followed by multiple T1-weighted images to assess the leakage of this 
agent into the extracellular space over several minutes. Again, the rate of pooling of the 
contrast agent is then used to quantify the degree of BBB permeability. Figure 4 
demonstrates results from our recent study in 4 sample patients. The horizontal axis 
represents the number of days post-surgery with each time point representing an MRI 
Figure 4: Examples of longitudinal Ktrans measurements in tumor patients follow the 
monteris procedure using the DCE method. More detail provided in the text 


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measurement. The vertical axis represents the Ktrans around the tumor, a measure of BBB 
breakdown. In 3 out of the 4 cases represented there is evidence of BBB breakdown seen 
as an elevated Ktrans in the first 2 weeks following surgery. In the case of monteris07 there 
was a smaller shift in the BBB that lasted for a longer period of time. The unusual marked 
increase in Ktrans seen at the later time points in monteris09 was due to tumor recurrence.   
 
We will employ both methods (DSC and DCE) to determine the degree, extent, and 
duration of BBB leakage in the peritumoral region after MLA procedure. This will generate 
a dynamic and temporal map of BBB disruption in the area surrounding the post MLA 
tumor. Our long-term goal is to correlate these maps with patients’ treatment outcome, 
when we have a larger database of treated patients, to determine whether patients with 
larger degree and extent of BBB leakage also derive more benefit from early 
chemotherapy. 
 
1.7 
Biomarkers of BBB Disruption 
 
The coagulative necrosis and BBB disruption induced by MLA share several parallels with 
classical brain injuries (e.g. traumatic, surgical, ischemic, or pathologic brain damage), 
albeit in a more controlled setting. Several serum biomarkers have been identified, and in 
some cases extensively validated, in large number of patients with various forms of brain 
injuries. The compromised BBB after brain injuries allows CNS-specific factors released 
by damaged CNS cells to escape into the peripheral circulation where they can be detected 
using highly sensitive and specific detection techniques such as ultrasensitive ELISA, 
antibody arrays and HPLC/mass spectroscopy. The temporal profile of serum levels of 
these brain-specific factors (e.g. S100B, GFAP, brain-specific enolase or BSE) can provide 
information about the duration and degree of BBB disruption[43-53], irrespective of the 
type of brain injuries.  
 
We will measure serum levels of these 4 brain-specific factors (S100B, GFAP, and BSE) 
immediately before and after MLA and then during the 6 weeks post-MLA, using 
ultrasensitive ELISA. If MLA results in sustained BBB disruption, we expect that the level 
of at least one of these biomarkers will increase precipitously soon after MLA and well 
above that caused by recurrent GBM, and that the increase will persist for several days to 
weeks. Antibody microarray can substitute ELISA if higher detection sensitivity is needed. 
We chose these biomarkers as they have previously been validated in brain trauma, 
recognizing that other brain specific factors may be more reliable.  
 
1.8 
MLA and Immune Activation 
 
One of the most striking findings from the Washington University experience with MLA 
is the delayed timing, degree, and persistence of peritumoral enhancement following 
treatment. Specifically, strong enhancement is observed several days after treatment and 
persists beyond 6 weeks. We hypothesize that these imaging findings are due to persistent 
disruption of the BBB, which is compounded and maintained by an enhanced immune 
infiltrate in the peritumoral area. Clinically, we have observed improved patient outcomes 
in our adult retrospective MLA series (Table 1) and hypothesize that this is in part due to 


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an MLA-induced anti-tumor immune response. CNS immunosurveillance is likely distinct 
when compared to other tissues with clearly defined secondary lymphoid structures[54]. 
However, CNS antigen presentation is thought to occur when antigens drain to the 
ipsilateral cervical lymph node chain[54]. Thus, it is possible that MLA disrupts the BBB 
such that tumor antigens, either native or heat denatured, have greater access to cervical 
draining lymphoid tissue, thereby stimulating an anti-glioma immune response that (a) 
prolongs patient survival and (b) leads to persistent contrast enhancement characteristic of 
this treatment.  The potential ability of MLA to augment anti-glioma immune responses is 
particularly compelling because patients with glioma are known to present with a broad 
range of immunological dysfunction [55].  
 
Table 1 
MLA Experience in Patients with GBM at 
Siteman Cancer Center in the Past 12 Months 
Age at MLA 
Diagnosis 
Date of 
MLA 
Chemotherapy  
(<3 wks after MLA) 
PFS 
(months) 
Alive 
1  73 
GBM 
11-4-11 
Yes 
12 
Yes 
2  64 
GBM 
12-27-11 
Yes 
10 
Yes 
3  34 
High grade, 
likely GBM 
01-03-12 
Yes 
Not yet 
progressed 
Yes 
4  46 
High grade, 
likely GBM 
01-16-12 
Yes 
11 
Yes 
5  72 
GBM 
02-10-12 
Yes 
Not yet 
progressed 
Yes 
6  68 
GBM 
07-06-12 
Yes 
Not yet 
progressed 
Yes 
 
1.9 
Study Rationale 
 
By employing a combination of advanced MRI techniques and correlative serum 
biomarkers of BBB disruption, we plan to develop a powerful, first of its kind clinical 
algorithm in pediatrics whereby we can measure and identify the window of maximal BBB 
disruption post MLA to 1) allow for an alternative to surgery in incompletely resected 
tumors, 2) allow for optimal chemotherapeutic dosing to achieve the greatest benefits and 
the least systemic side effects and 3) distinguish subsequent tumor progression from long-
term MLA treatment effects. Preliminary data in adult imaging studies have shown that the 
BBB disruption lasts for several weeks following treatment before returning to a low 
baseline. This pilot therapeutic study will provide preliminary validation in pediatric 
patients. 
 
Although we have a large armamentarium of cytotoxic drugs with potential activity against 
brain tumors, the vast majority has poor CNS penetration limiting their usefulness. Our 
proposed use of MLA to achieve both cytoreduction and increased permeability of the 
peritumoral BBB has the potential to be practice changing and will allow us to test many 
drugs that have not shown promise due to poor BBB penetration. The innovative algorithm 


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to detect and measure MLA-induced peritumoral BBB compromise may also be applied 
using other treatments. 
 
Recent work has collectively demonstrated striking immune dysregulation in patients with 
GBM, including T cell lymphopenia and anergy, cytokine dysregulation, and increased 
regulatory T cell (Treg) populations among others, which reflect immunologic compromise 
and functional impairment[54-57]. However, a growing list of potential tumor antigens has 
been identified, suggesting that tumor-specific recognition by immune cells may be 
biologically relevant and therapeutically exploitable[55, 58]. Therefore, given the immune 
dysfunction characterized in GBM patients, approaches that potentiate the anti-glioma 
immune response are particularly exciting. A priori, because the immune system has 
evolved to recognize a tremendous diversity of antigenic epitopes in vertebrates, immune-
potentiating efforts – with MLA being one potential approach – may be especially effective 
at targeting the heterogeneity that defines GBM. 
 
 
2.0 
OBJECTIVES 
 
2.1 
Primary Objectives 
 
1. To determine progression-free survival (PFS) and overall survival (OS) of patients 
with new diagnosis glioma who are candidates for MLA (Arm A).  
 
2. To determine 6-month progression-free survival (6PFS) in patients who receive 
doxorubicin immediately following MLA and low-dose etoposide as maintenance 
therapy in patients who are candidates for MLA with any type relapsed malignant 
brain tumors (Arm B).  
 
3. To evaluate quality of life (QOL) using Karnofsky or Lansky performance status in 
patients following MLA and in patients who receive doxorubicin and maintenance 
etoposide after MLA. 
 
2.2 
Secondary Objectives 
 
1. To determine MR imaging correlates of peritumoral BBB disruption after MLA. 
 
2. To identify serum biomarkers of peritumoral BBB disruption after MLA, which can 
be used to establish peritumoral permeability scores. 
 
3. To determine the predictive value of the peritumoral permeability score for patient 
outcome as measured by 6PFS rate. 
 
