Prognosis and Therapeutic Biomarkers for Glioblastoma Patients
Conditions: Glioblastoma
Sponsor: Baylor Research Institute

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March 7, 2017 
 
Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
Title: Prognosis and therapeutic biomarkers for glioblastoma patients (for research only) 
 
Investigators:  Principal Investigator, other co-investigators  
 
PI:  
Dr. Ekokobe Fonkem 
Co-PI:  Dr. Fengfei Wang 
 
 
1. Introduction/Background 
The purpose of this study is to investigate if the potential biomarkers identified could be used for 
facilitating the diagnosis and prognosis of patients with glioblastoma (GBM).  
 
GBM, accounting for 50% of central nerve system (CNS) tumors, is a deadly disease with no effective 
curable therapy. To improve the quality of patients’ life and enhance the therapy response, Optune was 
developed and approved by the US Food and Drug Administration (FDA) for treating recurrent GBM (1-
9). Now both Optune and Adjuvant TMZ plus Optune treatment are considered as a standard strategy for 
maintenance therapy to extend patients’ life after surgery and chemotherapy (2, 6). Currently, diagnosis 
and prognosis of GBM rely on magnetic resonance imaging (MRI), computed tomography (CT), and 
biopsy (10). These diagnosis and prognosis processes for patients with GBM are expensive, difficult, and 
inaccurate. Biopsy of brain tissues from GBM patients is a very aggressive procedure, and it is extremely 
difficult to re-biopsy. By analyzing previously published data derived from microarray and next 
generation sequencing as well as the experimental data generated from our lab, we have identified a group 
of genes that have potential to be used as biomarkers for GBM prognosis.   
 
To ascertain if the potential biomarkers could be used to facilitate the diagnosis of patients with GBM, the 
goal of this proposed study is to determine the expression levels of selected biomarkers in GBM tissues 
and the cerebrospinal fluid (CSF) from patients with GBM receiving different types of treatment against 
controls.  
 
 
2. Significance 
There is no effective therapy for GBM.  
There is a critical need for early 
diagnostic and prognostic biomarkers for 
GBM. The completion of this study will 
provide 
a 
potential 
biomarker 
for 
diagnosis and prognosis of GBM patients. 
 
 
3. Objectives & Specific Aims 
The objective of this proposed study is to 
determine the expression levels of a panel 
of markers including CD133, CD44, ABCC3, TNFRSF1A, AKT1, IDH2, and MGMT in GBM tissues 
and the CSF, blood, and saliva from patients with GBM receiving different types of treatment as 
described in Figure 1 and the non-brain tumor patients (controls). Our aims are: 1) To compare the 
differences of CD44 between the control group and the combined cancer groups at baseline; and 2) To 
compare the levels of CD44 and other markers among the 3 different groups such as control vs. GBM 
patients and Optune vs. Optune + TMZ after 8 weeks of treatment. Exploratory analysis of all other 
markers will be performed.  
 

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March 7, 2017 
 
Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
4. Methodology 
 
4.1 Study Design.   
To reach our goal, a small clinical study will be performed with the support from NovoCure Inc. In this 
study, 30 participants will be enrolled in which 10 non-brain tumor patients (e.g., patients will have other 
types of minor neurological disorder(s) that necessitate a lumbar puncture e.g., headache, epilepsy or 
neurodegenerative disorders such as Alzheimer’s disease or Multiple sclerosis or Parkinson’s disease) 
will be used as the control. As shown in Figure 1, 20 patients (n=20) with GBM will be divided into two 
groups; patients in group 3 will receive the combination of TMZ (150-200 mg/m2 orally every day; 5 
consecutive days per 28-day treatment cycle for 6 cycles) with Optune and patients in group 2 will 
receive Optune only. (The GBM patients will be monitored for tumor size using MRI and CT scan before 
and after each type of treatment, which is standard of care for all GBM patients.) We will collect the CSF 
(5 ml), blood (2 ml), and saliva (5 ml) from each participant as indicated in Figure 2 before and after 
treatment (Optune alone or Optune plus TMZ for detection of biomarkers. These samples will be used for 
detection of selected biomarkers at gene (RNA and DNA) and protein levels. CSF is standard of care for 
all GBM patients, we will ask to store the leftover tumor tissues for our research.  
 