2.3 
Exploratory Objective 
 
To determine the effects of MLA treatment on patient’s tumor-specific immune responses 


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and immunological correlates.   
 
 
3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria – Arm A 
 
1. Presumed pediatric gliomas (grades I-IV) on MRI that are determined to be candidates 
for MLA by the treating neurosurgeon.  
 
2. Age 3 to ≤ 21  
 
3. Karnofsky/Lansky performance status ≥ 60%, see Appendix 1. 
 
3.2 
Inclusion Criteria – Arm B 
 
1. Recurrent pediatric brain tumors determined candidates for MLA as determined by the 
treating neurosurgeon. 
 
2. Unequivocal evidence of tumor progression by MRI (see Section 12). 
 
3. There must be an interval of at least 12 weeks from the completion of radiotherapy to 
study registration except if there is unequivocal evidence for tumor recurrence per 
RANO criteria (see Section 12). When the interval is less than 12 weeks from the 
completion of radiotherapy, the use of PET scan is allowed to differentiate between 
evidence of tumor recurrence and pseudoprogression. 
 
4. Recurrent lesions with dimension and contour that are determined by the treating 
neurosurgeon to be appropriate for MLA.   
 
5. Age 3 to ≤ 21  
 
6. Karnofsky/Lansky performance status ≥ 60%, see Appendix 1. 
 
7. Adequate cardiac function as determined by a shortening fraction ≥ 27% or left 
ventricular ejection fraction ≥ 50% by echocardiogram within the past 1 year prior to 
registration. 
 
8. Prior anthracycline therapy does not exceed 200 mg/m2 total cumulative dose. 
 
9. Adequate bone marrow and hepatic function as defined below (must be within 7 days 
of MLA): 
a. Absolute neutrophil count (ANC) > 1000/mcl (G-CSF is allowed) 
b. Platelets ≥ 100 K/cumm 
c. Hemoglobin ≥ 9 g/dL (pRBC transfusion +/- ESA are allowed) 
d. ALT ≤ 3 x ULN 


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e. AST ≤ 3 x ULN 
f. ALP ≤ 3 x ULN.  If ALP is > 3 x ULN, GGT must be checked and be ≤ 3 x ULN. 
g. Bilirubin ≤ 2 x ULN 
 
10. At the time of registration, patient must have recovered from the toxic effects of prior 
therapy to no more than grade 1 toxicity. 
 
11. At the time of registration, patient must be at least 4 weeks from other prior cytotoxic 
chemotherapy. 
 
12. Women of childbearing potential and men must agree to use adequate contraception 
(hormonal or barrier method of birth control, abstinence) prior to study entry and for 
the duration of study participation.  Should a woman become pregnant or suspect she 
is pregnant while participating in this study, she must inform her treating physician 
immediately. 
 
13. Ability to understand and willingness to sign an IRB approved written informed 
consent document (or that of legally authorized representative, if applicable). 
 
3.3 
Exclusion Criteria – Arm A 
 
1. Currently receiving or scheduled to receive any other therapies intended to treat the 
newly diagnosed glioma prior to MLA and the first post-MLA blood collection for 
correlative studies. 
 
2. Multi-focal or metastatic disease. 
 
3. Pregnant and/or breastfeeding.  Premenopausal women must have a negative serum or 
urine pregnancy test within 14 days of study entry. 
 
4. Inability to undergo MRI due to personal or medical reasons. 
 
5. Known history of HIV or autoimmune diseases requiring immunosuppressant drugs. 
 
3.4 
Exclusion Criteria – Arm B 
 
1. Prior treatment with bevacizumab within 12 weeks of study entry. 
 
2. Previous treatment with complete cumulative doses of daunorubicin, idarubicin, and/or 
other anthracyclines and anthracenediones that is equivalent to a total dose of > 200 
mg/m2 doxorubicin. 
 
3. More than 2 prior relapses (not counting the current relapse being treated on this study). 
 
4. Currently receiving any other investigational agents that are intended as treatments of 
the relapsed tumor. 


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5. Multi-focal or metastatic disease. 
 
6. A history of allergic reactions attributed to compounds of similar chemical or biologic 
composition to doxorubicin or other agents used in the study. 
 
7. Uncontrolled intercurrent illness including, but not limited to, ongoing or active 
infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, recent heart attack within the previous 12 months or severe heart problems, 
or psychiatric illness/social situations that would limit compliance with study 
requirements. 
 
8. Pregnant and/or breastfeeding.  Premenopausal women must have a negative serum or 
urine pregnancy test within 14 days of study entry. 
 
9. Inability to undergo MRI due to personal or medical reasons. 
 
10. Known history of HIV or autoimmune diseases requiring immunosuppressant drugs. 
 
3.5 
Inclusion of Women and Minorities 
 
Males and females and members of all races and ethnic groups are eligible for this trial. 
 
 
4.0 
REGISTRATION PROCEDURES  
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center OnCore database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility by collecting the information listed below: 
 
1. Registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 


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4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
All patients must be registered through the Siteman Cancer Center OnCore database.  
Registration in the SCC database will be the last step of the patient registration process.  
Once the patient has been registered in the SCC database, s/he will be considered registered 
to the study. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  All data 
will be recorded with this identification number on the appropriate CRFs. 
 
 
5.0 
TREATMENT PLAN 
 
5.1 
Study Summary 
 
5.1.1 Arm A 
 
Six patients enrolled to this study will be in Arm A; patients may be enrolled to 
Arm A at any time throughout the lifetime of the study. Arm A patients will 
undergo MLA and will be followed with standard of care therapy at the discretion 
of the treating physician. 
 
Arm A patients will undergo DSC and DCE-MRI at the following time points along 
with standard of care imaging: 
 no more than 3 weeks prior to MLA (OPTIONAL) 
 within approximately 4 days after MLA 
 2-4 weeks after MLA 
 Every 12 weeks (+/- 7 days) for the first year or until disease progression 
(see Section 9.1)  
 
Arm A patients will have 7 ml of blood drawn at the following time points: 
 Before MLA; this sample can be collected any time during the 3 days before 
or the day of the procedure until the start of the procedure 
 Within approximately 3 days after MLA 
 2-4 weeks (+/- 3 days) after MLA (at time of DSC/DCE-MRI) 
 Every 12 weeks (+/- 7 days) at time points correlating with MRIs for the 
first year or until disease progression 
 
The purpose of these blood draws is to measure serum levels of brain-specific 
factors (S100B, GFAP, and BSE) and cytokine levels. PBMC (for the pre- and post-
MLA samples) will also be isolated to determine the phenotypes and functions of 
peripheral blood immune cells following MLA (see Section 9.2-9.4). 
 
5.1.2 Arm B 


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Six patients enrolled to this study will be in Arm B; patients may be enrolled to 
Arm B at any time throughout the lifetime of the study.  Arm B patients will 
undergo MLA and will begin doxorubicin (as described in Section 5.4) within 
approximately 7 days following MLA (range 2-14 days)   
 
Arm B patients will undergo DSC and DCE-MRI at the following time points along 
with standard of care imaging: 
 no more than 3 weeks prior to MLA (OPTIONAL) 
 within approximately 4 days after MLA 
 2-4 weeks  after MLA 
 every 8 weeks (+/- 7 days) until 2 years have elapsed or disease progression 
(see Section 9.1), whichever comes first  
 
Arm B patients will have 7 mL blood drawn at the following time points: 
 before MLA; this sample can be collected any time during the 3 days before 
or the day of the procedure until the start of the procedure 
 within approximately 3 days after MLA 
 weekly (+/- 3 days) before (on the same day as) chemotherapy for the 6 
weeks post-MLA 
 every 8 weeks (+/- 7 days) at time points correlating with MRIs for the first 
2 years or until disease progression, whichever comes first 
 
The purpose of these blood draws is to measure serum levels of brain-specific 
factors (S100B, GFAP, and BSE) and cytokine levels. PBMC (for the pre- and post-
MLA samples) will also be isolated to determine the phenotypes and functions of 
peripheral blood immune cells following MLA (see Section 9.2 -9.4). This is in 
addition to blood samples for routine laboratory prior to chemotherapy and surgery. 
During the weeks of DSC/DCE-MRI, biomarker lab must be drawn on the same 
day as DSC/DCE-MRI (prior to the scan). 
 
5.2 
MRI-Guided Laser Ablation 
 
MLA is a minimally invasive laser surgery currently FDA approved for cytoreductive 
treatment of brain tumors, both primary and metastatic [25].  MLA employs a small 
incision in the scalp and skull, through which a thin laser probe is inserted and guided by 
MR imaging to the core of a tumor mass where it delivers hyperthermic ablation from the 
core to the rim. The maximal temperature in the core can reach greater than 70oC resulting 
in coagulative necrosis. 
 
5.3 
Premedication Administration 
 
Because doxorubicin is emetogenic, prophylactic use of antiemetics will be used. 
 
 


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5.4 
Agent Administration 
 
Arm B only: Doxorubicin will be given intravenously on an outpatient basis weekly for 6 
weeks at a dose of 25 mg/m2 over 5-30 minutes.  Doxorubicin will be held for an ANC of 
<500/mcl or platelets <50 K/cumm. Following the 6 weeks of doxorubicin, etoposide at 50 
mg/m2/day will be given orally for 21 days of each 28-day cycle to begin once ANC ≥ 
1000/mcl and platelets ≥ 100 K/cumm. Etoposide will be held for ANC of < 500/mcl or 
platelets < 50 K/cumm.  Patients may receive up to 24 cycles of etoposide. 
 
5.5 
Evaluability Criteria 
 
All patients are evaluable for toxicity.  Patients are evaluated from first receiving study 
treatment until a 30-day follow up after the conclusion of treatment or death.  Patients on 
Arm A will be followed for a minimum of 6 months or death. 
 
All patients in Arms B are evaluable for efficacy after receiving at least 2 doses of 
doxorubicin. 
 
5.6 
General Concomitant Medication and Supportive Care Guidelines 
 
Subjects in Arm B should not receive medications that may interact with doxorubicin or 
etoposide. Refer to the product labels for details.  Therapies excluded during the conduct 
of this trial include in Arm B include other chemotherapy agents, hormonal therapy for 
cancer, and other tumor-targeted therapies including but not limited to radiotherapy.  
Therapeutic use of hematopoietic colony-stimulating factors is permitted following for 
ANC < 500/mcl and should not be given within 24 hours prior to or following doxorubicin. 
 
5.7 
Women of Childbearing Potential 
 
Women of childbearing potential (defined as women with regular menses, women with 
amenorrhea, women with irregular cycles, women using a contraceptive method that 
precludes withdrawal bleeding, and women who have had a tubal ligation) are required to 
have a negative serum pregnancy test within 14 days prior to MLA.   
 
Female and male patients (along with their female partners) are required to use two forms 
of acceptable contraception, including one barrier method, during participation in the study 
and for 2 months following the last dose of chemotherapy.  
 
If a patient is suspected to be pregnant, chemotherapy should be immediately discontinued.  
In addition a positive urine test must be confirmed by a serum pregnancy test.  If it is 
confirmed that the patient is not pregnant, the patient may resume dosing. 
 
If a female patient or female partner of a male patient becomes pregnant during therapy or 
within 2 months after the last dose of chemotherapy, the investigator must be notified in 
order to facilitate outcome follow-up. 


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5.8 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment on Arm B with 
etoposide may continue for 2 years or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.9 
Duration of Follow-up 
 
Patients in Arm A will be followed every 12 weeks after the 6-week (or 10-week if done) 
post-MLA scan until 1 year have elapsed since patient registration or until death, whichever 
occurs first. Following the first year, patients will continue to be followed every 6 months 
or at event occurrence for 5 years after patient registration or until death, whichever occurs 
first.   Patients in Arm B will be followed every 8 weeks after the 6-week (or 10-week if 
done) post-MLA scan until 2 years have elapsed since patient registration or until 
progression, whichever occurs first.  Following the first two years, patients will continue 
to be followed every 6 months or at event occurrence for 5 years after patient registration 
or until death, whichever occurs first.   Follow-up consists of collection of Karnofsky 
performance status, DSC/DCE-MRI, and blood for biomarkers until disease progression 
(or 2 years of elapsed since patient registration or death, whichever occurs first).  After 
documented disease progression, vital status and subsequent treatment will be obtained 
every 12 weeks for the remainder of the 2 years or until death, whichever occurs first.  
Patients removed from study for unacceptable adverse events that are possibly, probably, 
or definitely related to study procedures will be followed until resolution or stabilization 
of the adverse event.  “Stabilization” is defined as remaining at a consistent CTCAE 
version 4.0 grade of the event for two consecutive assessments. 


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6.0 
DOSE DELAYS/DOSE MODIFICATIONS (ARM B) 
 
The following algorithms are meant as guidelines only; adjustments to chemotherapy may be made 
at the discretion of the treating physician. 
 
Renal Dysfunction: 
CrCl 10 to 50 ml/min: administer 75% of etoposide dose 
CrCl <10ml/min: administer 50% of etoposide dose 
 
Congestive heart failure: 
Given the low dose and short duration of doxorubicin treatment, we do not anticipate significant 
cardiac toxicity. However, if a prolongation of the QTc interval (>0.48 sec), a decrease in ejection 
fraction to <50%, or a decrease in left ventricular shortening fraction to <27%, doxorubicin should 
be omitted one week, any existing electrolyte or micronutrient deficiencies corrected, and the tests 
repeated.  If the abnormalities persist, doxorubicin treatment should be permanently discontinued. 
Missed doses due to cardiac toxicity will not be made up.  
 
Hematologic abnormalities: 
Hold etoposide for ANC <500/mcl or platelet < 50 K/cumm.  May restart when ANC ≥ 1000/mcl 
and platelet > 100 K/cumm.  Etoposide may be restarted at full dose or reduced dose at the 
discretion of the treating physician.  Missed doses will not be made up. 
 
Baseline 
Permissible 
ANC 
Subsequent ANC 
Growth Factor Support, 
Treatment Delay and 
Follow-up Labs 
Dose Reduction of 
Doxorubicin Based on Rates 
of Abnormal Lab Recovery 
 
 
 
ANC ≥ 
1000/mcl 
  
ANC ≥ 1000/mcl 
None 
None 
ANC < 1000/mcl 
but > 500/mcl 
 
 
G-CSF 5 mcg/kg SQ or IV 
qday x up to 5 days. Check 
CBC daily. When ANC ≥ 
1000/mcl, restart 
treatment.  
None if ANC recovers to ≥ 
1000/mcl within 5 doses of G-
CSF. 
Decrease to 20 mg/m2 if ANC 
recovers to ≥ 1000/mcl after 5 
doses of G-CSF. 
Decrease to 15 mg/m2 if ANC 
takes > 14 days to become 
permissible. 
ANC < 500/mcl  
 
G-CSF 5 mcg/kg SQ or IV 
qday x up to 5 days. Check 
CBC daily. When ANC ≥ 
1000/mcl, restart 
treatment. 
Decrease to 20 mg/m2 if ANC 
recovers to ≥ 1000/mcl within 
5 doses of G-CSF. 
Decrease to 15 mg/m2 if ANC 
recovers to ≥ 1000/mcl after 5 
doses of G-CSF. 
Discontinue treatment if ANC 
takes > 21 days to become 
permissible. 