4.2 Subject Selection/Participants.   
The participants in both non-brain tumor group and GBM groups (male and/or female, there is no 
preference at this time) should be over 22 years old without pregnancy, or an infectious disease (cold, flu, 
HIV, etc.) or a blood disorder (e.g., lack of platelets, anemia, thrombosis) or vascular malformations. For 
controls, only patients evaluated for the neurological disorders whose evaluation required spinal taps at 
both the Neurology clinic and the Brain Tumor clinics will be used for this study. This is an exploratory 
pilot study. Based on others previous experiences, 30 subjects (10 subjects in each group) will allow us to 
estimate the levels of selected biomarkers in the body fluids of different groups of patients and the 
potential value of these selected biomarkers for GBM prognosis. 
 
4.3 Procedures/Methods.  
For the non-brain tumor control group, 5 ml of CSF, 2 ml of blood, and 5 ml of saliva will be collected one 
time from each participant. For the GBM patients, patients will first receive surgery to remove the tumor 
(the tissue will be used for diagnosis and we will use the left over for the biomarker detection) followed by 
radiation and/or chemotherapy according to patients’ health conditions. (CSF are standard of care for these 
patients.) Thereafter, the physician will discuss with the patients and help patients pick their FDA approved 
treatment options to receive Optune or Optune plus TMZ.  
 
All of the GBM patients will be asked for blood, saliva, and CSF withdraws at three time points as indicated 
in Figure 2 (the first time point is at after surgery +/- 1 week and before the combination of radiation and 
chemotherapy; the second time point is immediately before starting Optune/TMZ treatment +/- 3 days [at 6 
weeks after the combination treatment]; and the third time point is at 8 months after the Optune/TMZ 
treatment +/- 1 month). The samples collected will be used to determine the levels of selected potential 
biomarkers at protein, DNA/RNA levels. These data will be used to assess the efficacy of the treatments for 
GBM patients and the correlation of the efficacies of the treatments with the expression levels of testing 
biomarkers in their CSF, blood, and saliva.  
 
Method for saliva collection: Patients should be consented on day one and come back for blood and saliva 
withdrawal. For saliva collection, participants will be instructed to fast overnight before saliva collection. 
Teeth brushing should occur at least one hour prior to saliva collection. Saliva collection should (ideally) 
occur between 8:00 am and 10:00 am. Upon arrival at the clinic, patients should be instructed to rinse 
mouth with deionized (DI) water for thirty seconds (30 s) to remove any residual foreign particles (e.g., 
food) and/or mucus from the mouth prior to saliva collection. Water used to rinse mouth can be spit out 
and discarded. Wait one minute after rinsing mouth before beginning saliva collection. Saliva will be 
collected from both “normal patients” and “patients with the disease” using the passive drool method (i.e., 

160280 
 
March 7, 2017 
 
Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
non-chemically stimulated, whole saliva collection), as previously described. Patients should be informed 
that we strictly want to collect saliva, mucous and/or phlegm should not be collected. After the one 
minute waiting period, start a timer and instruct patients to think about eating their favorite food while 
making a chewing motion with their jaw (these actions help stimulate saliva production). Patients should 
pool saliva under their tongue and passively “drool” saliva through a drinking straw (cut to a length of 
approximately 2 inches (11) into a 15 mL centrifuge tube. Please keep collection tube in a cup filled with 
ice (crushed ice is preferable) during saliva collection to help preserve biomolecules. Final volume of 
saliva collected should be ~ 5 mL (no less than 3 mL). Upon completion of saliva collection, stop timer 
and record total time taken to collect the sample (this time will be used to estimate saliva production 
rates). Immediately after saliva collection, transfer 15 mL centrifuge tube to a -80 ˚C freezer for storage. 
Patients may resume a normal eating schedule immediately after completion of saliva collection. 
 
Subject recruitment & enrollment plan 
For GBM patients, there are over 60 GBM patients enrolled at Baylor Scott & White Health each 
year. The coordinators will screen patients’ files to find potential participants of patients with GBM. 
The patients are at least 22 years old and without pregnancy, or an infectious disease (cold, flu, HIV, 
etc.) or a blood disorder (e.g., lack of platelets, anemia, thrombosis) or vascular malformations will be 
selected and interviewed for participating this study after discussing treatment options with their 
physicians.  
 