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Baseline 
Permissible 
Platelets  
Subsequent 
Platelet count 
Treatment Delay and 
Follow-up lab§ 
Dose Reduction of 
Doxorubicin Based on Rates 
of Abnormal Lab Recovery 
 
 
 
Platelet ≥ 100 
K/cumm 
  
Plt ≥ 50 K/cumm 
None 
None 
Plt < 50  K/cumm 
but ≥ 10 K/cumm 
 
Hold treatment. Check 
CBC twice a week. When 
plt ≥ 50 K/cumm, restart 
treatment. 
Transfuse as needed to 
keep plt ≥ 20 K/cumm 
Decrease to 20 mg/m2 if plt 
recovers to ≥ 50 K/cumm 
within 7 days. 
Decrease to 15 mg/m2 if plt 
recovers to ≥ 50 K/cumm 
within 21 days. 
Discontinue treatment if plt 
takes > 21 days to recover to ≥ 
50 K/cumm 
Plt ≤ 10 K/cumm 
Hold treatment. Check 
CBC twice a week. 
Transfuse as needed to 
keep plt ≥ 20,000. 
When plt ≥ 100  K/cumm 
without transfusion, restart 
treatment. 
Decrease to 15 mg/m2 if plt 
recovers to ≥ 50 K/cumm 
within 21 days. 
Discontinue treatment if plt 
takes > 21 days to recover to ≥ 
50 K/cumm 
 
§ All follow-up labs for dose modifications are +/- 3 days. 
 
Hyperbilirubinemia: 
Direct Bilirubin: 
2 – 2.99 mg/dL 
Reduce etoposide and doxorubicin dose by 50% 
3 – 4.99 mg/dL 
Reduce etoposide and doxorubicin dose by 75% 
 ≥ 5 mg/dL 
 
Hold etoposide and doxorubicin 
 
 
7.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
7.1 
Adverse Events (AEs) 
 
Definition: any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading: the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website:  http://www.hhs.gov/ohrp/policy/advevntguid.html 
 
 


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7.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (“possibly related” means 
there is a reasonable possibility that the incident, experience, or outcome may have 
been caused by the procedures involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
7.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
7.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 
7.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event.  
 
7.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 


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 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
7.7 
Timeframe for Reporting Required Events  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment for patients in Arm B and for 30 days following the MLA for patients in Arm A.  
For patients in Arm A, adverse events (of any grade) considered possibly, probably, or 
definitely related to the MLA need be reported.  Adverse events that are possibly, probably, 
or definitely related to study procedures will be followed until resolution or stabilization 
of the event.  “Stabilization” is defined as remaining at a consistent CTCAE version 4.0 
grade of the event for two consecutive assessments. For Arm B, all grade 3 and higher 
events regardless of attribution are to be reported. 
 
 
8.0 
PHARMACEUTICAL INFORMATION 
 
8.1 
Doxorubicin (Adriamycin) 
 
8.1.1 Doxorubicin Description 
 
Doxorubicin is a cytotoxic anthracycline antibiotic isolated from cultures of 
Streptomyces 
peucetius 
var. 
caesius. 
 
Doxorubicin 
consists 
of 
a 
naphthacenequinone nucleus linked through a glycosidic bond at ring atom 7 to 
amino sugar, daunosamine.  Chemically, doxorubicin hydrochloride is (8S,10S)-
10-[(3-Amino-2,3,6-tirdeoxy-a-L-lyxo-hexopyranosyl)-oxy]-8-glycoloyl-
7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione 
hydrochloride. 
 
Chemical name: C27H29NO11•HCl 
Molecular weight: 579.99 
 
8.1.2 Clinical Pharmacology 
 
The cytotoxic effect of doxorubicin on malignant cells and its toxic effects on 
various organs are thought to be related to nucleotide base intercalation and cell 
membrane lipid binding activities of doxorubicin.  Intercalation inhibits nucleotide 
replication and action of DNA and RNA polymerases.  The interaction of 
doxorubicin with topoisomerase II to form DNA-cleavable complexes appears to 
be an important mechanism of doxorubicin cytocidal activity. 
  


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8.1.3 Pharmacokinetics and Drug Metabolism 
 
Pharmacokinetic studies, determined in patients with various types of tumors 
undergoing either single or multi-agent therapy, have shown that doxorubicin 
follows a mulitphasic disposition after intravenous injection.  The initial 
distributive half-life of approximately 5 minutes suggests rapid tissue uptake of 
doxorubicin, while its slow elimination from tissues is reflected by a terminal half-
life of 20 to 48 hours. 
 
8.1.4 Supplier(s) 
 
Doxorubicin is commercially available. 
 
8.1.5 Dosage Form and Preparation 
 
Doxorubicin for Injection is supplied as a sterile red-orange lyophilized powder in 
single dose flip-top vials in the following package strengths: 10 mg vial, 20 mg 
vial, 50 mg vial. 
 
8.1.6 Storage and Stability 
 
Store unreconstituted vial at 20º to 25º C.  Retain in carton until time of use.  
Discard unused portion. 
 
After adding the diluent, the vial should be shaken and the contents allowed to 
dissolve.  The reconstituted solution is stable for 7 days at room temperature and 
under normal room light and 15 days under refrigeration (2º to 8º C).  It should be 
protected from exposure to sunlight. 
 
8.1.7 Administration 
 
When possible, to reduce the risk of developing cardiotoxicity in patients receiving 
doxorubicin after stopping treatment with other cardiotoxic agents, especially those 
with long half-lives such as trastuzumab, doxorubicin-based therapy should be 
delayed until the other agents have cleared from the circulation. 
 
Care in the administration of doxorubicin will reduce the chance of perivenous 
infiltration.  It may also decrease the chance of local reactions such as urticaria and 
erythematous streaking.  On intravenous administration of doxorubicin, 
extravasation may occur with or without an accompanying burning or stinging 
sensation, even if blood returns well on aspiration of the infusion needle.  If any 
signs or symptoms of extravasation have occurred, the injection or infusion should 
be immediately terminated and restarted in another vein. 
 
Doxorubicin will be administered during this study at a dose of 25 mg/m2 weekly 
for 6 weeks. 


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8.1.8 Special Handling Instructions 
 
Caregivers should be counseled to take precautions (such as wearing latex gloves) 
to prevent contact with the patient’s urine and other body fluids for at least 5 days 
after each treatment. 
 
8.2 
Etoposide (VP-16) 
 
8.2.1 Clinical Pharmacology 
 
A semisynthetic derivative of podophyllotoxin that forms a complex with 
topoisomerase II and DNA which results in single and double strand DNA breaks. 
Its main effect appears to be in the S and G2 phase of the cell cycle. 
 
8.2.2 Pharmacokinetics and Drug Metabolism 
 
The initial t½ is 1.5 hours and the mean terminal half-life is 4 to 11 hours. It is 
primarily excreted in the urine. In children, approximately 55% of the dose is 
excreted in the urine as etoposide in 24 hours. The mean renal clearance of 
etoposide is 7 to 10 mL/min/m² or about 35% of the total body clearance over a 
dose range of 80 to 600 mg/m². Etoposide, therefore, is cleared by both renal and 
non renal processes, i.e., metabolism and biliary excretion. The effect of renal 
disease on plasma etoposide clearance is not known. Biliary excretion appears to 
be a minor route of etoposide elimination. Only 6% or less of an intravenous dose 
is recovered in the bile as etoposide. Metabolism accounts for most of the non renal 
clearance of etoposide.  The maximum plasma concentration and area under the 
concentration time curve (AUC) exhibit a high degree of patient variability. 
Etoposide is highly bound to plasma proteins (~94%), primarily serum albumin. 
Pharmacodynamic studies have shown that etoposide systemic exposure is related 
to toxicity. Preliminary data suggests that systemic exposure for unbound etoposide 
correlates better than total (bound and unbound) etoposide. There is poor diffusion 
into the CSF < 5%.   Cmax and AUC values for orally administered etoposide 
capsules consistently fall in the same range as the Cmax and AUC values for an 
intravenous dose of one-half the size of the oral dose. The overall mean value of 
oral capsule bioavailability is approximately 50% (range 25-75%). 
 
8.2.3 Supplier(s) 
 
 
Etoposide is commercially available. 
 
8.2.4 Dosage Form and Preparation 
 
Gelcaps: Etoposide is available as 50 mg pink capsules. Each liquid filled, soft 
gelatin capsule contains 50 mg of Etoposide in a vehicle consisting of citric acid, 
glycerin, purified water, and polyethylene glycol 400. The soft gelatin capsules 


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contain gelatin, glycerin, sorbitol, purified water, and parabens (ethyl and propyl) 
with the following dye system: iron oxide (red) and titanium dioxide; the capsules 
are printed with edible ink. 
 
Injection for oral use:  The Injection may be used orally for those children too young 
or unable to swallow capsules: 
Etoposide for Injection is available in sterile multiple dose vials. The pH of the 
clear, nearly colorless to yellow liquid is 3 to 4. Each mL contains 20 mg etoposide, 
2 mg citric acid, 30mg benzyl alcohol, 80 mg modified polysorbate 80/tween 80, 
650 mg polyethylene glycol 300, and 30.5 percent (v/v) alcohol. Vial headspace 
contains nitrogen. 
 
8.2.5 Storage and Stability 
 
Etoposide capsules must be stored under refrigeration (2°-8°C or 36°- 46°F).The 
capsules are stable until the expiration date on the package. 
 
Etoposide phosphate must be stored under refrigeration 2°-8°C (36°-46°F). 
Unopened vials of etoposide phosphate are stable until the expiration date on the 
package when stored at controlled room temperature (20˚-25˚C or 68˚-77˚F). 
 
8.2.6 Administration 
 
Gelcaps: Doses (up to 400 mg/day) can be given as a single once daily dose; doses 
>400 mg should be given in 2-4 divided doses.  For the purposes of this protocol, 
dosing will be once daily as the 400 mg/day limit will not be exceeded. 
 
Injection for oral use: For oral administration in children too young to take the 
capsules, the parenteral product can be used orally. A 1:1 dilution can be made by 
adding equal parts of normal saline and etoposide injection for a 10 mg/mL 
concentration which is stable for 22 days in Burron plastic oral syringes 
(polypropylene) either exposed or protected from light (McLeod HL, Relling MV. 
Stability of etoposide solution for oral use. Am J Hosp Pharm. 1992:49;2784-5). 
This dilution can be administered directly to be followed by sour candy or gum, or 
can be further diluted immediately prior to administration with fruit juice. 
Etoposide injection can be mixed in orange juice, apple juice, or lemonade. 
Etoposide injection diluted this way in fruit juice must be consumed immediately 
as it tends to precipitate. 
 
Undiluted etoposide injection (20 mg/mL) has been tested and is stable in a plastic 
syringe for 5 days when stored at room temperature under fluorescent light. 
 
When etoposide is stored in plastic, there is potential for cracking of rigid plastic 
containers and of leaching of diethylhexyl phthalate (DEHP) from polyvinyl 
chloride (PVC) containers.  Based on information provided by Bristol-Meyers 
Squibb Division of Oncology, undiluted etoposide appears to crack syringes made 


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of an acrylic-based plastic, polycarbonates, polyethylene glycol 300 or ABS 
(polymer produced by combining acrylonitrile, butadiene and styrene). Storing 
etoposide 10 mg/mL in polypropylene syringes should not pose a cracking risk or 
result in leaching of DEHP. 
 
Concentrations need to be 0.4 mg/mL or less to substantially enhance taste. 
Etoposide stays in solution at 0.4 mg/mL for up to 3 hours in one of the above 
juices. If storage is required add 1 mL of undiluted etoposide injection (20 mg/mL) 
or 2 mL of the 10 mg/mL dilution above to 50 mL- of juice (1 & 2/3 ounces) for a 
concentration of 0.4 mg/ml, which is stable for up to 3 hours but should be 
consumed as soon as possible after mixing. 
 
8.2.7 Special Handling Instructions 
 
Use proper chemotherapy handling techniques. 
 
 
9.0 
CORRELATIVE STUDIES 
 
9.1 
DCE and DSC-MRI 
 
All patients will undergo DSC and DCE-MRI at the following time points: 
 no more than 3 weeks prior to MLA (OPTIONAL) 
 within approximately 4 days after MLA 
 2-4 weeks (+/- 3 days) after MLA 
 Every 12 weeks (+/- 7 days) following the last MRI for the first year or until disease 
progression for Arm A; every 8 weeks (+/- 7 days) until 2 years have elapsed or 
until disease progression, whichever comes first, for Arm B. 
 
9.2 
Blood for Correlative Studies 
 
Patients will have 7 mL blood for serum drawn at the following time points: 
 before MLA; this sample can be collected any time during the 3 day before or the 
day of the procedure until the start of the procedure 
 within approximately 3 days after MLA 
 weeks 2-4 post-MLA (Arm A only, at time of DSC/DCE-MRI) 
 weekly (+/- 3 days) prior to (on the same day as) doxorubicin (Arm B only) 
 Every 12 weeks (+/- 7 days) for the first year or until disease progression for Arm 
A; every 8 weeks (+/- 7 days) until 2 years have elapsed or disease progression, 
whichever comes first, for Arm B. Blood draws will be drawn one same days as 
MRIs, prior to scan. 
 
A minimum of 2 ml will be drawn and placed in a red top tube and a minimum of 5 ml will 
be collected into a green top (heparin) tube. Specimens should be immediately transported 
to the Tissue Procurement Core (TPC) for processing.  
 
 


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9.3 
Blood for Serum Biomarkers of BBB Disruption 
 
For protein-based serum biomarker assays 0.5 to 1 ml serum will be to be aliquoted and 
stored at -80oC until used for ELISA analysis or for plasma microRNA-based assays in the 
Tran lab.  
 
9.4 
Blood for Phenotyping of Peripheral Blood Immune Cells 
 
The remainder of serum will stored at -80oC until used for the Meso Scale Discovery 
(MSD)-based cytokine quantitation, which will be performed in the Immune Monitoring 
Core of the Center for Human Immunology and Immunotherapy Programs. Serum will be 
used in the MSD immunoassays to assess immunoregulatory cytokine and chemokine 
profiles pre- and post-treatment over time.   
 
Blood obtained and stored for comparison of immune profiles of peripheral blood cells 
before and after MLA, provided there is no evidence of significant treatment-induced 
cytopenias. Blood will be Ficoll separated and the mononuclear cell population (PBMC) 
isolated per standard protocol through the TPC [59, 60]. The PBMC isolated from blood 
obtained prior to MLA will be cryopreserved for later analysis.  The isolated monocytes 
will be incubated with IL-4 and GM-CSF to produce dendritic cells (DC)[60] which will 
then be cryopreserved for analysis of tumor-specific functional activity of peripheral blood 
immune cells detailed in section 9.4 below.  To determine the phenotype of peripheral 
blood immune cells, following the 4-week interval, cryopreserved PBMC will be thawed 
and stained with labeled antibodies to CD4, CD8, CD45, CCR7, CD27, CD28, and CD62, 
as well as to markers of T cell hypofunctionality (PD-1, CTLA-4, LAG-3, TIM3, and 
ICOS).  Specifically, we will compare levels of CD8+PD-1+LAG-3+ T cells between MLA-
treated and untreated patients, as this cell population has been shown to mark tumor-
specific CD8+ T cells.  Cells will be assessed by flow cytometry in the CHiiPs immune 
monitoring core.  
 
Due to the limited blood volumes that may be available for some patients, our ability to 
conduct these cellular analyses may be limited.  In this event, we will substitute quantitative 
PCR for lymphocyte cytokines—such as IFN- and TNF---from mRNA generated from 
PBMC to assess lymphocyte functional status.   
 
9.5 
OPTIONAL Biopsy for Tumor-Specific Functional Activity of Peripheral 
Blood Immune Cells 
 
The patients in both arms may have a biopsy immediately before MLA.  Specimens not 
used for histologic diagnosis will be cryopreserved in the tumor bank for later processing.  
Should our studies by either flow cytometry or qRT-PCR described in Section 9.4 show 
evidence of lymphocyte activation, then after PBMC samples have been obtained, we will 
test T cell reactivity in an in vitro stimulation assay. Dendritic cells matured from 
monocytes as described in Section 9.3 above will be incubated with freeze-thawed biopsy 
specimen lysate for 2 days and subsequently incubated with T cells purified from PBMC 


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using CD3/CD8-positive microbeads.  Five days after stimulation, T cell reactivity will be 
assessed by ELISPOT assay for IFN-γ stimulation.  This study will test whether MLA 
stimulates increased tumor-specific T cell recognition and activation.  As in Section 9.4, 
the amount of blood drawn from some patients may be limited and could limit or ability to 
conduct these functional assays.   
 