For the participants in the control group, there are several hundreds of patients with neurological 
disease (e.g., patients with Alzheimer’s or multiple sclerosis or Parkinson’s disease) enrolled at 
Baylor Scott & White Health yearly. CSF and blood draws are required for diagnosis of these 
patients. As CSF draw is standard of care, we will be able to find those individuals from enrolled 
patients who have other neurological disorders such as Alzheimer’s or multiple sclerosis and use the 
leftover CSF for our research. Thus, there is no need for advertisement. The coordinators will screen 
patients’ files to find potential participants of patients with non-brain tumor and then the coordinators 
will interview those individuals and recruit them for this study. As soon as the project is approved by 
the IRB committee, we will start to recruit patients for this study.  
 
Although there is little risk for patients who participant in this study, we still ask all participants to 
sign an Informed Consent form. All participants will be given an IRB approved Informed Consent 
form by their doctors before they enter in this study.  
 
Incentives for participation/compensation 
The participants will be paid for giving their blood and CSF for the tests ($25 each time per test) after 
each visit. The payment will be made by from Baylor Scott & White Research Institute and it is 
considered taxable income. The participants must be eligible to be paid in the United States and 
willing to complete all the necessary tax/legal paperwork to receive this payment.  
 
Study procedures for individual visits/interventions and/or interactions 
Optune, a Novo treating tumor field (NovoTTF) device, is approved by FDA for treating patients with 
relapsed GBM. Recent studies indicate that it is possible for people with newly diagnosed GBM 
living longer when Optune is used together with the chemotherapy TMZ.  
The control group will have no interaction with anything as they will not receive any anticancer 
treatment and all participants without brain tumor will be signed up in this group.  
 
Data collection variables  
The time points for data and sample collection will follow the schedule as indicated in Figure 2. 
The first point will be served as the baseline; the second point will be the intermediate; and the third 
point will be the endpoint for the biomarker study of using CSF samples.  The data generated from 
the patients’ samples will be kept in the lab. As these data do not contain any privacy information, 
there will be no privacy concern. 

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March 7, 2017 
 
Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
 
The data for the tumor sizes and patients’ general performs will be a part of the standard care. Data 
collection forms can be Excel spreadsheets or created in Microsoft Access (the Data Management 
group can create these for the PI). 
 
For privacy concerns, all the information about participants from this study will only be shown to 
the people working on the study. The results of this study may be published in a scientific book or 
journal.  If this is done, the name of any participants will not be used. All information about 
participants from this research project will be kept in a locked office. Information that is kept on 
computers will be kept safe from access by people who should not see it. Sensitive information is 
not allowed to transfer or distribute to the third party without permission.  All investigators who 
deal with collected tissue samples on this application have taken or will take the respective 
institutional “human subject protection” training for this project. Only Key Study Personnel (KSP) 
will have access to collected data and that it will be housed in secure environment. The data will not 
be destroyed at least for 6 year.  
 
Tumor tissues will be collected from patients for diagnosis and future research e.g., evaluating the 
levels of biomarkers selected. The tissues will be snap-frozen (fresh-frozen, FF) and then stored at -
80 or liquid nitrogen. Alternatively, it could be formalin-fixed paraffin-embedded (FFPE) for long 
time storage (10 years). All samples are expected to be used for diagnosis, thus, though the original 
samples may be labeled with patients’ information. However, the patients’ information will not be 
released to the researchers. The pathologist will change the label to GBM with numbers only.  
 
The 5 ml of CSF collected from each patient each time will be centrifuged at 3500 rpm at 4oC for 
10 min to remove any possible blood contamination and kept in the dry ice or stored at -80°C 
immediately. The samples will be used for determining the protein levels of selected potential 
markers.  All the samples will be labeled with number system without PHI information and the 
samples are expected to be analyzed within 12 months. The amount of samples collected in this 
study is very limited and we do not expect any sample left after the study. 
 
For the privacy concerns, only publishable data will be shared by the third party. All samples will 
not be transferred from Baylor Scott & White Health to a non –Scott & White Health laboratory for 
analysis. In case, there is a need to transfer the materials from Baylor Scott & White Health to a 
third party for sample analysis, permission. A Materials Transfer Agreement (MTA) or Data Use 
Agreement (DUA) will be required before any materials or data can be shared. Medical Research 
Building (MRB), an MTA is also required. The Office of Research Business Development can 
assist with development of these documents   
 
Subject withdrawal 
The patients have the right to leave the study anytime. The data collected will be used for the analysis 
as long as the data are meaningful. Otherwise, more patients will be enrolled.  
 
Reporting adverse events or other significant events 
As both Optune and TMZ are FDA approved treatment options for GBM patients, there are no 
significant adverse events caused by these treatment. In an unlikely case, there are any significant 
events; it should be reported to IRB immediately.  
  