9.6 
Quality of Life Assessments 
 
Patients in both arms will undergo QOL assessments in the form of evaluation of 
Karnofsky/Lansky performance status at each physician visit and documented in the note. 
 
 


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10.0 
STUDY CALENDAR 
 
10.1 
Arm A Study Calendar 
 
 
Baseline1 
MLA 
3 Days 
Post-MLA 
2-4 Weeks 
Post-MLA 
Every 12 Wks Thereafter 
for 12 months Post-MLA4 
F/U10 
Informed consent 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
Physical exam incl. wt 
X 
 
X3 
X 
X9 
X11 
Karnofsky/Lansky 
X 
 
X 
X 
X9 
X11 
CBC 
X 
 
 
 
 
 
CMP 
X 
 
 
 
 
 
Urine or serum βHCG 
X7 
 
 
 
 
 
MRI with DCE/DSC 
X8 
 
X2 
X2 
X9 
X11 
Biopsy 
 
X8 
 
 
 
 
Blood to measure brain-
specific factors 
(Sections 9.2-9.4) 
X 
 
X2 
X2 
X9 
X11 
Adverse event assessment5 
 
X 
X 
X 
X 
X 
1. No more than 21 days prior to MLA. 
2. +/3 days 
3. Weight not required 
4. Patients will be followed every 12 weeks from the Week 4 MRI until 1year has elapsed since MLA or progression, whichever occurs first.  After 
progression, vital status and subsequent treatment will be recorded. 
5. Through one year or off protocol. 
6. To be obtained with MRI.  
7. Females of childbearing potential 
8. Optional 
9. +/- 7 days 
10. F/U every 6 months or at event occurrence for 5 years or until death, whichever occurs first 
11. +/- 2 weeks 
 
 


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10.2 
Arm B Study Calendar 
 
 
B/L1 
MLA 
3 Days 
Post-MLA 
Wk
14 
Wk
24 
Wk
34 
Wk
44 
Wk
54 
Wk
64 
Monthly5 
F/U10 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
X 
 
Physical exam incl. wt 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X15 
Karnofsky/Lansky 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X15 
CBC 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
CMP 
X 
 
 
X 
 
 
X 
 
 
X 
 
Urine or serum βHCG 
X12 
 
 
 
 
 
 
 
 
 
 
Echocardiogram 
X2 
 
 
 
 
 
 
 
 
 
 
MRI with DCE/DSC 
X14 
 
X 
 
X11 
 
 
 
 
X5,6 
 
Biopsy 
 
X14 
 
 
 
 
 
 
 
 
 
Blood to measure brain-
specific factors 
(Section 9.2-9.4) 
 
X3 
X 
X7 
X7 
X7 
X7 
X7 
X7 
X5,6 
 
Doxorubicin8 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
Etoposide13 
 
 
 
 
 
 
 
 
 
X 
 
AE assessment9 
 
X ------------------------------------------------------------------------------------------- X 
1. No more than 21 days prior to MLA. 
2. No more than 1 year prior to registration. 
3. May be collected any time during the 3 days before or the day of the procedure until the start of the procedure. 
4. +/- 3 days 
5. +/- 7 days 
6. Every 8 weeks for the first 2 years or until disease progression, whichever occurs first. To coincide with MRI. After progression, vital status and subsequent treatment 
will be recorded. 
7. Same day as chemotherapy. 
8. To begin approximately 7 days after MLA. 
9. Through one year or off protocol 
10. Patients will be followed every 6 months or at event occurrence for 5 years or until death, whichever occurs first. 
11. To be obtained 2-4 weeks post-MLA 
12. Females of childbearing potential 
13. To be started at time of count recovery after the 6 weeks of treatment with doxorubicin has concluded. 
14. Optional 
15. +/- 2 weeks 


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11.0 
DATA SUBMISSION SCHEDULE 
 
Case report forms with appropriate source documentation will be completed according to the 
schedule listed in this section. 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
On-Study Form 
Prior to starting treatment 
MLA Form 
After procedure 
Biopsy Form 
After biopsy 
QOL Form 
(documentation of performance 
status) 
Weeks 1, 3, and 5 of doxorubicin 
With each cycle of etoposide 
Every 8 weeks for one year post-MLA 
Doxorubicin Form (Arm B) 
At the end of treatment with doxorubicin 
Etoposide Form (Arm B) 
At the end of each cycle of etoposide 
Biomarkers Form 
Refer to Section 9.2 
Adverse Events Form 
Continuous through 30 days after MLA (Arm A)  
Through 30 days after last dose of chemotherapy 
(Arm B) 
Treatment Summary Form 
(Arm B) 
Completion of treatment 
Follow Up Form 
Arm A: Every 12 weeks for one year post-MLA 
Arm B: Every 8 weeks for one year post-MLA 
Tumor Response Form 
With each MRI 
AE Reporting Form 
See Section 7.0 for reporting requirements 
 
 
12.0 
MEASUREMENT OF EFFECT 
 
12.1 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, patients should be re-evaluated for response every 12 
weeks on Arm A and every 8 weeks on Arm B.  The DCE/DSC-MRI scan obtained 
within approximately 3 days after MLA will be used as the baseline scan for 
determining response.  The DCE/DSC-MRI scans obtained at pre-MLA, post-MLA, 
and prior to 6 weeks post-MLA will not be assessed for disease response.   
 
Response and progression will be evaluated in this study using the updated response 
assessment criteria for high-grade gliomas: Response Assessment in Neuro-Oncology 
(RANO) working group guideline [JCO 28(11): 1963-1972, 2010]. 
 
Criteria for Determining First Progression Depending on Time From Initial Chemoradiotherapy 


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First Progression 
Definition 
Progressive disease < 12 
weeks after completion of 
chemoradiotherapy 
Progression can only be defined using diagnostic imaging if there is 
new enhancement outside of the radiation field (beyond the high-dose 
region or 80% isodose line) or if there is unequivocal evidence of 
viable tumor on histopathologic sampling (eg, solid tumor areas [ie, 
> 70% tumor cell nuclei in areas], high or progressive increase in 
MIB-1 proliferation index compared with prior biopsy, or evidence 
for histologic progression or increased anaplasia in tumor). Note: 
Given the difficulty of differentiating true progression from 
pseudoprogression, clinical decline alone, in the absence of 
radiographic or histologic confirmation of progression, will not be 
sufficient for definition of progressive disease in the first 12 weeks 
after completion of concurrent chemoradiotherapy.  
Progressive disease ≥ 12 
weeks 
after 
chemoradiotherapy 
completion 
1. New contrast-enhancing lesion outside of radiation field on 
decreasing, stable, or increasing doses of corticosteroids. 
2. Increase by ≥ 25% in the sum of the products of perpendicular 
diameters between the first postradiotherapy scan, or a 
subsequent scan with smaller tumor size, and the scan at 12 
weeks or later on stable or increasing doses of corticosteroids. 
3. Clinical 
deterioration 
not 
attributable 
to 
concurrent 
medication or comorbid conditions is sufficient to declare 
progression on current treatment but not for entry onto a 
clinical trial for recurrence.  
4. For patients receiving antiangiogenic therapy, significant 
increase in T2/FLAIR nonenhancing lesion may also be 
considered progressive disease. The increased T2/FLAIR 
must have occurred with the patient on stable or increasing 
doses of corticosteroids compared with baseline scan or best 
response after initiation of therapy and not be a result of 
comorbid events (eg, effects of radiation therapy, 
demyelination, 
ischemic 
injury, 
infection, 
seizures, 
postoperative changes, or other treatment effects).  
 