4.3 Power Calculation 
The goal of the study is to compare the differences in the CSF, blood and saliva levels of CD44 between 
1) the control group and the Optune alone and the TMZ + Optune group; and 2) Optune alone vs, TMZ + 
Optune alone.  
 

160280 
 
March 7, 2017 
 
Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
There is no previous literature on CD44 levels in GBM. To calculate a sample size, we are assuming that 
the baseline and post treatment levels of CD44 standard form in the CSF will be approximately 327±134 
ng/ml and 185±103, respectively, similar to those reported by Kawano et al. for head and neck 
cancer(12). One of the drawbacks of the report is that they only reported the median and do not specify if 
it is the standard deviation or range that they reported.  
 
For the primary goal, it is assumed that the median is similar to the mean and the reported value is the 
standard deviation. Further assumptions are: sample sizes of n=10 and n=20 (for the control and the 
cancer groups, respectively); significance level of 0.05; expected means of 327 and 185 (for the cancer 
and the control groups, respectively); common standard deviation of 115; independence of the sample 
units and normality of the observations. The power was calculated using a two sample t-test and it was 
found to be 87%. Power calculation was performed with PASS v.13.0.4. 
 
4.4 Data Analysis. 
Descriptive statistics will be provided. Categorical data will be reported as count (percentages). 
Continuous data will be reported as mean (standard deviation) or median (range) as appropriate. A two 
sample t-test will be used to compare the biomarker level between groups, unless the distributions are 
found to be non-symmetric. In that case, Wilcoxon-Rank-Sum tests will be used. 
 
5. Human Subjects 
Thirty individuals will take part in this study at Baylor Scott & White Healthcare. Body fluids we 
requested are considered to have concern of human subject. 
 
Risks (including physical, emotional, social, and/or privacy) 
Risks of Radiation – Diagnostic Test: Patients will have the same amount of radiation exposure regardless 
of whether the participant elects to take part in this research study or receives standard medical care.    
 
Risks of participation in the study for individuals using Optune alone or Optune plus TMZ: TMZ is 
commonly used to treat patients with GBM. Common side effects of patients using TMZ include nausea, 
vomiting, loss of appetite, constipation, diarrhea, skin rash, tiredness, weakness, dizziness, blurred vision, 
sleep problems (insomnia), unusual or unpleasant taste in your mouth, and headache. Optune is a FDA 
approved device for GBM patients as well. In a large clinical study, patients who received Optune for at 
least 18 hours a day responded better to treatment. Because Optune works differently, it does not cause 
many of the side effects associated with chemotherapy. The most common side effect reported is skin 
irritation occurring under the transducer array. These reactions go away shortly after the TMZ and/or 
Optune are stopped. We are studying possible risks and reactions related to the TMZ and Optune. Current 
studies show that both TMZ and Optune are very safe to be used for GBM patients and no risk related to 
the use of TMZ and/or Optune has been reported so far. 
 
Risks of blood and CSF withdrawals:   Withdrawal of CSF (5 ml), blood (2 ml), and saliva (5 ml) from 
patients is a standard practice in the hospital. In general we do not expect more than a minor risk for 
patients. However, patients after withdrawal of CSF may have back pain for short time, or bleeding (this 
only happens to the patients with bleeding disorder and these subjects are excluded from the study) or 
infection. The incidence of having infection is very rare as the procedure will be very clean. In the case of 
back pain, patients will receive pain killer such as nonsteroidal anti-inflammatory drugs (NSAIDs), or 
Ibuprofen to stop pain.  In the case of infection, patients will receive antibiotics.    
 
Risks of Genetic Testing: In this study, only a few genes will be tested and this test will only help doctors 
understand that certain therapies are better for a particular subset of patients. The results will NOT show 
whether the disease is caused by genetic abnormalities or linked to a particular patient. Thus patient and 
patient’s family members will not face any problems in obtaining insurance coverage. However, in case 
there is possible any kind of risk to patients, or patient’s family members, to keep this from happening, 

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Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
the results of this test will NOT be given to anyone outside the study staff. This means that the results will 
not be made available to patients, patients’ family members, patients’ private physician, patients’ 
employer, patients’ insurance company, and/or any other party unless required by law.   
 