 
 


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Criteria for Response Assessment Incorporating MRI and Clinical Factors (Adapted from JCO 
2010) 
 
Response 
Criteria 
Complete response 
 Requires all of the following: complete disappearance of all enhancing 
measurable and nonmeasurable disease sustained for at least 4 weeks. 
 No new lesions; stable or improved nonenhancing (T2/FLAIR) lesions. 
 Patients must be off corticosteroids (or on physiologic replacement doses 
only) and stable or improved clinically. Note: Patients with 
nonmeasurable disease only cannot have a complete response; the best 
response possible is stable disease.  
Partial response 
Requires all of the following:  
 ≥ 50% decrease compared with baseline in the sum of products of 
perpendicular diameters of all measurable enhancing lesions sustained 
for at least 4 weeks. 
 No progression of nonmeasurable disease. 
 Any new measureable lesion within the 3 cm radius of the rim of the 
MLA-treated recurrent tumor. † 
 Stable or improved nonenhancing (T2/FLAIR) lesions on same or lower 
dose of corticosteroids compared with baseline scan; the corticosteroid 
dose at the time of the scan evaluation should be no greater than the dose 
at time of baseline scan. 
 Stable or improved clinically. Note: Patients with nonmeasurable disease 
only cannot have a partial response; the best response possible is stable 
disease.  
Stable disease 
Requires all of the following:  
 Does not qualify for complete response, partial response, or progression. 
 Stable nonenhancing (T2/FLAIR) lesions on same or lower dose of 
corticosteroids compared with baseline scan. In the event that the 
corticosteroid dose was increased for new symptoms and signs without 
confirmation of disease progression on neuroimaging, and subsequent 
follow-up imaging shows that this increase in corticosteroids was 
required because of disease progression, the last scan considered to show 
stable disease will be the scan obtained when the corticosteroid dose was 
equivalent to the baseline dose.  
Progression 
Defined by any of the following:  
 ≥ 25% increase in sum of the products of perpendicular diameters of 
enhancing lesions compared with the smallest tumor measurement 
obtained either at baseline (if no decrease) or best response, on stable or 
increasing doses of corticosteroids*. 
 Significant increase in T2/FLAIR nonenhancing lesion on stable or 
increasing doses of corticosteroids compared with baseline scan or best 
response after initiation of therapy* not caused by comorbid events (e.g. 


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Response 
Criteria 
radiation therapy, demyelination, ischemic injury, infection, seizures, 
postoperative changes, or other treatment effects). 
 Any new measureable lesion within the 3 cm radius of the rim of the 
MLA-treated recurrent tumor. † 
 Clear clinical deterioration not attributable to other causes apart from the 
tumor (e.g. seizures, medication adverse effects, complications of 
therapy, cerebrovascular events, infection, and so on) or changes in 
corticosteroid dose. 
 Failure to return for evaluation as a result of death or deteriorating 
condition; or clear progression of nonmeasurable disease.  
 
NOTE. All measurable and nonmeasurable lesions must be assessed using the same 
techniques as at baseline. 
 
Abbreviations: MRI, magnetic resonance imaging; FLAIR, fluid-attenuated inversion 
recovery. 
 
* Stable doses of corticosteroids include patients not on corticosteroids.  
 
† If any new measurable lesion is outside the 3 cm radius of the rim of the MLA-treated 
recurrent tumor or is located in the contralateral hemisphere regardless of its distance from 
the rim of the MLA-treated tumor AND there is not any measurable lesion within the 3 cm 
radius, the patient will be considered to have progressive disease but will not be considered 
evaluable for PFS in this study, in which only local disease control or failure is measured. 
 
12.2 
Disease Parameters 
 
Measurable disease:  Bi-dimensionally measurable lesions with clearly defined margins 
by MRI scan.  All tumor measurements must be recorded in millimeters (or decimal 
fractions of centimeters).   
 
Non-measurable disease:  Uni-dimensionally measurable lesions or lesions with margins 
not clearly defined such as areas of T2/FLAIR signal abnormality or poorly defined 
enhancing abnormality.  
 
Note: For cystic lesions, the only measurable part is any enhancement area around the cyst 
that is clearly defined and bi-dimensionally measurable. The cyst itself should not be 
considered measurable or non-measureable disease. 
 
Target lesions:  All measurable lesions that are residual of the lesion treated with MLA or 
that are located within the 3 cm radius of the rim of the MLA-treated recurrent tumor should 
be identified as target lesions and recorded and measured.  Target lesions should be selected 
on the basis of their size (lesions with the longest diameter), but in addition should be those 
that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion, which can be measured reproducibly should be 
selected.  When there are too many measurable lesions, choose the largest 3 lesions as 
target lesions to follow.  The other measurable lesions should be considered evaluable for 


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the purpose of objective status determination. 
 
Non-target lesions:  All non-measurable lesions should be identified as non-target lesions 
and should also be recorded at baseline.  Measurements of these lesions are not required, 
but the presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow-up. 
 
12.3 
Methods for Evaluation of Measurable Disease 
 
All measurements should be taken and recorded in metric notation using a ruler.  All 
baseline evaluations should be performed as closely as possible to the beginning of 
treatment and never more than 2 weeks before the beginning of the treatment. 
 
Clinical lesions:  Clinical lesions will only be considered measurable on brain MRI when 
they are ≥ 5 mm diameter as assessed using a ruler. 
 
Histology:  This technique can be used to differentiate between partial responses (PR) and 
complete responses (CR) in rare cases when biopsy or surgical resection of a measureable 
lesion is clinically indicated.  
 
Perfusion/CBV: This advanced brain MRI technique can be used as an adjunct test to 
determine treatment response or disease status.  However, it should not be used as the 
primary or sole method to determine response or disease status. 
 
Brain FDG-PET coupled with head CT or brain MRI: This advanced metabolic 
imaging technique can be used as an adjunct test to determine response or disease status. 
However it should be used as the primary or sole method of determining response or 
disease status. 
 
12.3.1 Evaluation of Target Lesions 
 
Complete Response (CR):  Disappearance of all target lesions. 
 
Partial Response (PR):  ≥ 50% decrease compared with baseline in the sum of 
products of perpendicular diameters of all target lesions sustained for at least 4 
weeks. 
 
Progressive Disease (PD):  At least a 25% increase in the sum of products of 
perpendicular diameters of at least 1 target lesion, taking as reference the smallest 
sum of products of perpendicular diameters on study (this includes the baseline sum 
if that is the smallest on study).  
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum of products of 
perpendicular diameters while on study. 
 


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12.3.2 Evaluation of Non-Target Lesions 
 
Complete Response (CR):  Disappearance of all non-target lesions.  
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s). 
 
Progressive Disease (PD): Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions on stable or increasing doses 
of corticosteroids compared with baseline scan or best response after initiation of 
therapy* not caused by comorbid events (e.g. radiation therapy, demyelination, 
ischemic injury, infection, seizures, postoperative changes, or other treatment 
effects). Unequivocal progression should not normally trump target lesion status.  
It must be representative of overall disease status change, not a single lesion 
increase.     
 
Although a clear progression of “non-target” lesions only is exceptional, the 
opinion of the treating physician should prevail in such circumstances, and the 
progression status should be confirmed at a later time by the review panel (or 
Principal Investigator). 
 
12.3.3 Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the 
treatment until disease progression/recurrence (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started).  The 
patient's best response assignment will depend on the achievement of both 
measurement and confirmation criteria. 
 
Summary of the RANO Response Criteria (Adapted from JCO 2010) 
Criterion 
CR 
PR 
SD 
PD 
T1 gadolinium 
enhancing disease 
None 
≥ 50% ↓ 
< 50% ↓ but < 25% ↑ ≥ 25% ↑* 
T2/FLAIR 
Stable or ↓ Stable or ↓ Stable or ↓ 
↑* 
New lesion 
None 
None 
None 
Present* 
Corticosteroids 
None 
Stable or ↓ Stable or ↓ 
NA† 
Clinical status 
Stable or ↑ Stable or ↑ Stable or ↑ 
↓* 
Requirement for 
response 
All 
All 
All 
Any* 
Abbreviations: RANO, Response Assessment in Neuro-Oncology; CR, complete response; PR, 
partial response; SD, stable disease; PD, progressive disease; FLAIR, fluid-attenuated inversion 
recovery; NA, not applicable.  
* Progression occurs when this criterion is present.  
† Increase in corticosteroids alone will not be taken into account in determining progression in the 
absence of persistent clinical deterioration.  
 