Use of Genomic Testing Information: In this study, the samples will only be used for the specific regions 
related potential biomarkers for GBM; the results from this test will not include the full genomic 
information about any patient. This information from this test should NOT affect patients’ insurance 
coverage. Insurance companies in Texas may not use the results of genetic testing to deny anyone 
insurance coverage, cancel their coverage, or increase their premiums. Even so, to avoid any potential 
risk, we will do everything possible to make sure that the results of genomic testing are not given to 
anyone except the study staff. The results of the testing will not be given to patients or patients’ family, 
will not be placed in patients’ medical record, and will not be given to patients’ private physician, 
patients’ employer, patients’ insurance company, and/or any other party unless required by law. To 
further protect against the misuse of this information, patients’ name will be removed from the results 
before the results are shared with the sponsor of the study. 
 
Risks of Genomic Testing: The genomic tests in this study will not show that patient’s disease is caused 
by genetic abnormalities; patients’ family members will not face problems in obtaining insurance 
coverage for this disease. However, to keep this from happening, the results of this test will NOT be given 
to anyone outside the study staff. This means that these results will not be made available to the patients, 
patients’ family members, patients’ private physician, patients’ employer, patients’ insurance company or 
any other party unless required by law. 
 
Benefits to the Subjects (if none, indicate none) 
There will be no benefit to the subjects for participating in this study but future patients may benefit from 
this study.  
 
Informed Consent (indicate how it will be obtained, who will obtain it, and how it will be documented; if 
not practicable, justify why it is not and how forgoing consent will not adversely impact the subjects’ 
rights and welfare, included their right to the privacy of their medical records) 
Taking part in this study is voluntary. All participants will be given an informed consent form and the 
doctors will tell the participants about any new information that may affect their health, welfare. The 
participants may choose not to take part or may leave the study at any time. If the individuals agree to 
take part in the study and then decide against it, those individuals can withdraw from the study for any 
reason. Deciding not to be in the study, or leaving the study early, will not result in any penalty or loss of 
benefits that you would otherwise receive. 
 
Conflicts of Interest Disclosure(s) (i.e. indicate/specify any conflicts of interests and how they will be 
managed) 
The doctor may be an investigator in this research study.  If so, s/he is interested both in patients’ medical 
care and in the conduct of this research.  All participants signing up for this study or at any time during 
the research may discuss their medical care with another doctor who is not associated with this research 
project.  The patients are not under any obligation to take part in any research study offered by their 
doctors. There are no financial interests to influence the results.   
 
6. References 
 
1. 
Lacouture ME, Davis ME, Elzinga G, et al. Semin Oncol,41 Suppl 4:S1-14.2014. 
2. 
Wong ET, Lok E, Swanson KD. Cancer Med,4:383-91.2015. 
3. 
Kanner AA, Wong ET, Villano JL, Ram Z. Semin Oncol,41 Suppl 6:S25-34.2014. 
4. 
Vymazal J, Wong ET. Semin Oncol,41 Suppl 6:S14-24.2014. 
5. 
Elzinga G, Wong ET. Case Rep Neurol,6:109-15.2014. 

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Baylor Scott & White Health ~ Central Texas Division ~ Office of Academic Research Development                              
(rev. Dec. 2014) 
 
6. 
Wong ET, Lok E, Swanson KD, et al. Cancer Med,3:592-602.2014. 
7. 
Butowski N, Wong ET, Mehta MP, Wilson LK. Semin Oncol,40:S2-4.2013. 
8. 
Fonkem E, Wong ET. Expert Rev Neurother,12:895-9.2012. 
9. 
Stupp R, Wong ET, Kanner AA, et al. Eur J Cancer,48:2192-202.2012. 
10. 
Ryken TC, Aygun N, Morris J, et al. J Neurooncol,118:435-60.2014. 
11. 
Ferla R, Bonomi M, Otvos L, Jr., Surmacz E. BMC Cancer,11:303.2011. 
12. 
Kawano T, Yanoma S, Nakamura Y, et al. Am J Otolaryngol,26:308-13.2005. 
 
 
7. Attachments/Appendices  
(not included in protocol, but uploaded to the application packet in iRIS as separate documents) 
- 
CVs of all listed on the study 
- 
Informed consent form 
- 
Case report forms, Data collection forms 
- 
Recruitment materials (emails, flyers, posters) 
- 
Questionnaires, surveys, focus group questions 
- 
Grant (if project is funded by a grant, internal or external) 
- 
Other research funding (departmental funding, start-up funds, etc.) 
- 
Budget 
- 
Others