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12.3.4 Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from 
the time measurement criteria are met for CR or PR (whichever is first recorded) 
until the first date that recurrent or progressive disease is objectively documented 
(taking as reference for progressive disease the smallest measurements recorded 
since the treatment started). 
 
The duration of overall CR is measured from the time measurement criteria are first 
met for CR until the first date that progressive disease is objectively documented.  
 
Duration of stable disease:  Stable disease is measured from the start of the 
treatment until the criteria for progression are met, taking as reference the smallest 
measurements recorded since the treatment started, including the baseline 
measurements.  
 
12.3.5 Neurological Exam and Performance Status  
 
Patients will be graded using the Karnofsky Performance Status scale and their 
neurological function evaluated as improved, stable or deteriorated in addition to 
objective measurement of tumor size. These parameters will be used to determine 
the overall response assessment. 
 
12.3.6 Progression-Free Survival 
 
PFS is defined as the duration of time from start of treatment to time of progression 
or death, whichever occurs first. 
 
 
13.0 
DATA AND SAFETY MONITORING 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months after accrual has opened (if at least five patients have been enrolled) or one 
year after accrual has opened (if fewer than five patients have been enrolled at the six-month mark). 
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 


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 Study-wide target accrual and study-wide actual accrual 
 Protocol activation date 
 Average rate of accrual observed in year 1, year 2, and subsequent years 
 Expected accrual end date and accrual by arm 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities separated by arm 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
 
14.0 
STATISTICAL CONSIDERATIONS 
 
Since we do not have any preliminary data for MR imaging, serum biomarkers, etc, it is not feasible 
to estimate sample size or provide power estimates. Therefore, this study is considered as a pilot 
study and the data collected will be used to design future studies. We propose six patients each 
arm as this is considered to be reasonable to recruit within the available a time frame.  
 
In each arm, we will consider the following statistical analysis: 
 
For the primary endpoint, MR imaging will be collected at the time points: pre-MLA 
(OPTIONAL), post-MLA, two-four weeks after MLA and every twelve weeks in Arm A; pre-
MLA, post-MLA, two-four weeks after MLA and every eight weeks in Arm B. The linear 
regression model will used to investigate the correlation between MR imaging and peritumoral 
BBB disruption. To account for correlation among the repeated measures from the same patient, 
the longitudinal data will be analyzed with the use of linear generalized estimating equation (GEE). 
Whether the average measurements differ at the multiple time points will be evaluated through 
GEE model. Least-square means at each time points will be presented and standard errors will be 
calculated within the use of the GEE sandwich method when accounting for within-patient 
correlation.  
 
Since we do not know which biomarkers will have better correlation with the Ktrans data from 
DCE and DSC-MRI and patients’ survival outcome, we plan to determine the levels of all 3 
biomarkers in a blinded fashion. Once both the Ktrans and biomarker levels are available, we will 
determine which biomarkers have the closest correlation that is statistically significant with the 
Ktrans. Pearson correlation coefficient (r) will be determined for each biomarker and Ktrans value. 
Biomarkers with higher correlation coefficient (r approaching 1) will be given higher priority.  A 
minimum r=0.5 is required for inclusion for further analysis and will be used as a peritumoral 


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permeability score. This score will then be correlated with the patient outcome data (as measured 
by 6PFS rate) to determine whether it has a predictive value. 
 
PFS and OS will be estimated by Kaplan-Meier product limit method. The progression-free 
survival and overall survival probabilities at certain time points (e.g. 3 months, 6 months) will be 
presented as well. The difference in OS between Arm A and the historical control of bevacizumab 
alone will be compared by log-rank test. The quality of life (QOL) using Karnofsky or Lansky 
performance status will be characterized by summary statistics (mean, standard deviation and 
range). 
 
The tumor-specific immune responses are collected at pre and post-MLA treatment. Paired t-test 
will be used to determine the effects of MLA treatment on patient’s tumor-specific immune 
responses. Cox proportional hazard model will be used to determine the relationship between the 
duration of MLA-induced BBB disruption and PFS. 
 


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We would like to thank the Alvin J. Siteman Cancer Center at Washington University School of 
Medicine and Barnes-Jewish Hospital in St. Louis, Missouri, for the use of the Clinical Trials 
Core which provided protocol development service. The Siteman Cancer Center is supported in 
part by an NCI Cancer Center Support Grant #P30 CA91842. 
 
 


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APPENDIX 1: Karnofsky and Lansky Performance Status Scale 
 
Karnofsky 
Lansky 
Score 
Description 
Score 
Description 
100 
Normal,   no   complaints,   no 
evidence of disease 
100 
Fully active, normal. 
90 
Able to carry on normal activity, minor 
signs or symptoms of disease. 
90 
Minor  restrictions  in  physically 
strenuous activity. 
80 
Normal activity with effort; some signs 
or symptoms of disease. 
80 
Active, but tires more quickly 
70 
Cares for self, unable to carry on normal 
activity or do active work. 
70 
Both  greater  restriction  of  and less time 
spent in play activity. 
60 
Required occasional assistance, but is 
able to care for most of his/her needs. 
60 
Up   and   around,   but   minimal active play; 
keeps busy with quieter activities. 
50 
Requires considerable assistance  and  
frequent medical care. 
50 
Gets   dressed,   but   lies   around much of 
the day; no active play, able to participate in 
all quiet play and activities. 
40 
Disabled, requires special care and 
assistance. 
40 
Mostly   in   bed;   participates   in quiet 
activities. 
30 
Severely 
disabled, 
hospitalization  
indicated. Death not imminent. 
30 
In bed; needs assistance even for quiet 
play. 
20 
Very     sick,     hospitalization 
indicated.  Death not imminent. 
20 
Often    sleeping;    play    entirely limited 
to very passive activities. 
10 
Moribund,     fatal     processes 
progressing rapidly. 
10 
No play; does not get out of bed. 
 
 
 


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APPENDIX 2: PATIENT’S MEDICATION DIARY 
 
Today’s Date:    
 
 
 
 
Agent:  Etoposide 
Cycle:    
 
 
 
Patient Name:    
 
 
 
 
 
 
Study ID#:   
 
 
 
 
INSTRUCTIONS TO THE PATIENT: 
1. Complete one form for each cycle.  Take _____mg ( ___capsules) of etoposide as directed by your study doctor. 
2. Record the date, the number of capsules taken, and when you took them. 
3. If you forget to take it before bedtime, then do not take a dose that day.  Restart taking it the next day. 
4. If you have any questions or notice any side effects, please record them in the comments section.  Record the 
time if you should vomit. 
5. Please return the forms to your physician or your study coordinator when you go to your next appointment.  Please 
bring your unused study medications and/or empty bottles with you to each clinic visit so that a pill count can be 
done. 
Day 
Date 
What time was dose taken? 
# of capsules taken 
Comments 
1 
 
 
 
 
2 
 
 
 
 
3 
 
 
 
 
4 
 
 
 
 
5 
 
 
 
 
6 
 
 
 
 
7 
 
 
 
 
8 
 
 
 
 
9 
 
 
 
 
10 
 
 
 
 
11 
 
 
 
 
12 
 
 
 
 
13 
 
 
 
 
14 
 
 
 
 
15 
 
 
 
 
16 
 
 
 
 
17 
 
 
 
 
18 
 
 
 
 
19 
 
 
 
 
20 
 
 
 
 
21 
 
 
 
 
22 
 
 
 
 
23 
 
 
 
 
24 
 
 
 
 
25 
 
 
 
 
26 
 
 
 
 
27 
 
 
 
 
28